Preface In three decades or so of widespread use, antibiotics have wrought a revolution in the medical, veterinary, and ...
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Preface In three decades or so of widespread use, antibiotics have wrought a revolution in the medical, veterinary, and agricultural sciences; indeed, in all of the biological sciences. Early research on antibiotics was necessarily directed at the production, isolation, characterization, and pharmacology of this important class of natural products. It was soon apparent that microorganisms could develop resistance to antibiotics and that this resistance was due, at least in part, to the possession of enzymes that could, in some fashion, chemically modify the antibiotic. TO keep ahead (or even abreast) of antibiotic resistance, it is necessary either to constantly discover new antibiotics or to develop derivatives that are insensitive to the enzymes that cause inactivation of the natural compound. Both avenues have been tried. It is evident that the search for new antibiotics must eventually reach the point of diminishing returns and that the second approach offers the best hope of extendiag the life of an antibiotic. Research on the biosyntheses of antibiotics as well as on enzymatic means of degradation has received less emphasis than other aspects of antibiotic chemistry, but, as it has become apparent that a knowledge of biosynthetic pathways can assist in isolating intermediate compounds capable of being modified chemically, this area has received more attention. Similarly, degradative enzymes have been found that are capable of providing antibiotic derivatives which can be chemically modified, allowing production of large numbers of semisynthetic antibiotics. Other practical uses of antibiotic enzymology include the use of enzymes as analytical reagents in determining the concentrations of antibiotics in samples and in the effective removal of antibiotics from reaction mixtures by converting them to inactive compounds. Aside from the utilitarian aspects of antibiotic enzymology a principal driving force behind research on these enzymes is the intellectual curiosity as to the raison d'etre of antibiotic synthesis. The production of these secondary metabolites, which serve no evident function in the producing organism, requires large quantities of energy and considerable metabolic machinery. In some instances very complicated pathways involving 20-30 enzymes are required to synthesize an antibiotic. As the pathways are unraveled and the branch points with normal metabolic routes are established, light may be shed on the mechanisms of antibiotic synthesis. Even with the renewed interest in antibiotic enzymology, the extent and scope of research in this area are uneven, and the published results are scattered throughout the scientific literature. The aim of this volume is to collate in one source as much information concerning antibiotic xiii
xiv
PREFACE
enzymology as possible. The work is divided into three sections. The first is concerned with methods used in the study of antibiotics, and covers techniques from culturing the producing organism to various chromatographic methods to sophisticated physical techniques. The second and third sections are devoted to enzymes involved in antibiotic biosynthesis and antibiotic degradation and modification, respectively. In some cases the division between the second and third section is quite arbitrary because it is not always clear whether an enzyme belongs in a biosynthetic or degradative pathway. The coverage of enzymes represents the state of the art of antibiotic enzymology; the range extends from pure enzymes that have been sequenced to enzymes that have been studied only in crude extracts. Many other enzymes that act on antibiotics, antibiotic precursors, or antibiotic derivatives have been detected in extracts or whole cells. Most of these had to be omitted because of a paucity of information. It is evident that only a small part of antibiotic enzymology has reached the stage where it can be consolidated into a treatise of this kind, and it is hoped that this volume will serve as a stimulus for further research on the enzymes involved with this important class of compounds. I am indebted to many people for many ideas and suggestions, but I am especially indebted to investigators in pharmaceutical laboratories throughout the world for their ideas and contributions. JOHN H. HASH
Contributors to Volume X L I I I Article numbers are in parentheses following the names of contributors. Affiliations listed are current. ABBOTT ( 5 5 b ) , Fermentation Cambridge, Massachusetts Products Research, Lilly Research JOHN E. DOWDING (48), Department o] Biochemistry, The University o] WisLaboratories, Eli Lilly and Company, Indianapolis, Indiana consin, Madison, Wisconsin COLETTE DUEZ (53f), Institut de BotaE. P. ABRAHAM (29, 55a), Sir William nique, Service de Microbiologie, UniDunn School o] Pathology, University o] Ox/ord, Ox]ord, England versitg de Liege, Sart Tilman, Liege, ADORJAN ASZALOS (8), The Squibb InstiBelgium tute ]or Medical Research, New E. F. ELSTNER (37), Department o] BioBrunswick, New Jersey, and Princeton logical Organic Chemistry, Albert Ein,~tein Medical Center, Philadelphia, University, Princeton, New Jersey MOHINDER S. BATHALA (17), College o/ Pennsylvania Pharmacy, The Ohio State University, AMEI)EO A. FANTINI (2), Department o] Microbiology, Lederle Laboratories, Columbus, Ohio Pearl River, New York VLADIMIR BETINA (7), Department o] PATRICIA FAWCETT (29, 55a), Sir William Technical Microbiology and BiochemDunn School o] Pathology, University istry, Faculty o] Chemistry, Slovak o] Ox]ord, Ox]ord, England PoIytechnical University, Bratislava, HEINZ G. FLOSS (34), Department o] Czechoslovakia Medicinal Chemistry and PharmacogDONALD B. BORI)ERS (10), Department o] nosy, School o] Pharmacy and PharFermentation and Isolation, Lederle macal Sciencies, Purdue University, Laboratories, Pearl River, New York West La]ayette, Indiana EBERHARD BREUKER (41), Lohman and Company AG, Neu]elder Strasse, JEAN-MARIE FRERE (53f), Institut de Botanique, Service de Microbiologie, 219 Cuxhaven, Germany Universit~ de Liege, Sart Tilman, RICHA~ BaU~NEa (31), Institut ]iir BioLiege, Belgium chemische Technologie und Mikrobiologie, Technischen HochschuIe DAvm FROST (8), The Squibb Institute ]or Medical Research, New Brunswick, Wien, Wien Getreidemarkt, Austria New Jersey JOHN H. COATS (58), Research Laboratories, The Upjohn Company, Kala- DAVID S. FUKUDA (55b), Lilly Research Laboratories, Eli Lilly and Company, mazoo, Michigan Indianapolis, Indiana M. COLE (54a, 54b), Research Division, STEN GATENBECK (30), Division o] Pure Beecham Pharmaceuticals Betchand Applied Biochemistry, The Royal worth, Surrey, England Institute o] Technology, Stockholm, JOHN W. CORCORAN (33), Department o] Biochemistry, Northwestern UniverSwede~ sity School o] Medicine, Chicago, G. M. GAUCHER (40), Department o] Chemistry, The University of Calgary, Illinois Calgary, Alberta, Canada LYMAN C. CRAIG (16), The Rocke]eUer JEAN-MARIE GHUYSEN (53f), Institnt de University, New York, New York Botaniq~te, Service de Microbiologie, JULIAN DAVIES (3), Department o] BioUniversitg de Liege, Sart Tilman, chemistry, The University o/ WisconLihge, Belgium sin, Madison, Wisconsin ARNOLD L. DEMAIN (52), Department o] MICHAEL J. HAAS (48), Department o] Biochemistry, The University o] WisNutrition and Food Science, Massaconsin, Madison, Wisconsin chusetts Institute of Technology,
BERNARD J.
ix
X
CONTRIBUTORS TO VOLUME XLIII
Inc., Nutley, New Jersey Microbiology, Vanderbilt University, LESTER A. MITSCHER (17), College o/ Pharmacy, The Ohio State University, School o/ Medicine, Nashville, Columbus, Ohio Tennessee GERHARD HEINRICH (41), Case Western NORBERT NEUSS (20), Lilly Research Laboratories, Eli Lilly and Company, Reserve School o] Medicine, CleveIndianapolis, Indiana land, Ohio ULFERT HORNEMANN (34), Department CYNTHIA H. O'CALLAGHAN (5), Bacterial Chemotherapy Unit, Glaxo Research o] Medicinal Chemistry and PharmaLimited, Green/ord, Middlesex, cognosy, School o] Pharmacy and England Pharmacal Sciences, Purdue UniverSEAN C. O'CONNOR (14), Lilly Research sity, West La]ayette, Indiana Laboratories, Eli Lilly and Company, EDWARD INAMINE (52), Developmental Indianapolis, Indiana Microbiology Department, Merck Sharp and Dohme Research Labora- HENRY PAULUS (44), Department o/Biological Chemistry, Harvard Medical tories, Rahway, New Jersey School, Boston, Massachusetts KENNETH JOHNSON (53f), Biochemistry Laboratory, National Research Council D. PERLMAN (60, 61), School o] Pharmacy, The University o/ Wisconsin, o] Canada, Ottawa, Canada Madison, Wisconsin FREDERICK KAVANAGH (4), 231 Blue PETER PFAENDER (41), Institut ]~r BioRidge Road, Indianapolis, Indiana logische Chemie und ErntihrungswisL. A. KOMINEK (35), Fermentation Resenscha/t, Universitat Hohenheim, search and Development, The Upjohn Hohenheim, Germany Company, Kalamazoo, Michigan SHINICHI KONDO (11, 12), Institute o] BURTON M. POGELL (36), Department o/ Microbiology, St. Louis University Microbial Chemistry, Shinagawa-ku, School o] Medicine, St. Louis, MisTokyo, Japan souri ZOFIA KUR,.~_~o-BoRoWSKA (42), The Rocke]eller University, New York, JOHN N. PORTER (1), Department o] Microbiology, Lederle Laboratories, New York Pearl River, New York SCREN G. LALAND (43), Department o] Biochemistry, University o] Oslo, Oslo, M. H. RICHMOND (6, 53C, 53d), Department o/ Bacteriology, The Medical Norway School, University o/ Bristol, Bristol, SUNG G. LEE (45), The Rocke]eller UniEngland versity, New York, New York ROBLEY J. LIGHT (39), Department o] HANSPETER RIEDER (41), Institut ]i~r Biologische Chemie und ErntihrungswisChemistry, Florida Stale University, senscha]t, Universittit Hohenheim, Tallahasee, FloT~da Hohenheim, Germany FRITZ LIPMANN (45), The Rocke]elIer JOHN H. ROBERTSON (9), Fermentation University, New York, New York Research and Development, The UpF. LYNEN (38), Max-Planck-Institut ]~r john Company, Kalamazoo, Michigan Biochemie, Munich, Germany GARY G. MARCONI (13), Lilly Research MAX RSHR (31), Institut ]~r Biochemische Technologie und MikrobioIogie, Laboratories, Eli Lilly and Company, Technische Hochschule Wien, Wien, Indianapolis, Indiana Austria It. F. MEYER (35), Fermentation Research and Development, The Upjohn GORDON W. Ross (5, 53e), Glaxo Research Limited, Green]ord, Middlesex, Company, Kalamazoo, Michigan England PHmIP A. MILLER (46, 47), Department o] Microbiology, Ho]]mann-La Roche W. A. SAVIDGE (54a, 54b), Biochemical
JOHN H. HASH (46, 47), Department o/
CONTRIBUTORS TO VOLUME X L I I I
Services Unit, Beecham Pharmaceuticals, Betchworth, Surrey, England W. V. SHAW (57), Department of Biochemistry, University of Leicester, Leicester, England HOWARD SIEGEBMAN (18), Princeton Applied Research Corporation, Princeton, New Jersey MAHAVIR M. SIMLOT (41), Agricultural Experimental Station, University of Udaipur, Udaipur (Rajasthan), India GEORGE SLOMP (19), Fermentation Research and Development, The Upjohn Company, Kalamazoo, Michigan JOHN SOGN (16), The Rockefeller University, New York, New York THEODORE S. SOKOLOSKI (17), College of Pharmacy, The Ohio State University, Columbus, Ohio MARILYN K. SPEEDIE (34), School of Pharmacy, Oregon State University, Corvallis, Oregon BRIAN SPENCER (32), Department of Biochemistry, University of Dublin, Trinity College, Dublin, Ireland R. J. SUHADOLNIK (37, 59), Department of Biological Organic Chemistry, Albert Einstein Medical Center, Philadelphia, Pennsylvania DAVID ~. THATCHER (53a, 53b), Department of Molecular Biology, University
xi
of Edinburgh, Edinburgh, Scotland KivosHi T s w i (9, 15), Control Analyti-
cal Research and Development, The Upjohn Company, Kalamazoo, Michigan W. UEMATSU (59), Department of Biological Organic Chemistry, Albert Einstein Medical Center, Philadelphia, Pennsylvania HAMAO UMEZAWA (11, 12), Institute of Microbial Chemistry, Shinagawa-ku, Tokyo, Japan HUBERT VANDERHAEGHE (54a, 54c), Rega Institute, University of Leuven, Leuven, Belgium L. C. VINING (56), Department of Biology, Dalhonsie University, Halifax, Nova Scotia, Canada GiiTNTEB VOGEL (38, 39), Max-PIanckInstitute ]iir Biologie, Tubingen, Germany JAMES B. WALKER (21, 22, 23, 24, 25, 26, 27, 28, 49, 50, 51), Department of Bio-
chemistry, Rice University, Houston, Texas MAR6ABET S. WALKER (50, 51), Department of Biochemistry, Rice University, Houston, Texas TRINE-LIsE ZIMMER (43), Department of Biochemistry, University of Oslo, OsIo, Norway
METHODS IN ENZYMOLOGY EDITED
BY
Sidney P. Colowick and N a t h a n O. Kaplan DEPARTMENT OF C H E M I S T R Y
VANDERBILT UNIVERSITY
UNIVERSITY OF CALIFORNIA
SCHOOL OF MEDICINE
AT SAN DIEGO
NASHVILLE, T E N N E S S E E
LA JOLLA, CALIFORNIA
I. II. III. IV. V. VI.
Preparation and Assay of Enzymes Preparation and Assay of Enzymes Preparation and Assay of Substrates Special Techniques for the Enzymologist Preparation and Assay of Enzymes Preparation and Assay of Enzymes (Continued) Preparation and Assay of Substrates Special Techniques VII. Cumulative Subject Index
XV
METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF
Sidney P. Colowick
Nathan 0. Kaplan
VOLUMEVIII. Complex Carbohydrates
Edited by ELIZABETH F. NEUFELD AND VICTOR GINSBURG VOLUME IX. Carbohydrate Metabolism
Edited by WILLIS A. WOOD VOLUME X. Oxidation and Phosphorylation
Edited by RONALDW. ESTABROOKAND MAYNARDE. PULLMAN VOLUME XI. Enzyme Structure
Edited by C. H. W. HIRS VOLUME XII. Nucleic Acids (Parts A and B)
Edited by LAWRENCEGROSSMANAND KIVIE MOLDAVE VOLUME XIII. Citric Acid Cycle
Edited by J. M. LOWENSTEIN VOLUME XIV. Lipids Edited by J. M. LOWENSTEIN VOLUME XV. Steroids and Terpenoids
Edited by RAYMOND B. CLAYTON VOLUME XVI. Fast Reactions
Edited by KENNETH KUSTIN VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B) Edited by HERBERTTABORANDCELIAWHITE TABOR VOLUMEXVIII. Vitamins and Coenzymes (Parts A, B, and C)
Edited by DONALDB. MCCORMICKAND LEMUELD. WRIGHT VOLUME XIX. Protcolytic Enzymes
Edited by GERTRUDEE. PERLMANNAND LASZLOLORAND VOLUME XX. Nucleic Acids and Protein Synthesis (Part C)
Edited by KIVIE MOLDAVEAND LAWRENCEGROSSMAN xvii
xviii
METHODS
IN
ENZYMOLOGY
VOLUME XXI. Nucleic Acids (Part D)
Edited by LAWRENCEGROSSMANAND KIVIE MOLDAVE VOLUME XXII. Enzyme Purification and Related Techniques Edited by WILLIAMB. JAKOBY VOLUME XXIII. Photosynthesis (Part A)
Edited by ANTHONY SAN PIETRO VOLUMEXXlV. Photosynthesis and Nitrogen Fixation (Part B) Edited by ANTHONY SAN PIETRO VOLUME XXV. Enzyme Structure (Part B)
Edited by C. H. W. HIRS AND SERGEN. TIMASHEFF VOLUME XXVI. Enzyme Structure (Part C)
Edited by C. H. W. HIRS AND SERGEN. TIMASHEFF VOLUME XXVII. Enzyme Structure (Part D)
Edited by C. H. W. HIRS AND SERGEN. TIMASHEFF VOLUME XXVIII. Complex Carbohydrates (Part B)
Edited by VICTOR GINSBURG VOLUMEXXIX. Nucleic Acids and Protein Synthesis (Part E)
Edited by LAWRENCEGROSSMANAND KIVlE MOLDAVE
VOLUMEXXX.
Nucleic Acids and Protein Synthesis (Part F)
Edited by KIVlE MOLDAVEAND LAWRENCEGROSSMAN VOLUME XXXI, Biomembranes (Part A) Edited by SIDNEY FLEISCHERAND LESTER PACKER VOLUME XXXII. Biomembranes (Part B)
Edited by SIDNEY FLEISCHERAND LESTER PACKER VOLUMEXXXIII. Cumulative Subject Index Volumes I-XXX
Edited by MARTHAG. DENNIS AND EDWARDA. DENNIS VOLUMEXXXIV. Affinity Techniques (Enzyme Purification: Part B) Edited by WILLIAMB. JAKOBYAND MEIR WILCHEK
METHODS IN ENZYMOLOGY
xix
VOLUME XXXV. Lipids (Part B) Edited by JOHN M. LOWENSTEIN VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones)
Edited by BERT W. O'MALLEYAND JOEL G. HARDMAN VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones)
Edited by BERT W. O'MALLEYANDJOEL G. HARDMAN VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides) Edited by JOEL G. HARDMANANDBERT W. O'MALLEY VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems) Edited by JOEL G. HARDMANANDBERT W. O'MALLEY VOLUMEXL. Hormone Action (Part E: Nuclear Structure and Function)
Edited by BERT W. O'MALLEYAND JOEL G. HARDMAN VOLUME XLI. Carbohydrate Metabolism (Part B)
Edited by W. A. WOOD VOLUME XLII. Carbohydrate Metabolism (Part C)
Edited by W. A. WooD VOLUME XLIII. Antibiotics
Edited by JOHN H. HASH
[1]
ANTIBIOTIC-PRODUCING MICROORGANISMS
3
[1] Cultural Conditions for Antibiotic-Producing Microorganisms By
JOHN N. PORTER
I. II. III. IV. V.
Introduction . . . . . . . . . . . . . . . . . . Culture Maintenance . . . . . . . . . . . . . . . . Culture Preservation . . . . . . . . . . . . . . . . . Antibiotic Production in Liquid Culture . . . . . . . . . . Selected Antibiotic Fermentations by Fungi . . . . . . . . . A. Cephalosporin C . . . . . . . . . . . . . . . . B. Griseofulvin . . . . . . . . . . . . . . . . . C. Penicillin . . . . . . . : . . . . . . . . . . VI. Selected Antibiotic Fermentations by Eubacteria . . . . . . . A. Bacitracin . . . . . . . . . . . . . . . . . . B. Polymyxins . . . . . . . . . . . . . . . . . VII. Selected Antibiotic Fermentations by Actinomycetes . . . . . . A. Chloralnphenicol . . . . . . . . . . . . . . . . B. Chlortetracycline . . . . . . . . . . . . . . . . C. Erythromycin . . . . . . . . . . . . . . . . . D. Gentamicin . . . . . . . . . . . . . . . . . E. Neomycin . . . . . . . . . . . . . . . . . . F. Streptomycin . . . . . . . . . . . . . . . . .
3 3 8 II 14 14 15 16 16 16 17 18 18 19 20 21
22 23
I. I n t r o d u c t i o n Of t h e a p p r o x i m a t e l y 3000 k n o w n a n t i b i o t i c s a b o u t 70% are d e r i v e d f r o m a c t i n o m y c e t e s , e s p e c i a l l y species of Streptomyces, 2 0 % f r o m fungi, a n d 10% f r o m e u b a c t e r i a . M o s t of the p r o d u c i n g o r g a n i s m s , b u t b y no m e a n s all, were i s o l a t e d f r o m soils in p r o g r a m s designed to d i s c o v e r new a n t i b i o t i c s . Some of t h e m o r e p r o m i n e n t c u l t u r e collections f r o m which m a n y of these o r g a n i s m s are o b t a i n a b l e a r e t h e following: A T C C - A m e r i c a n T y p e C u l t u r e Collection, R o c k v i l l e , M a r y l a n d ; C B S - - C e n traalbureau voor Schimmelcultures, Baarn, The Netherlands ; CMI--Comm o n w e a l t h M y c o l o g i c a l I n s t i t u t e , K e w , E n g l a n d ; I F O - - I n s t i t u t e for F e r m e n t a t i o n , O s a k a , J a p a n ; N C I B - - N a t i o n a l C o l l e c t i o n of I n d u s t r i a l Bacteria, Aberdeen, Scotland ; NRRL--Northern Utilization Research and D e v e l o p m e n t D i v i s i o n , U.S. D e p a r t m e n t of A g r i c u l t u r e , P e o r i a , Illinois. II. Culture Maintenance
The g r o w t h r e q u i r e m e n t s of m i c r o o r g a n i s m s cover a v e r y wide r a n g e of conditions. H o w e v e r , few a n t i b i o t i c p r o d u c e r s are p a r t i c u l a r l y f a s t i d i ous, a n d m o s t can be r e a d i l y c u l t i v a t e d b y t h e p r o p e r selection of m e d i a a n d o t h e r e n v i r o n m e n t a l factors.
4
METHODS FOR T H E STUDY OF ANTIBIOTICS
[1]
Culture Media. Solid media for maintaining eubacteria commonly contain proteins or peptones as sources of both carbon and nitrogen. Media for growing fungi, on the other hand, more often contain a carbohydrate to supply carbon, and nitrates or ammonium salts to supply nitrogen. Actinomycetes, which are related to the eubacteria but not to fungi, are ordinarily grown on media containing both a carbohydrate and an organic nitrogen source. Since spores have more prolonged viability than vegetative cells, a maintenance medium should encourage sporulation rather than vegetative growth. Czapek's agar, for example, serves as a good medium for species of Penicillium and Aspergillus, fungi which grow sparsely on this medium but sporulate readily. I n the preparation of media containing agar, the media should be heated for 15-30 min at 100 ° in order to melt the agar before dispensing. Sterilization of small volumes of media, such as in test tubes and small flasks, is carried out in an autoclave at 120 ° for 15 min at 15 psi. Large volumes require longer sterilization times. Formulations for some of the most commonly used agar media are given below. Some m a y be obtained commercially and should be prepared as directed on the container. M E D I A FOR MAINTAINING FUNGI 1,2
Potato dextrose agar Potatoes, peeled and sliced Distilled water
300 g 1000 ml
Boil the potatoes or heat momentarily in 500 ml of water in an autoclave at 121°. Filter through cheesecloth. Make up the volume to 1000 ml and add Glucose Agar
20 g 15 g
Heat to boiling, mix, then dispense and autoclave. Czapek's solution agar NaNOa K~HPO4 MgSO4 -7H20 KCI FeSO4 •7H20 Agar Distilled water to volume
(g/liter) 3 1 0.5 0.5 0.01 15
Melt and add sucrose 30 g/liter, pH not adjusted. 1F. A. Weiss, in "Manual of Microbiological Methods," p. 99. McGraw-Hill, New York, 1957. *W. C. Haynes, L. J. Wickerham, and C. W. Hesseltine, Appl. Microbiol. 3, 361 (1955).
[1]
ANTIBIOTIC-PRODUCING MICROORGANISMS
5
Malt extract agar
Malt extract Glucose Peptone Agar Distilled water to volume p H n o t adjusted
(g/liter) 20 20 1 20
MEDIA FOR MAINTAINING EUBACTERIA3
Nutrient agar
(g/liter) 3 5 15
Beef extract Peptone Agar The addition of yeast extract (5 g/liter) is optional. TG Y a#ar
Tryptone Yeast extract Glucose K~HPO4 Agar Adjust to pH 7.0
(g/liter) 5 5 1 1 2O
MEDIA FOR MAINTAINING ACTINOMYCETES4'5
Yeast extract agar
Yeast extract Malt extract Glucose Agar Distilled water to volume Adjust to pH 7.3 with NaOH
(g/liter) 4 10 4 20
3 The American Type Culture Collection. Catalogue of Strains, 10th ed., 1972. T. G. Pridham, P. Anderson, C. Foley, L. A. Lindenfelser, C. W. Hesseltine, and R. G. Benedict, Antibiot. Annu. 1956/1957, p. 947 (1957). 5S. T. Williams and T. Cross, in "Methods in Microbiology" (J. R. Norris and D. W. Ribbons, eds.), Vol. 4, p. 295. Academic Press, New York, 1971.
6
METHODS FOR THE STUDY OF ANTIBIOTICS
[1]
Tomato paste oatmeal agar
(g/liter) Solution 1 Heinz baby oatmeal Contadina fancy tomato paste Add to 500 ml of boiling tap water Solution 2 Agar Tap water
20 20
15 500 ml
Melt agar by steaming at 100° for 15-20 min. Mix the two solutions, steam at 100 ° for 10 rain, dispense, and sterilize. No pH adjustment. Oatmeal agar
Oatmeal Agar
(g/liter) 20 18
Cook or steam the oatmeal in 1 liter of distilled water for 20 min. Filter through cheesecloth. Add water to restore volume to 1 liter. Add 1 ml of trace salts solution. Adjust pH to 7.2 with NaOH and add agar. Pridham and Gottlieb trace salts solution in g/liter: CuSO4.5H20, 0.64; FeSO4.7H~O, 0.11; MnC12- 4H~O, 0.79; ZnS04.7H~O, 0.15. Bennett's agar
Yeast extract Beef extract N-Z Amine A (Casein digest: Sheffield Farms) Glucose Agar Distilled water to volume
(g/liter) 1 1 2 10 15
Trypticase-yeast extract agar 6 (Recommended for thermophilic actinomycetes)
Trypticase Yeast extract Sucrose Dung extract Molasses MgSO4 - 7H20 FeSO4.7H~O Microelement solution Agar Distilled water to volume Adjust pH 7.0-7.2
(g/liter) 5 3 5 5 ml 5 ml 0.5 0.01 1 ml 20
Dung extract: suspend 25% dried sheep manure in tap water, autoclave for 30 min, filter, refrigerate under toluene. M. D. Tendler and P. R. Burkholder, Appl. Microbiol. 9, 394 (1961).
[1]
ANTIBIOTIC-PRODUCING MICROORGANISMS
7
Microelement stock solution (per ml): Fe [as Fe(NH4)2(SO4)2], 1.0 mg; Zn (as ZnSO4), 1.0 mg; ]V[n (as MnSO4), 0.5 mg; Cu (as CuSO~), 0.08 rag; Co (as COSO4), 0.1 rag; B (as H3BO3), 0.1 mg. N - Z amine-starch-glucose medium ~
(Recommended for maintaining Micromonospora)
Glucose Soluble starch Yeast extract N-Z Amine A (Casein digest; Sheffield Farms) CaCO~ Agar Distilled water to volume
(g/liter) 10 20 5 5 1 15
Temperature. Most antibiotic-producing microorganisms are mesophilic; that is, the optimum temperature for growth is somewhere in the range of 23-37 °. A few, particularly those from marine sources, are psychrophilic and have an optimum temperature below 20 °. Antibiotics are also known to be produced by certain thermophilic actinomycetes with optima above 50 °. Agar media on which thermophiles are grown tend to dry out rapidly, and sufficient moisture should be provided during the period of incubation. The majority of fungi grow well at room temperature, ordinarily 20-22 °, and when incubators are used the temperature is usually set at 23-25 °. Some fungi are thermotolerant and grow satisfactorily above 30 °, but most do not. Actinomycetes and eubacteria may also be grown at these temperatures, but actinomycetes in particular are normally incubated at a somewhat higher temperature, e.g., 28 °. While pathogenic bacteria frequently require incubation temperatures of 37 ° , most antibiotic-producing strains are grown at temperatures of 32 ° or lower. pH. Fungi grow well in acid environments, but they are not restricted to them. Most will grow normally within a pH range of 4.5 to 8.0. However, the range for sporulation is generally narrower than for growth. M a n y bacteria and actinomycetes, on the other hand, will not grow at a pH much below 5.5-6.0 and the optimum lies between 6.5 and 7.5. In fact, an alkaline environment tends to encourage actinomycetes. The pH changes during growth depend on a number of factors, the most important of which are the buffering capacity of the medium and the type of metabolites produced. Aeration. Practically all antibiotic producers are highly aerobic and grow on the surface of agar and liquid media. As will be pointed out later, oxygen must be provided in deep fermentations. Light. Most fungi grow and sporulate well in the light but some sporu-
8
METHODS FOR THE STUDY O F ANTIBIOTICS
[1]
late better in the dark. It is a safer procedure to avoid incubating cultures in sunlight, however, because certain actinomycetes in particular will not grow or will not sporulate in the presence of light. III. Culture Preservation
Periodic subculture of strains to fresh media may lead to a loss of the ability to produee antibiotics or to other biosynthetic changes. Frequent transfer may also allow low-producing strains to become predominant and in this case it is necessary to plate out the culture and reisolate the desired colony type. Storing cultures at refrigerator temperatures or under oil will reduce or eliminate the possibility of deterioration. A safer method than refrigeration for preserving viability is simply to place fresh agar slants in a freezer, with the caution that repeated freezing and thawing is detrimental to survival. Culture collections, particularly the larger ones, use the lyophil technique because of reliability and the fact that large numbers of cultures can be stored in a relatively small space. Some nonsporulating cultures, however, may be lost by using this method. In these cases storage at very low temperature under liquid nitrogen is the most dependable procedure. Periodic Trans]er. Mycelial organisms, such as fungi and Streptomyces, are grown on test tube slants composed of an agar medium that favors sporulation. Transfers of spore masses are then made periodically by means of a wire loop to fresh slants of the same medium. With nonsporulating cultures, transfers should be made at frequent intervals from young, actively growing marginal areas where these are evident. In making the transfer, a portion of the agar supporting the mycelium should also be dug out and the material on the loop smeared on the surface of the new slant. Cultures should be incubated at an appropriate temperature until they reach maturity and then stored at about 5 ° . Soil Culture. 4,%s For actinomycetes, a loamy soil at neutral or slightly alkaline pH is preferred. If the soil is acid, add 1 g of CaCO3 per 100 g of soil and if lacking in organic matter add 0.25 g of dried blood or casein at the same rate. Enough water should be added to reach a level of about 60% of the maximum water-holding capacity. Place in tubes or flasks, plug with cotton, and autoclave at 15 psi for 1 hr or, alternatively, four times for 30 min each on alternate days. Inoculate with a ' S. A. Waksman, "The Actinomycetes," Vol. I, pp. 26-27. Williams & Wilkins, Baltimore, Maryland, 1959. s D. I. Fennell, Bot. Rev. 26, 79 (1960).
[1]
ANTIBIOTIC-PRODUCING MICROORGANISMS
9
spore suspension, substrate mycelium or young broth culture and incubate at the optimal temperature for a particular culture, preferably with occasional shaking. Allow soil to desiccate after growth. For fungi, inoculate 5 g of orchard loam soil (20% moisture) with 1 ml of a heavy aqueous suspension of conidia. Dry at room temperature and store in the refrigerator. If growth rather than simply retention of spores is required, then larger inocula or more soil moisture should be used followed by a period of incubation. Mineral Oil. s This method is used with agar slants and has been preferred for some nonsporulating cultures which are not particularly amenable to lyophilization. Autoclave a medicinal grade mineral oil at 121 ° for 2 hr and dry in an oven at 170 ° for 1-2 hr. Cover slants completely with a layer at least 1 cm deep of the sterile oil. None of the agar should remain uncovered. Cultures are then stored at the same temperature at which unsealed cultures are normally stored. Freezing. Grow cultures on a medium conducive to sporulation if possible. When mature, place in a deep freeze unit at approximately --22% Prevent unnecessary thawing since repeated thawing and freezing reduces viability. Liqzdd Nitrogen. The method of storing cultures under liquid nitrogen as described below is essentially that of Hwang2 Nonsporulating filamentous cultures are grown on appropriate agar media in petri dishes. Upon maturity cut agar plugs with a No. 3 cork borer and transfer the plugs to sterile cotton-plugged glass vials by means of a sterile spatula. Add 0.8 ml of sterile 10% glycerol or 10% dimethyl sulfoxide. Remove the cotton plugs and heat-seal the ampules. The ampules may be cooled rapidly by plunging directly into liquid nitrogen or slowly frozen to --35 ° and then rapidly to --196 ° by immersing in liquid nitrogen or by the sudden introduction of cold nitrogen gas into the cooling chamber. Store in a liquid nitrogen refrigerator at --150 ° to --196 °. To retain maximum viability the cultures should be retrieved by thawing for 0.5-1 min in an agitated water bath at 38-40 ° . Sporulating cultures may be preserved by the same method, except that harvested spores rather than agar plugs are suspended in 10-15% glycerol or dimethyl sulfoxide. Lyophilization. The basic steps in this process involve the preparation of a dense suspension of cells in a selected fluid, freezing the suspension in vials or ampules in a dry ice bath, evacuating the vials while frozen until the contents are completly desiccated, and then hermetically sealing the evacuated containers. There are several modifications of the 9S. Hwang, Mycologia 60, 613 (1968).
10
METHODS FOR THE STUDY OF ANTIBIOTICS
[1]
lyophil process and the equipment to carry it out from which the investigator may choose in selecting the method most suitable to his particular problem. With some suggested possible variations the procedure outlined below is that of Raper and Alexander 1° and has been in use at the Northern Regional Research Laboratory, Peoria, Illinois, and in other laboratories for many years. Grow fungi and actinomycetes on appropriate maintenance media in test tubes or petri dishes until abundant spores are produced, normally a matter of 7-10 days. Place approximately 0.25 ml of a sterile suspending liquid in a plugged and sterilized agglutination tube and add spores to make a dense suspension. Nonsporulating strains can be homogenized in the suspending medium before freeze drying. For bacteria, wash down the culture growing on an agar slant with the suspending agent to form a dense suspension of cells or centrifuge about 40 ml of a liquid culture, decant, and take up the pellet in the suspending fluid. Some of the more popular suspending media, to be used only after sterilization, are beef serum, double-strength skim milk, 10% high molecular weight dextran, or a gelatin/sucrose solution (5%/5%). Use a micropipette to dispense about 0.05 ml of suspension into each of four sterile lyophil tubes or ampules. Micropipettes can be drawn from 10-mm glass Pyrex tubing. Lyophil tubes may be made of 4-inch lengths of 6-ram Pyrex glass tubing, sealed at one end and lightly fire-polished at the other, plugged with cotton, and sterilized. After adding a suspension to a lyophil tube replace the cotton plug, burn off the excess cotton, and push the cotton about 0.5 inch into the tube. Attach lyophil tubes to a manifold by means of rubber sleeves. An important and frequently overlooked step at this point is "degassing." Enough vacuum should now be used to bubble off most of the air present in the material before freezing. Turn off the vacuum and lower the manifold into a bath of dry ice in either Methyl Cellosolve or 95% ethanol. When the tubes are completely frozen, raise them above the surface of the bath, where a temperature of about --10 ° is maintained, and begin evacuation by means of a vacuum pump. Best results are obtained with a vacuum between 200 and 500 ~m of mercury although some investigators using other apparatus recommend 70 to 100 ~m. The time of drying may be from less than 2 to more than 18 hr, depending on the nature of the organism and the preference of the investigator. By some methods water vapor removed from frozen preparations is taken up in a column of anhydrous calcium sulfate. 1oK. B. Raper and D. F. Alexander, MycoIogia 37, 499 (1945).
[1]
ANTIBIOTIC-PRODUCING MICROORGANISMS
ll
When the pellets are dry continue evacuation at room temperature for 0.5 hour. Then seal off the tubes under vacuum with a gas-oxygen torch. Refrigerated storage of lyophilized preparations is preferable to storage at room temperature.
IV. Antibiotic Production in Liquid Culture If only small quantities of metabotites are desired, it is customary to grow cultures in selected liquid media in flasks placed on a reciprocating or rotary shaking apparatus. Occasional use is also made of stationary cultivation on liquid media and on solid substrates, such as agar, bran, corn meal. When larger quantities of metabolites are needed it is necessary to scale up the fermentation into stirred, aerated bottles or tanks. In this case the conditions which have first been established in flasks may have to be modified to produce maximum results in larger volumes of media. The procedures and culture media described in this section are for the most part those pertaining to antibiotic production in shake flasks. In general, microorganisms can be grown in the same media minus agar that are employed for culture maintenance. Other environmental conditions, such as temperature, pH, also apply. However, these conditions, especially media, do not necessarily promote a desirable level of antibiotic activity. It may be necessary, therefore, to undertake a systematic study of a number of environmental factors before such a level is achieved when dealing with an organism for which appropriate conditions have not been established. If a strain selection and improvement program is undertaken, it should be kept in mind that selected strains sometimes require conditions somewhat different from those which gave superior results with the original strain. Inoculum. Inoculation of a very limited number of fermentation flasks can be accomplished directly from agar slants to fermentation media. Add several milliliters of sterile tap water or saline solution (0.8% NaC1) to the slant and vigorously scrape the surface with a wire loop to make a suspension of spores and vegetative cells. At this point the use of a vortex test tube mixer is sometimes employed in order to enhance the uniformity of the cell suspension, but splashing of the contents onto the plug or cap should be avoided. Using a pipette with a large tip orifice deliver the suspension into liquid media at the rate of about 3% v/v. It is not advisable to initiate a fermentation by digging out a small piece of agar and adding this to the fermentation flask. If more than two or three fermentation flasks are to be used it will
12
METHODS FOR T H E STUDY OF ANTIBIOTICS
[1]
be necessary to employ an intervening liquid inoculum step to provide enough volume. Practical medium/flask relationships are 30 ml of medium in a 250-ml Erlenmeyer flask or 100 ml in a 500-ml flask. Inoculate these flasks as described above and place on a shaker at a suitable temperature: normally 25 ° for fungi, 28-37 ° for eubacteria, and 28 ° for actinomycetes. Incubate long enough to provide a young actively growing inoculum for the fermentation step: in most cases overnight for eubacteria and 36-72 hours for fungi and actinomycetes. Levels of inoculum used for antibiotic fermentations vary, but 3-5% v / v is a useful range. Fermentation, The word fermentation is now commonly employed in relation to antibiotic production under the modern broad definition of the term, namely, the production of metabolites by microorganisms on culture media. The most important environmental conditions to be considered in carrying Out a successful antibiotic fermentation are medium, aeration, pH, and temperature. Each culture passes through the stages of growth, product formation, and senescence and the goal of fermentation improvement is to increase and extend the period of biosynthesis-that is, to achieve and maintain a high level of the desired active principle. Practically all antibiotic producers are highly aerobic organisms. Aeration must be provided, and this is done by placing flasks on a rotary or reciprocating shaking machine for the duration of the fermentation. In addition to the speed of shaking, increased aeration can be obtained by using flasks with indentations or by reducing the volume in each flask. Neomycin is an example of an antibiotic produced in higher yield under conditions of increased aeration. In deep fermentations, air is supplied both by vigorous stirring and by the introduction of air at the bottom of the fermentor. Foaming in tank fermentations then frequently becomes a problem and can be controlled by the addition of one of several agents, a popular example of which is 3% octadecanol in lard oil. However, this may be toxic to some organisms, causing depressed yields, and another type of agent should be used. Most antibiotic fermentations start at a pH close to neutrality, drop to about 6.0, and then rise to 8.0 or above as the carbohydrate becomes exhausted. Maximum antibiotic production often occurs during the earlier stages of this rise in pH. The inclusion of phosphates and CaCO3 in culture media exerts a buffering and stabilizing effect on the fermentation. Occasionally, sterile NaOH or H2S04 is added during the course of the fermentation to maintain a particular pH range. In griseofulvin production, glucose syrup is added at intervals to keep the pH from rising much above 7.0, the optimum for production of the antibiotic. The media used in antibiotic production originate from laboratory
[1]
ANTIBIOTIC-PRODUCING MICROORGANISMS
13
studies whose purpose is to elicit maximum yields from a particular strain of organism. Such studies may also have the additional purpose of obtaining significant yields of one particular component from among two or more components which a culture is capable of making. A wide variety of substrates from which to choose is available. Carbon sources may include such carbohydrates as the various sugars, starch, or glycerol and a number of plant oils. Commonly used nitrogen sources are amino acids, casein and milk products, animal and plant meals, corn steep liquor, meat extract, and various peptones. Growth-promoting substances, such as distillers' solubles or yeast extract, may be added. Sodium chloride, di- and monopotassium phosphate, magnesium sulfate, sodium nitrate and other salts may be included for supplementary or buffering purposes. However, many of the natural products contain enough material to satisfy any requirements for trace elements. Some representative media for specific antibiotic fermentations are given at the end of this account. In addition to these, there are general purpose media which have been found empirically useful in screening programs to discover new antibiotics because diverse species of microorganisms are capable of producing acceptable antibiotic levels on them. These media can frequently be used for both inoculum and fermentation purposes, and some are suitable for all three major groups of antibioticproducing microorganisms. Warren et al. 1~ listed a number of general purpose media, the following four examples of which appeared to them to be superior. Medium A-4
Soybean meal Glucose NaC1 CaCO~
(g/liter) 10 10 5 1
Medium A-~h
Soybean meal Glucose Curbay BG Glycerol NaCI CaCO3
(g/liter) 15 15 5 2.5 5 1
11It. B. Warren, Jr., J. F. Prokop, and W. E. Grundy, Antibiot. Chemother. (Washington, D.C.) 5, 6 (1955).
14
METHODS FOR THE STUDY OF ANTIBIOTICS
[1]
Medium A-9 (g/liter) 5 10 20
Peptone Glucose Molasses
Medium A-l$ Soybean meal Corn steep liquor Dextrin NaC1 CaCO, K2HPO4
(g/liter) 10 20 10 5 2 2
V. Selected A n t i b i o t i c F e r m e n t a t i o n s b y F u n g i
A. Cephalosporin C Organism. Cephalosporium acremonium grown on C z a p e k - D o x agar with 5 % lactose s u b s t i t u t e d for sucrose.
Media Inoculum medium TM Corn steep solids Ammonium acetate Sucrose Adjust to pH 7.2
(g/liter) 11 4.4 20
Complex medium TM Meat extract Fish meal Corn steep solids Ammonium acetate Sucrose Glucose n~Methionine Rotary shaker, 220 rpm, 28°
(g/liter) 10 10 2.5 2 36 9 0.5
12A. L. Demain and J. F. Newkirk, Appl. Microbiol. 10, 321 (1962).
[1]
ANTIBIOTIC-PRODUCING MICROORGANISMS
15
Defined medium 13 Sucrose Glucose DL-Methionine Oleic acid (NH4)~SO4 KH~PO4 K2HPO4 Na~S04 MgS04" 7H20 Fe(NH4) ~(S04)2.6H20 CaC12 MnSO~ ZnSO4.7H20 CuSO4 •5H20 Distilled water to volume
(g/liter) 36 27 5 1.5 7.5 15 21 O. 75 0.18 0.15 0.06 0.03 0.03 0. 0075
Autoclave glucose separately; adjust initial pH to 7.4; optimum yields are obtained when the flask capacity is 5 times the volume of the medium.
B. G r i s e o f u l v i n Organism.
Penicillium
spp.,
especially
P.
patulum
Bainier-Thom
gr o wn on C z a p e k - D o x agar.
Media Corn steep-lactose medium 14 Corn steep liquor Lactose KC1 KH2PO, CaCO3
(g/liter) (to give 0.15% N) 70 1 4 8
Inoculate with 1-10% of a well-grown vegetative inoculum; incubate on a shaker at 25 ° for 10-i2 days.
Corn steep-glucose medium (for tank fermentation)14 Corn steep liquor CaCO~ KH~PO4 KCI Antifoam agent
(g/liter) (to give 0. 175% N) 4 4 1.5 0.05
Adjust the pH to 5.5 before sterilization inoculate with 10% vegetative inoculum. Glucose syrup is added after the ninth hour at a rate of 0.75 liter per 400 liters of medium per hour to maintain the pH as close to 7.0 as possible. 13A. L. Demain, J. F. Newkirk, and D. Hendlin, J. Bacteriol. 85, 339 (1963). " A . Rhodes, in "Progress in Industrial Microbiology" (D. J. D. Hockenhull, ed.), Vol. IV, p. 165. Gordon & Breach, New York, 1964.
16
METHODS FOR THE STUDY OF ANTIBIOTICS
[1]
C. P e n i c i l l i n Organism. Penicillium chrysogenum. Media Complex medium 15 Lactose Corn steep solids KH2PO4 MgSO4 •7H20 NaNOs ZnS04 •7H~O CaCOs Dow-Corning silicone antifoam
(g/liter) 20 20 0.5 0.25 3 0.04 2.75 2 drops
Adjust to initial pH of 4.7; incubate on a shaker at 22-24 °.
Defined medium 18 Lactose Glucose Starch Acetic acid Citric acid Phenylacetic acid (NH4) 2SO4 Ethylamine Distilled water to volume
(g/liter) 30 10 15 2.5 10 0.5 5 3
VI. Selected Antibiotic Fermentations by Eubacteria
A. B a c i t r a c i n Organism. Bacillus subtilis m a i n t a i n e d on n u t r i e n t agar. Media Complex medium 17 Defatted soya flour Sucrose (NH4)~SO4 CaCO3
(g/liter) 70 12 2 2
lS B. W. Churchill and J. F. Stauffer, in "Biogenesis of Antibiotic Substances" (Z. Vanek and Z. Host£lek, eds.), p. 43. Academic Press, New York, 1965. 1~D. J. D. Hockenhull, in "Biochemistry of Industrial Microorganisms" (C. Rainbow and A. H. Rose, eds.), p. 227. Academic Press, New York, 1963. " J . Ziffer, U.S. Patent 2,813,061 (1957).
[ll
ANTIBIOTIC-PRODUCING MICROORGANISMS
17
Inoculate with 0.5% by volume of a 20-24 hr culture of an appropriate strain of
B. subtilis; incubate at 28 ° on a rotary shaker, 225 rpm, for 45 hr. Defined medium is (g/liter) L-Glutamic acid Glucose Citric acid K2HPO4 KH2PO4 MgSO4 • 7H~O MnSO4- 4H~O FeSO4.7H20 Distilled water to volume
l0 5 1 0.5 0.5 0.2 0.01 0.01
Adjust to p H 6.8-7.0 with N a O H before sterilization; sterilize glucose separately in solution and add aseptically. Inoculate each flask from an agar slant or with 48-hr submerged growth. Incubate at 28 ° on a rotary shaker, 220 rpm.
B. P o l y m y x i n s
Organism. Bacillus polymyxa m a i n t a i n e d o n n u t r i e n t a g a r . Media Nutrient broth-glucose medium (polymyxins A, B, and E)19 (g/liter) N u t r i e n t broth MnSO4 Glucose (NH4) 2HPO4
100 ml 0.02 30 6
For inoculum grow the culture in nutrient broth for 18-24 hr at 37°; use at 5 % v / v . Incubate flasks as static shallow layers or on a shaker at 22-28 ° for 20-24 hr.
Corn steep-glucose medium (polymyxin D)20 (g/liter) Corn steep liquor solids Glucose CaCO3
20 40 10
Inoculate with 2.5-4% v / v liquid inoculum grown 18 hr at 30 °. Incub'~te flasks on a shaker for 4 days at 30 °. is D. Hendlin, Arch. Biochem. Biophys. 24, 435 (1949). 1~G. C. Ainsworth and C. G. Pope, U.S. Patent 2,695,261 (1954). ,0 R. G. Benedict and F. H. Stodola, U.S. Patent 2,771,397 (1956).
18
METHODS FOR THE STUDY OF ANTIBIOTICS
[1]
Defined medium (polymyxin B) ~1 (g/liter) 5
Glucose
(NH4)~S04
1.5
MgSO4.7H20 NaC1 CaCI~ FeSO4.7H~O ZnSO4 MnSO4.4H20 Potassium phosphate Biotin
0.2 0.1 0.1 0.01 0.01 0. 0075 40 ml of 0.5 M, pH 7.5 0.5 gg
Heat to boiling and filter before autoclaving; add glucose as a 50% solution aseptically after sterilization. Incubate without shaking at 30°. The following starter medium for this fermentation is suggested: (in g/liter) glucose, 10; (NH4)2S04, 20; Bacto yeast extract, 5; K~HP04, 2.6; MgSO4.7H~O, 0.5; NaC1, 0.05; FeSO4.7H~O, 0.01. V I I . Selected A n t i b i o t i c F e r m e n t a t i o n s b y A c t i n o m y c e t e s A. C h l o r a m p h e n i c o l
Organism. S t r e p t o m y c e s venezuelae. Media Complex medium ~2 Glycerol Tryptone NaC1 B.Y. fermentation solubles Distilled water to volume
(g/liter) 10 5 5 5
Defined medium 2~ Glycerol Serine NaC1 Sodium lactate K~HPO4 -3H~O KH2PO4 MgSO4.7H20 Distilled water to volume
(g/liter) 10 5 3 11 2.8 1.4 2
Adjust above media to pH 7.0; inocula can be prepared on the same media; incubate inocula on a shaker for 3 days at 26°. 21H. Paulus and E. Gray, J. Biol. Chem. 239, 865 (1964). = M. Legator and D. Gottlieb, Antibiot. Chemother. (Washington, D.C.) 35 809 (1953).
[1]
ANTIBIOTIC-PRODUCING MICROORGANISMS
19
B. Chlortetracycline Organism. S t r e p t o m y c e s aureo]aciens. Media Inoculum medium 2~ Sucrose Corn steep liquor CaCOa (NH,)2SO4
(g/liter) 30 20 7 2
I n c u b a t e a t 26.5 ° for 24 hr on a r o t a r y shaker, 185 r p m ; use at the rate of 4 % v / v .
Starch-corn steep medium 23 (g/liter) 55 25 9 5 1.5 0.06 0.05 0.1 Add individually to flasks at the rate of 20 m l / l i t e r
Starch Corn steep liquor CaCO3 (N H4) 2SO~ NH~C1 FeSO~- 7H20 MnSO~. 4H20 ZnSO4 •7H~O Lard oil
Defined medium 24 CaCO8 (NH4)~SO4 NH4CI MgCl~_ • 6H~O KC1 H3PO4 FeS04.7H20 ZnSO4.7H20 MnSO4 •4H20 CoC12.6H~O Starch (or sucrose) L-Histidine I,-Methionine Lard oil
(g/liter) 9 5 1.5 2 1.3 0.4 0.06 0.1 0.05 0. 005 55 0.8 0.8 Add individually to flasks at the rate of 20 m l / l i t e r
I n c u b a t e at 26.5 ° on a r o t a r y shaker, 185 rpm. 23j. j. Goodman, M. Matrishin, R. W. Young, and J. R. D. McCormick, J. Bacteriol. 78, 492 (1959). ~*J. R. D. McCormick, N. O. Sjolander, S. Johnson, and A. P. Doerschuk, J. Bacteriol. 77, 475 (1959).
20
METHODS FOR THE STUDY OF ANTIBIOTICS
[1]
C. E r y t h r o m y c i n
Organism. Streptomyces erythreus grown on nutrient agar or on the following sporulation medium at 28-37 ° for 10-14 days (light inhibits sporulation). Media S p o r u l a t i o n m e d i u m 25 E
Starch Glucose Tryptone Betaine Curbay BG K2HPO4 NaC1 CaCl~ Mineral mixture Agar Deionized water to volume
(g/liter) 75 5 5 0.5 2 0.2 10 0.08 2 ml 20
Mineral mixture (g/liter) in deionized water: MgSO,.7H~O, 100; FeSO,.7H20 2; ZnSO4.7H20, 1; CuSO,. 5H20, 0.5; MnSO4.5H,O, 0.4; CoCl~. 6H20, 0.1; conc. HC1, 1 ml l n o c u l u m m e d i u m 25
Glucose Sucrose Bactotryptone Bacto yeast extract
(g/liter) 5 10 5 2.5
Incubate 48-72 hr on a shaker at 30-32 °. Complex m e d i u m ~5
Starch Glucose Soybean meal Corn steep solids Yeast NaC1 CaCO8 CoC12.6H20
(g/liter) 25 25 47 10 5 5 2 Trace
~5W. M. Stark and R. L. Smith, in "Progress in Industrial Microbiology" (D. J. D. Hockenhull, ed.), Vol. III, p. 211. Wiley (Interscience), New York, 1961.
[1]
ANTIBIOTIC-PRODUCING MICROORGANISMS
21
Defined medium ~ (g/liter) 2.5 5 0.5 0.02 0.05 O. 001 0. 001 3 68.4 7.5 1.8
K2HP04 (sterilized separately) NaC1 MgSO4 • 7H~O FeSO4 - 7H~O ZnSO4.7H20 MnCI2- 4H20 CoCI~. 6H~O CaCO8 Sucrose Glycine L-Tyrosine Deionized water to volume Original pH of 7.8 is left unadjusted.
D. Gentamicin
Organism. M i c r o m o n o s p o r a purpurea m a i n t a i n e d o n a g a r s l a n t s cont a i n i n g 1 % y e a s t e x t r a c t , 1 % glucose, a n d 0 . 3 % C a C 0 3 . Media Inoculum medium ~e Beef extract Tryptone Glucose Soluble starch Yeast extract CaCOs
(g/liter) 3 5 1 24 5 4
Incubate on a rotary shaker, 280 rpm, for 4-5 days at 37°; transfer to the same medium (5% v / v ) and incubate at 28 ° for 72 hr; use at a level of 5% v / v .
Fermentation media 2G,27 Yeast extract Glucose CaCOa
(g/liter) 10 10 1
Soybean meal Glucose CaCO~
(g/liter) 30 40 4
Incubate for 5-6 days on a rotary shaker at 26 °. 2, M. J. Weinstein, G. M. Luedemann, E. M. Oden, and G. H. Wagman, Antimicrob. Ag. Chemother. 1963, 1 (1964). 2, G. M. Luedemann and M. J. Weinstein, U.S. Patent 3,091,572 (1963).
22
METHODS FOR THE S T U D Y
OF ANTIBIOTICS
[1]
E. Neomycin
Organism. S t r e p t o m y c e s /radiae. Media Inoculum medium ~8 Glucose N-Z amine A (casein digest: Sheffield F a r m s ) (NH4) 2HPO4 MgS04.7H~O FeSO4 •7H20 CuSO4 •5H20 CaC03 Deionized water to volume
(g/liter) 10 10 5 0.5 0.05 0.05 10
Sterilize the glucose separately as a 50 % stock solution and add to the medium before inoculation; adjust p H to 7.2-7.3 prior to sterilization, I n c u b a t e at 25--27 ° for 1-2 days on a r o t a r y shaker and use at a level of 3 % v / v .
Complex medium ~8 (g/liter) Soybean meal 25 Glucose 10 Brewer's yeast 5 NaC1 5 2 CaCO3 Deionized water to volume Sterilize glucose separately; m a i n t a i n fermentation p H above 7.2 with sterile K O H . M a x i m u m yields occur in a b o u t 5 days at 25-27 ° and under conditions of high aeration (shake flasks w i t h baffles on a rotary shaker at 350 rpm).
Defined medium ~s (g/liter) Glucose 10 L-Histidine 10 (NH4) ~HPO4 1 MgSO4 •7H20 0.5 FeSO4 • 7H~O 0.05 CuSO4.5H20 0.05 CaCO3 10 Deionized water to volume Other f e r m e n t a t i o n conditions remain as above. 2s O. K. Sebek, Arch. Biochem. Biophys. 57, 71 (1955).
[1]
ANTIBIOTIC-PRODUCING MICROORGANISMS
23
F. Streptomycin Organism. S t r e p t o m y c e s griseus. Media Inoculum medium 2~ (g/liter) 10 l0 10 6
Glucose Enzymic hydrolyzate of casein NaC1 Meat extract Distilled water to volume Incubate on a shaker for 48 hr at 28°. Complex medium 29
(g/liter) 25 40 5 2.5
Glucose Soybean meal Distillers dried solubles NaC1 Adjust to pH 7.3-7.5 before sterilization Defined medium 3° Glucose MgS04.7H20 Sodium citrate Glycine NaCl CaCl~- 6H20 KH2PO4 Fe Mg Cu Zn Mo Distilled water to volume
(g/liter) 20 10 10 5 5 0.5 0.5 (mg/liter) ~} as sulfates 0.1 as sodium molybdate
Adjust to pH 7.3 before sterilization. 2gD. J. D. Hockenhull, K. H. Fantes, M. Herbert, and B. Whitehead, J. Gen. Microbiol. 10, 353 (1954). C. G. C. Chesters and G. N. Rolinson, J. Gen. Microbiol. 5, 559 (1951).
24
METHODS FOR THE STUDY OF ANTIBIOTICS
[2]
[2] S t r a i n D e v e l o p m e n t
By
AMEDEO A. FANTINI
I. II. III. IV. V.
Introduction . . . . . . . . . . . . . . . . . . Fermentation Conditions . . . . . . . . . . . . . . . Assay for Antibiotic Yield . . . . . . . . . . . . . . Preparation of Suspension for Mutation Treatment . . . . . . . Mutation Techniques . . . . . . . . . . . . . . . . A. Ultraviolet Light (UV) Irradiation . . . . . . . . . . . B. Ionizing Radiation . . . . . . . . . . . . . . . C. Alkylating Agents . . . . . . . . . . . . . . . . D. Other Mutagens . . . . . . . . . . . . . . . . E. Combined Action . . . . . . . . . . . . . . . . VI. Selection from Mutagen-Treated Populations . . . . . . . . .
24 25 26 26 27 29 31 32 35 36 36
I. I n t r o d u c t i o n Strain development, or strain improvement, is a key factor in the development of an antibiotic as a potential therapeutic agent. Before any satisfactory biological testing and chemical characterization of a new antibiotic entity can be accomplished, adequate amounts of the material obviously must be available. As a rule, however, it soon becomes apparent t h a t the producing culture is synthesizing totally inadequate amounts of the antibiotic; thus, the problem of increasing yields becomes one of primary importance, and a pressing one. This discussion will deal with cultures producing low levels of potentially new antibiotics, and the "how to" aspects of improving these strains. B y strain improvement is meant the obtaining of microbial isolates possessing certain desirable characteristics for solving a specific problem. The improvement is only one of utility for the microbiologist, and the culture itself is not necessarily improved from the standpoints of viability, stability, sporulation, pigmentation, or other factors. Further, some of the mutants obtained m a y bear little resemblance to the original parent culture from which they were derived, occasionally creating a problem for the taxonomist. While increasing yields is usually one of the first tasks to be accomplished, it is not the only problem encountered; others include the elimination of unwanted antibiotic components where mixtures are produced, the elimination of undesirable pigments, the selection of mutants with improved growth characteristics, lessened foaming in fermentors, mutants resistant to phage, or mutants synthesizing a modified antibiotic mole-
[2]
STRAIN DEVELOPMENT
25
cule. Whatever the specific problem, its solution comes under the heading of strain development. Simply stated, strain development involves mutation and selection of mutants. For a successful strain improvement program, however, some additional aspects should be considered before starting with mutation techniques; most are obvious and basic, but to ignore them would lessen the effectiveness of the program. They may be listed as follows: fermentation conditions; assay for antibiotic yields; preparation of sample for mutation treatment; mutation techniques; selection from mutagentreated populations. It may also be mentioned at this point that while strain improvement has been applied to innumerable antibiotic-producing cultures over the years, very little has actually been published on the subject. Partly, this is due to the fact that antibiotics are manufactured by a very competitive pharmaceutical industry, so that much of the technical information and data on yields has been considered confidential information. The unavailability of this information, however, should not be considered a serious loss, as much of it would probably be very specific for a particular organism and product. Thus, for an investigator embarking on a strain development program for a novel antibiotic, it may well be more advantageous not to be too influenced by specific techniques which may be useless for a culture for which they were not developed. What may be considered a classic in this field is the work of Backus and Stauffer1 with Penicillium chrysogenum at the University of Wisconsin. This paper is important not only because it reports an increase in penicillin yields 40 times greater than those produced by the parent culture, but primarily because it presents an overall view of the techniques and approach followed over a 10-year period.
II. Fermentation Conditions
These will not be discussed in any detail here; suffice it to say that with a mutation program a parallel study should be initiated to develop satisfactory fermentation conditions. These include seed inoculum, fermentation medium, pH of medium, temperature of incubation, and aeration. In the early stages of improving yields, the above factors are extremely important, and frequently marked gains are made by a study of fermentation conditions alone. In addition, since improvements by mutation are often small, potenM. P. Backus and J. F. Stauffer, Mycologia 47, 429 (1955).
26
METHODS FOR THE STUDY OF ANTIBIOTICS
[2]
tially improved mutants will go undetected unless satisfactory fermentation conditions are available. Further, a continuing study of fermentation conditions should be done as an important part of a strain development program, as new mutants will be found that will perform better under conditions other than those originally developed for the parent culture. III. Assay for Antibiotic Yield A reasonably sensitive assay system must be available at the start, as improvements in yields are frequently reflected by relatively small increases in the diameter of a zone of inhibition against a sensitive organism; thus, small changes may well go undetected even though they may represent a 2-fold or better yield improvement. In any case, the assay procedure should be relatively simple, and above all it should not be a limiting factor for the number of mutagentreated isolates that can be fermentation-tested. IV. Preparation of Suspension for Mutation Treatment For efficiency, a suspension of cells or spores lends itself well to mutagenic treatment, subsequent dilution, and plating. 1. Bacterial suspensions of single cells may be prepared by growing in a liquid medium and then subculturing in fresh medium so as to obtain an actively growing population. 2. Spore suspensions from Streptomyces are prepared by adding 5-10 ml of sterile distilled water to a slant and scraping its surface with a pipette; the addition of 0.05% of a suitable wetting agent to the water will result in a more satisfactory suspension. Mycelial fragments are removed by filtering through Whatman No. 2 filter paper held in a small glass funnel. All manipulations should be done aseptically and with sterile equipment; this recommendation will not be mentioned further. 3. For fungi, essentially the same procedure is employed, but mycelial fragments may be removed more readily by filtering through glass wool held in a cylindrical filter tube, such as Kimax No. 46170. 4. Occasionally a microorganism does not produce spores or a mutant has lost its ability to sporulate. Satisfactory mycelial suspensions may be prepared by scraping the surface of a slant in 10 ml of water as mentioned above, and transferring the entire volume to a relatively narrow sterile tube. The coarser particles are allowed to settle out for a few minutes, and a 1-ml volume can then be pipetted from the supernatant and diluted in 9 ml of water. 5. For more difficult cases, where tough or leathery growth is pro-
[2]
STRAIN DEVELOPMENT
27
duced, the latter is transferred in 3-5 ml of water to a tissue homogenizer. The grinding procedure must be restricted to very few strokes, or viability will be greatly reduced. This method will produce fine suspensions devoid of large particles. 6. For certain asporogenous fungi, particularly those exhibiting a rapid filamentous growth, the hyphal tips may be exposed to a mutagen more advantageously than the entire colony growing on a plate. This may be readily accomplished by placing a sterile square of cellophane (avoid water-proofed cellophane) or other porous membrane 3-4 cm wide on the agar surface of a nutrient medium; the center of the square is inoculated with the fungus, and, after an appropriate period of incubation, the growing front of the colony will grow beyond the edge of the cellophane. When the hyphal tips reach a 2-5-ram length beyond this front, the cellophane may be peeled from the surface of the plate, leaving behind the delicate hyphal tips for exposure to mutagenic treatment. 7. Certain spores produced by microorganisms can be very resistant structures. Suspensions of these are made as described above, but in order to make the DNA more accessible to the mutagen it is preferable to pregerminate these spores in a suitable liquid medium prior to treatment. When a germ tube twice the size of the spore is microscopically visible, the suspension is ready for mutagenic treatment. This germination time should not be overextended. 8. Pregerminating spores (or dividing single cells) prior to or during the application of the mutagen has also been employed by some investigators even when good suspensions of uninucleated spores were available, on the assumption that the replicating DNA would be more sensitive to the mutagenic treatment. Unless a definite advantage, or a specific requirement due to the nature of the mutagen, is obvious, the use of germinating spores of filamentous organisms will introduce difficulties in the eventual selection of mutants. Dividing cells must be employed, however, when purine-pyrimidine analogs are used as mutagens, so that they may be incorporated into the replicating DNA molecule. In another application, a mutagenic treatment has been applied at various time intervals to a synchronized cell population, so as to make single-stranded DNA available to the mutagen in a sequential fashion during the replication cycle. 2 V. Mutation Techniques A microbial culture is composed of a dynamic population of cells, and from the standpoint of searching for improved antibiotic producers 2 F. J. Ryan and S. D. Cetrulo, Biochem. Biophys. Res. Commun. 12, 445 (1963).
28
METHODS FOR THE STUDY OF ANTIBIOTICS
[2]
it is an advantage that such populations do not remain homogeneous for many generations. Some gains can thus frequently be made in the early stages of a strain development program by simply plating suitable dilutions of the culture and isolating a few of the resultant colonies for fermentation testing. Unfortunately, many mutations, whether induced or spontaneous, are not "gain" mutations; thus, some improved producers may, with time, show signs of lessened antibiotic productivity. In such cases it will frequently be useful to plate appropriate dilutions and isolate a number of the resulting colonies for recovery of the original improved isolate. In any case, if any significant improvements are to be made, the introduction of mutagenic agents becomes necessary. While the chemical changes brought about by the interaction of some physical and chemical mutagens with chromosomal DNA are known, in practice it is not possible to recommend a specific agent that will prove successful in solving a problem like improving the ability of a culture to synthesize greater quantities of an antibiotic molecule. For one reason, the chemical nature of the DNA molecule is such that too many common denominators exist as targets for a mutagen, and thus "directed" mutation of a specific site presents a difficult problem. And perhaps even more important, as Demain ~ pointed out, we are ignorant of the genetic and enzymic changes brought about in a cell as the result of the rare "successful" mutations. Over the years, a number of agents have been described as mutagenic, and it is outside the scope of this article to discuss the application of most of these agents or their mode of action. A paper by Auerbach 4 presents an interesting background history of the better known mutagens, and Heslot ~ has reviewed the principal groups of mutagens and their mode of action. It is usually not necessary to employ many different mutagens in order to conduct a successful strain development program. Actually some agents that have found wide application in the elucidation of the nature of the gene have not been particularly useful in strain development work. Further, most studies of mutagens have been based on the induction of reverse mutations; effectiveness in such mutation induction may not have a parallel in inducing the type of mutant sought in strain development. A. L. Demain, in "Fermentation TechnologyToday" (Proc. 4th Int. Ferm. Syrup.) (G. Terui, ed.), p. 239. Soc. Fermentation Technology,Japan, 1972. 4C. Auerbach, in "Chemical Mutagens; Principles and Methods for Their Detection" (A. Hollaender, ed.), Vol. 3, p. 1. Plenum, New York, 1973. s H. Heslot, Proc. Symp. Radiat. Radloisotop. Ind. Microorganisms, p. 13. Int. Atomic Energy Agency,Vienna, 1971.
[2]
STRAIN DEVELOPMENT
29
Thus, since we do not know what kind of change in the genetic material will bring about the desired effect, selection of a mutagenic treatment should be based on practical considerations: (a) the procedure should not be excessively hazardous (many mutagens are carcinogenic), (b) the techniques should be relatively simple, and (c) equipment and chemicals should be readily available. A magic mutagen for strain development has not yet been found, and what has been effective for culture A may very well be much less so for culture B. Thus it is important to be flexible, and a different mutagen should be applied when few or no improved mutants are obtained. The application of a few mutagenic techniques will now be described.
A. Ultraviolet Light (UV) Irradiation This is probably one of the most frequently employed methods of mutation induction, particularly at the start of a strain development program. An ordinary germicidal lamp equipped with a mercury vapor tube (or tubes) rated to emit radiation of 2537 A wavelength is commonly used. Since the absorption spectrum for DNA (2600 A) is close to this value, this has been suggested as the cause of the mutagenie effect of UV. Absorption of UV by DNA appears to cause bonding of adjacent thymine bases (dimerization) on the same DNA polynucleotide chain. The resulting distortion of the DNA may then cause additional crosslinking of thymine on opposite strands and eventual mutation as the result of base pair substitutions. Since an enzyme requiring visible light as an energy source has been found to repair (by splitting pyrimidine dimers) the lethal and mutagenic effects of UV irradiation (photoreaetivation), all manipulations should be conducted under a limited light source such as a 25-W yellow or red bulb. Suitable spore or cell suspensions are prepared as described earlier; a volume of 10 ml is adequate. In order to determine the effect of irradiation on survival, a count must be made before UV treatment. While some investigators find it convenient to do cell counts by means of a hemaeytometer or Petroff-Hausser counting chamber, determination of the viable count by plating of suitable dilutions is to be preferred. In addition, isolation of a few colonies from this untreated population and eventual testing for their antibiotic-producing ability may uncover a naturally occurring improved mutant. For a viable count then, 1.0 ml of the suspension is diluted serially 1"10 (10-1 dilution) in saline or water with Tween 80 (0.05%) to 10-8; more or fewer dilutions may be required depending on the density of
30
METHODS FOR THE STUDY OF ANTIBIOTICS
[2]
the suspension. A 0.1-ml volume from dilution tubes 10.4 to 10-s is then spread on the surface of plates containing an appropriate agar medium; at least 2 plates should be used per dilution. The remainder of the suspension (about 9 ml) is then placed in a petri plate in preparation for UV irradiation. To establish percentage survivals for different exposure times, the distance from UV lamp to the surface of the suspension is maintained constant; a good starting point would be 20 cm. It is important to standardize one's conditions of treatment right at the start, as changes may then be more accurately made based on the results obtained. Further, changes in sensitivity or resistance to UV irradiation may be more readily detected. A range of exposures of 15, 30, 45, and 60 sec for Streptomyces should make possible the selection of an exposure time that will result in a 95-99% kill. UV exposure times should be controlled by opening and closing the petri plate cover, not by turning the lamp on and off. The UV rays will not significantly penetrate the glass or plastic covers for the short working periods here involved. During the exposure times, the suspension should be agitated by gently rotating the plate. At time 0, the cover is removed and then replaced at 15 sec (or other preselected time); a 1.0-ml volume is then pipetted from the plate and into a tube containing 9.0 ml of water and labeled 15 sec (10-1). The operation is repeated for an additional 15 sec, and again a 1.0-ml volume is diluted and labeled 30 sec (10 -1) and so on as outlined. Additional dilutions and subsequent platings should then be carried out; obviously, the greater the UV exposure the fewer the dilutions required to obtain well-spaced colonies following incubation. A reasonable initial plating scheme for a member of the streptomyceres may follow the pattern shown in Table I. All plates should then be incubated at a suitable temperature and in reasonable darkness so as to avoid possible photoreactivation. After colonies develop (3-6 mm in diameter), counts should be made for all exposure times wherever possible. The percentage survival for each exposure period may then be calculated. While a 1-5% rate of survival has been found more useful for obtaining mutants than greater survival values, it should be kept in mind that such data are based on reverse mutation studies and may not be particularly significant for the "gain" mutations sought in strain development. It may thus be worthwhile to occasionally isolate colonies from UV treatments allowing greater or lesser survival rates. In any case, subsequent UV treatments utilizing the survival data
[2]
STRAIN
DEVELOPMENT
31
TABLE I U V EXPOSURE AND INITIAL PLATING SCHEME FOR
Streptomyces
Dilutions UV exposure (sec)
10 -x
10 - z
0 15 30 45
60
x
10 - a
10 -4
10-a
10-~
10-7
10-s
X x
x x
X x x
x
Xa x x x
X
x x x
x
x
x
a x Represents dilution and plating to be done; at least two or more plates should be used per dilution.
obtained, can be designed to more specifically produce a desired percentage of survivors, which with proper dilutions will give counts of 10-60 colonies per plate. At this point, the number of plates employed should be increased so that a reasonably large number of colonies will be available for isolation. It should not be assumed that a high kill rate is a general prerequisite for high mutation efficiency; while it may be true for some mutagens, it is definitely not the case for others.
B. Ionizing Radiation X-Rays, y-rays, a-particles, and fast neutrons are effective forms of radiation for mutation induction. The term ionization refers to their common property of dissipating energy in matter by causing ionization of atoms.
X-Rays are produced by commercially available machines, van de Graaff generators, linear accelerators, and synchrotrons. -/-Rays are given off by radioactive elements such as cobalt-60, and have wavelengths corresponding to very short X-rays. Fast neutrons are usually obtained from a cyclotron, atomic pile, or indirectly from a van de Graaff generator. As mentioned earlier, the selection of a mutagenic treatment must be based on practical considerations. It is obvious that most of the above equipment for generation of ionizing radiation is not readily available from a local scientific supply house; arrangements can usually be made, however, with a university or other institute for irradiation of selected samples. The unit of measurement for X- and -/-rays is the roentgen (r) ; for neutrons, the rad is employed as the unit of measure.
32
METHODS FOR THE STUDY OF ANTIBIOTICS
[2]
In contrast to the selective absorption of UV by DNA, ionizing radiation may act directly on chemical bonds, or indirectly by producing free radicals from organic molecules or water, which in turn affect the DNA molecule. The entire mechanism, however, is not completely understood. While X-rays have been shown to produce point mutations, they are capable of producing macrolesions as well (deletions, inversions, translocations) .6 X-Rays have been employed rather extensively in strain development work, and their application seems to have earned them the reputation for inducing useful mutations where other agents have failed. Preparation of a cell or spore suspension for X-ray treatment is the same as described earlier for UV; a volume of about 5 ml is usually placed in a special vial to fit the generating equipment employed. A 2-ml sample of the untreated suspension should be set aside to determine viable counts and calculation of percentage survival for the irradiated portion of the suspension. After irradiation, the suspension is diluted and plated on an appropriate medium. Dilutions of 10-1 to 10-4 are usually satisfactory for the purpose, and a number of plates (10 or more) should be used for each dilution so that recovery of a substantial number of colonies may be anticipated. In the case of a first trial with X-irradiation, plating of the treated suspension may be done on a limited number of plates (2 per dilution) and dilutions extended from 10-1 to 10-e, while the remainder of the irradiated suspension is maintained under refrigeration. As colonies become evident on the plates, a dilution may be selected which will give counts of 10-80 colonies per plate; a final plating may then be done based on this information. Thus, if 10-3 appears optimal, platings should be done at 10-2, 10-8, and 10-4, using several plates. Doses of 100i000-150,000 r have been found satisfactory for Streptomyces species; the percentage survival may be expected to be in the range of 0.1 to 10.
C. Alkylating Agents A number of compounds belongs to this group of agents. They possess one or more alkyl groups capable of being transferred to other molecules. Some of these compounds are sulfonates, ethyleneimines, certain lactones, epoxides, nitrogen mustards, and nitroso compounds. While some alkylate F. J. DeSerres, H. V. Mailing, and B. B. Webber, Brookhaven Syrup. Biol. 20,
56 (1967).
[2]
STRAIN DEVELOPMENT
33
ing agents are carcinostatic, some are also carcinogenic, and safety precautions must be applied in pipetting (use a safety bulb) and disposal. It is beyond the scope of this article to present procedures for the application of many of the alkylating agents as mutagens; while a number have been shown to be effective mutagens in various genetic studies, probably two, the nitrogen mustards and the nitroso compounds, have had the widest application in strain development programs. Nitrogen Mustards. Of these, the most commonly employed is methylbis(fl-chloroethyl)amine, commonly referred to as "HN2." Other members of this family have been labeled "HN," " H N I , " and "HN3" from the wartime code for mustard gas of " H " ; the numbers refer to the replacement of the methyl groups of trimethylamine by one or more 2-chloroethyl groups. A convenient procedure, representing a modification of the technique reported by Roegner et al., 7 is described below. Care should be taken that the mutagenic treatment be done in test tubes large enough to conveniently hold a total volume of 10 ml without danger of spilling; small flasks may be more conveniently used for the purpose. The following reagents should be prepared: Methyl-bis(fl-chloroethyl)amine (HN2) at 1% in sterile distilled water (do not sterilize HN2) NaHC03 at 2%, sterile Decontaminating solution, consisting of: NaHC03 7.0 g and glycine 6.0 g per liter of distilled water Portions of the decontaminating solution should be distributed in tubes, 9 ml per tube, and sterilized; it will be used to end the action of the HN2, and for serial dilutions of the treated spore suspension. The remainder should be kept available for use in case of an accidental spill, and for submerging glassware on completion of the procedure. A spore or cell suspension should be prepared in the usual manner (6-7-ml volume), diluted 1:5, and 5.0-ml aliquots distributed into 5 small flasks (or test tubes) labeled 1 to 5 (Table II). Some investigators prefer pregermination of the spore suspension. The 2% N a H C Q solution is added to each flask at the volumes indicated in the table. The HN2 is then added to 4 of the flasks as shown in the table; flask No. 1 is the control. The reaction is usually allowed to proceed for 30 min, preferably with slight agitation (reciprocating shaker). At this time, 1.0-ml volumes are removed from each reaction flask, and diluted 1:10 in decontaminating solution tubes. This will end the reaction; additional serial dilutions and 7 F. R. Roegner, M. A. Stahmann, and J. F. Stauffer, Amer. J. Bot. 41, 1 (1954).
34
METHODS FOR THE STUDY OF ANTIBIOTICS
T A B L E II PROCEDURE FOR N I T R O G E N M U S T A R D
Spore suspension Flask no. (ml) 1 2 3 4 5
5.0 5.0 5.0 5.0 5.0
[2]
( H N 2 ) ~ TREATMENT
2% NaHCO3 (ml)
1% HN2 (ml)
HN2 Conc. (%)
Exposure time (rain)
5.0 4.9 4.75 4.5 4.0
0 0.1 0.25 0.5 1.0
0 0.01 0. 025 0.05 0.1
30 30 30 30 30
• HN2 = methyl-bis(fl-chloroethyl)amine. platings on suitable media are then completed. The 0.05 and 0.1~o H N 2 levels frequently produce a reasonably satisfactory balance between number of survivors and improved mutants in Streptomyces. Exposure time or H N 2 concentration m a y be increased if necessary; killing of spores is usually rather rapid, and an increase in morphological variants is frequently observed. N-Methyl-N'-nitro-N-nitrosoguanidine ( N T G ) . This compound deserves special mention as a very effective mutagen, s,9 Its action has been attributed to its decomposition prSducts (diazoalkanes)~o; this decomposition appears to occur more readily at alkaline pH. Until fairly recently it was believed t h a t N T G was more effective as a mutagen in dividing cells; thus, spore suspensions were usually pregerminated and N T G was then added during a set incubation period. Now a procedure has been described for Streptomyces coelicolor 11 which differs somewhat in detail from those applied to other bacteria and fungi, and the results obtained from reversion studies show no marked differences between germinated or ungerminated spore suspensions. A stock solution of N T G should be newly prepared for each experiment and kept at low temperature to prevent its premature decomposition. The buffer system to be employed (Tris buffer is convenient for this purpose) should be adjusted to p H 9.0 and sterilized. A filtered spore suspension is prepared in the usual manner, centrifuged at 1000 g for 10 rain, and resuspended in 2.5 ml of buffer. A 1.0-ml volume of the suspension is serially diluted 1:10, and plated as a control 8E. A. Adelberg, M. Mandel, and G. C. C. Chen, Biochem. Biophys. Res. Commun. 18, 788 (1965). 9 E . Cerdh-Olmedo and P. C. ttanawalt, Biochlm. Biophys. Acta 142, 450 (1967). 1oE. Cerd~-Olmedo and P. C. Hanawalt, Mol. Gen. Genet. 101, 191 (1968). ,1V. Delic, D. A. Hopwood, and E. J. Friend, Murat. Res. 9, 167 (1970).
[2]
STRAIN DEVELOPMENT
35
for viable counts. Another 1.0 ml volume of the spore suspension is added to 9.0 ml of NTG solution in a small flask; the solution is prepared so as to give a final concentration of 1 mg/ml. This is considered time 0, and the mixture is incubated in a water bath (28-30°), preferably with gentle agitation. At times 0.5, 1.0, 1.5, and 2.0 hr, 2.0-ml volumes of the reaction mixture are withdrawn from the flask, immediately centrifuged in sterile tubes, and resuspended in 2.0 ml of water; this ends the reaction. Since the spores are exposed to NTG during the centrifugation period, this time should be recorded and considered part of the total treatment time. Dilutions are then made from each exposure time, and plating is done on appropriate media. The procedure described should prove effective with most S t r e p t o m y ces species; NTG concentrations as high as 3.0 mg/ml were reported by Delic et al. ~ in the above study. An exploratory run will provide useful data for establishing more suitable NTG concentrations and exposure times for the particular organism under investigation. NTG levels for other microorganisms have usually been lower than those reported above; for E. coli, a 30-min exposure at 0.1 mg/ml pH 6 has been found very effective,8 and for Saccharomyces cerevisiae, a 30-rain exposure at 0.7 mgfml pH 4 has been reported as quite suitable. 12 A potentially useful property of NTG is that it has been reported to act preferentially at certain sites on the genome, inducing closely linked multiple mutations; this was reported for synchronized E. coli cultures. ~3 A comparable approach applied to an antibiotic producing organism may prove helpful in the development of a less random approach to strain development.
D. Other Mutagens A number of other agents has been reported as effective mutagens in various genetic studies; some of the better known are base analogs, hydroxylamine, nitrous acid, and the acridines. These agents have not been applied widely to strain improvement, possibly because they seem to induce primarily mutations of the microlesion type, whereas multiple mutations or macrolesions among survivors appear more likely to have a positive advantage in strain improvement. 14 12F. K. Zimmerman, R. Schwaier, and V. von Laer, Z. Vererbungslehre 97, 68 (1965). 1~N. Guerola, J. L. Ingraham, and E. Cerd£-Olmedo, Nature (London) New Biol. 230, 122 (1971). ~' D. A. Hopwood, in "Actinomycetales: Characteristics and Practical Importance" (G. Sykes and F. A. Skinner, ed.), p. 131. Academic Press, New York, 1973.
36
METHODS FOR THE STUDY OF ANTIBIOTICS
[2]
E. Combined Action More efficient mutagenesis has been reported in a few cases by the application of two different agents. Improved erythromycin 15 and chlortetracycline TM producers were obtained by combined treatment with ethyleneimine plus UV irradiation. A synergistic effect has also been reported when UV-treated bacterial cells are grown in the presence of caffeine. T M It has been suggested that caffeine interferes with the excision repair of pyrimidine dimers from the UV-irradiated DNA. The procedure is essentially as described for UV treatment, but the irradiated suspension is plated on a medium supplemented with 500 ~g/ml of caffeine. The nonirradiated control suspension should also be plated on caffeine-supplemented plates in order to determine a nontoxic concentration. The 500 ~g/ml level appears to be effective for E. coli and Streptomyces. More recently a procedure that significantly improves the percentage of mutants among survivors was described by Townsend et al. TM for Streptomyces. In this case, near UV light in the presence of the photosensitizing chemical, 8-methoxypsoralen was employed. A longwave UV lamp (emitting at >3000 A), commonly referred to as "black light," is set up as previously described for UV treatments. The 8-methoxypsoralen is dissolved in ethanol at 1 mg/ml, and then diluted 1:10 in a water suspension of spores; exposure times of 10-60 min appear to be adequate. The suspension is then irradiated for periods of 0, 5, 10, and 20 min; dilutions and plating are done as mentioned earlier. The authors report that this procedure may be a useful alternative to NTG treatment, as it appears to be less specific as to site of action (at least in E. coli) and less hazardous to use. VI. Selection from Mutagen-Treated Populations After a microbial population has been treated with a mutagen, a number of the survivors will have to be tested for the desired improvement. Two questions will immediately arise: (a) how many colonies should be isolated for testing, and (b) which ones should be isolated. Statistical approaches have been suggested in answer to (a) which might indicate the probability of obtaining an improved mutant from 1~S. I. Alikhanian and V. G. Zhdanov, Dold. Acad. Naulc. SSSR Set. Biol. 125, 1353 (1959). 1, S. I. Alikhanian and N. B. Romanova, Antibiotiki (Moscow) 10, 11'13 (1965). 1~D. M. Shankel, J. Bacteriol. 84, 410 (1962). 18K. Shimada and Y. Takagi, Biochim. Biophys. Acta 145, 763 (1967). ~*M. E. Townsend, H. M. Wright, and D. A. I-Iopwood, J. Appl. Bacteriol. 34, 799 (1971).
[2]
STRAIN DEVELOPMENT
37
a certain number of colonies isolated and tested. However, since the induced improvements are usually rather small and the experimental error and the variability of testing and assaying can be of considerable magnitude, such approaches have not been practical. The answer to (a) then, will be dictated by the practical limiting factors of personnel, incubator and shaker space, and time. The answer to (b), which colonies should be isolated in the search for mutants with the desired characteristics among the many nonmutated ones, or among those which were changed negatively, is a perplexing one, and frequently only a random laborious approach will succeed. The techniques described below have generally been successful, but the technique of choice is the one that will result in the isolation of the greater number of gain mutations over the total number of isolates tested over a period of time. 1. The simplest and most laborious approach is the random isolation of a set number of colonies from a mutagen-treated population. Frequently, especially at the start of a strain improvement program, no other choice may be apparent. 2. Selection of colonies can be based on obvious morphological differences. This approach at least gives the investigator the impression that he is being selective, and a pattern will eventually develop from the testing of these colonies that will at least suggest that certain morphological types are poorer antibiotic producers than others and can thus be avoided. 3. Selection of improved antibiotic-producing colonies has been made by the application of various "overlay techniques." Essentially the procedure consists of flooding the plates containing the treated colonies with a thin layer (4 ml) of soft nutrient agar (0.7%) seeded with a sensitive organism. After incubation, inhibition zones of different diameter will suggest which colonies are better producers, or at least indicate which are obviously poorer. Colony diameter of the producing organism must be considered in evaluating zone sizes. A modification of this technique by Dulaney and Dulaney 2° makes use of a cellophane sheet between the colonies and the seeded agar layer. In a somewhat different approach, single colonies are grown on agar plugs 21 (or a plug of agar is cut around a colony:2), which are then placed on a medium seeded with a sensitive organism; the resulting zones of inhibition are then used as an index of the antibiotic-producing potential of each colony. The application of the various overlay techniques has been found very :~ E. L. Dulaney and D. D. Dulaney, Trans. N.Y. Acad. Sci. 29, 782 (1967). 21T. Ichikawa, M. Date, T. Ishikura, and A. Osaki, Folia Microbiol. (Prague) 16, 218 (1971). 22R. C. Pittenger and E. McCoy, J. Bacteriol. 65, 56 (1953).
38
METHODS FOR THE STUDY OF ANTIBIOTICS
[2]
advantageous by some investigators in selecting improved antibiotic producers; others have reported poor correlation when isolates selected by this method were eventually tested in shaker flasks. In any case, the following suggestions will help avoid pitfalls: (a) If the antibiotic is not bactericidal, the colonies to be overlayered should first be replica-plated 2~ to another set of plates before flooding with the test organism. Failure to do so will result in isolates contaminated with the test organism. Alternatively, the cellophane technique should be employed. (b) Frequently the resulting zones of inhibition are too large; obviously a less sensitive organism should be used, or a plating medium less favorable to antibiotic elaboration should be employed, or the thickness of the seeded layer should be increased. 4. Selection of nutritional mutants (auxotrophs). While mutation to the auxotrophic state has frequently been observed to have a negative effect on antibiotic yields, cases of improved productivity have also been reported? 4 This approach is worth investigating, as auxotrophs may be readily recognized and isolated. One such procedure makes use of the replica-plating technique, and consists of replicating colonies resulting from a mutation treatment to a defined medium on which the parent culture is capable of growing. Comparison of the master with the replica plates following incubation will indicate which colonies are auxotrophic mutants by their inability to grow on the defined medium. Various procedures have been described for increasing the percent of auxotrophs among survivors of a treated population. Only two will be mentioned here: Filtration enrichment technique for fungi,2~ and the penicillin technique for bacteria. 26,~7 Basically, both approaches result in the selective elimination of many of the prototrophic survivors with consequent improvement in the ease of recovery of auxotrophs. 5. Selection of prototrophs derived from auxotrophs (revertants). These may be readily selected by, plating a mutagen-treated auxotroph (spore or cell suspension) on a defined medium; only revertants will be able to grow. Some revertants may be due to partial reverse mutation; others to suppression of the original mutation by mutation at a different locus. The reversion approach has been applied with some success in increasing yields of chlortetracycline~° and actinomycin28 A discussion of J. Lederberg and E. M. Lederberg, J. Baclerlol. 63, 399 (1952). E. L. Dulaney, Ann. N.Y. Acad. Sci. 60, 155 (1954). ~ N. Fries, Nature (London) 159, 199 (1947). B. D. Davis, J. Amer. Chem. Soc. 70, 4267 (1948). 2,L. Gorini and H. Kaufman, Science 131, 604 (1960). 2~M. Polsinelli, A. Albertini, G. Cassani, and O. Ciferri, J. Gen. Microbiol. 39, 239 (1965).
[2]
STRAIN DEVELOPMENT
39
the possible biosynthetic pathways and relative value of these approaches has been written by Demain. 29 6. Selection of mutants resistant to toxic analogs of precursors can be an effective method for obtaining isolates capable of synthesizing increased levels of antibiotic. The procedure has been applied in the selection of improved vitamin producers ~° and consists of exposing a microbial population to a gradient concentration of an antimetabolite that is inhibitory to the growth of the cells; survivors usually consist of some mutants capable of producing increased yields of the metabolite. A similar approach was applied by Elander e t al. 31 for the isolation of improved producers of the antibiotic pyrrolnitrin, n-Tryptophan is a direct precursor for this antibiotic but impractical to use in large-scale fermentations; an effort was made to isolate analog-resistant mutants no longer subject to feedback inhibition of the end product. Gradient plates were employed in conjunction with the following D-tryptophan analogs: 5-fluorotryptophan, 6-fluorotryptophan, and 5-methyltryptophan. By isolating colonies resistant to these analogs, a mutant was eventually selected that produced 2-3 times more antibiotic than the parent culture, and in addition D-tryptophan was no longer stimulatory to pyrrolnitrin yields. This approach has particular potential for selection of mutants where something is known of the biosynthesis of the antibiotic. 7. Selection of mutants producing only one antibiotic entity where a mixture is commonly produced by the parent culture. Biosynthesis of antibiotic mixtures is frequently encountered; the mixtures may be made up of related chemical structures, or they may be totally different. The problem is to produce the desired antibiotic component exclusively, or at least to preferentially synthesize more of the desired one and less of the others. Essentially two approaches are employed to achieve the desired result: mutation, and/or manipulation of fermentation conditions. Mutation was proved to be successful in obtaining isolates capable of preferentially synthesizing various components of the nebramycin antibiotic group2 2 Similarly, the investigation of various fermentation conditions (carbon and nitrogen sources, temperature, pH, aeration, carbon to nitrogen ratios) has been useful in favoring production of a desired antibiotic entity. Further, certain components in a mixture may be synthesized relatively early or late during the fermentation period; thus, '~ A. L. Demain, Advan. Appl. Microbiol. 16, 177 (1973). 30G. H. Scherr and M. E. Rafelson, J. Appl. Bacteriol. 25, 187 (1962). ~1R. P. Elander, J. A. Mabe, R. L. Hamill, and M. Gorman, Folia Microbiol. (Prague) 16, 156 (1971). W. M. Stark, N. G. Knox, and R. M. Wilgus, Folia Microbiol. (Prague) 16, 205 (1971).
40
METHODS FOR THE STUDY OF ANTIBIOTICS
[2]
by judicious monitoring of time or pH conditions, improved yields of a specific component may be obtained by harvesting at appropriate times. The prevailing pH values during the fermentation period can also be very important in affecting the ratio of components in a mixture as their stability may be quite different. Thus, various approaches are available for the solution of this rather challenging problem. A complicating difficulty is that the presence or absence of specific antibiotic components will have to be confirmed by chromatography or some other analytical procedure. Since this could be a limiting factor in evaluating numerous isolates, it is usually well worth searching for a differential assay as a preliminary screen. Differences in the chemical structure of the antibiotics in a mixture are occasionally reflected by differences in the sensitivity of various test organisms. These differences may be absolute, such as complete resistance or sensitivity, or only partial, but they can prove useful in detecting changes in the composition of mixtures. 8. In the search for mutants with desired biosynthetic capabilities, the investigator should be aware of the possibility that a genetic modification of the antibiotic-producing culture may well result in the production of a modified antibiotic. In some cases the new molecule may well have improved therapeutic value~3; more frequently, it will be found to be less desirable than the antibiotic produced by the parent culture. In concluding, it must be stated that strain development is frequently a laborious and time-consuming challenge, and every avenue should be explored which may lead toward a more selective isolation of "gain" mutations. The utilization of more than one mutagen is necessary as no single chemical or physical agent can be said to be more effective than another in inducing productive strains. Further, a change in mutagenic agents should be made not only when its effectiveness seems lessened, but also at random. It is important to build a strain improvement program around more than just one improved key mutant, so that if a problem develops mutation work can continue with another improved isolate. Finally, it may be well to repeat that strain improvement has been a key factor for the manyfold antibiotic yield increases which have made possible the development of antibiotics as significant therapeutic agents and economic products. As a possible criticism, it may be said that the application of the empirical approach in strain development has been so successful over the u j . R. D. McCormick, N. O. Sjolander, U. Hirsch, E. Jensen, and A. P. Doerschuk, J. Amer. Chem. Soc. 79, 4561 (1957).
[3]
METHODS FOR STUDY OF ANTIBIOTIC RESISTANCE PLASMIDS
41
years that industrial laboratories seem to have been reluctant to investigate the potential benefits to be derived from the application of more sophisticated genetic studies.
[3] Genetic Methods for the Study of Antibiotic Resistance Plasmids B y JULIAN DAVIES I. Introduction . . . . . . . . . . . . . . . . II. Genetic Transfer of Resistance Plasmids . . . . . . . . A. Conjugation . . . . . . . . . . . . . . . B. Transduction . . . . . . . . . . . . . . . C. Transformation . . . . . . . . . . . . . . III. Enhanced Segregation of Resistance Plasmids by Curing Agents A. Acridine Orange, Quinacrine, and Ethidium Bromide Curing B. Sodium Dodecyl Sulfate . . . . . . . . . . . . C. Mitomycin . . . . . . . . . . . . . . . . IV. Compatibility Properties of R Plasmids . . . . . . . . A. Determination of fi Character . . . . . . . . . . B. Testing for Compatibility of R Factors . . . . . . . .
. . . . .
. . . . .
. . . . . . . . . .
41 43 43 45 48 49 51 51 52 52 54 54
I. I n t r o d u c t i o n T h e extensive use of c h e m o t h e r a p e u t i c agents has p r o v i d e d a n e n v i r o n m e n t conducive to the selection of b a c t e r i a l s t r a i n s I r e s i s t a n t to these agents. Since t h e i r discovery in J a p a n in the 1950's, the occurrence of b a c t e r i a r e s i s t a n t to a n t i m i c r o b i a l agents in clinical s i t u a t i o n s has been reported t h r o u g h o u t the world, a n d it is n o w clear t h a t the presence of such r e s i s t a n t b a c t e r i a can, u n d e r m a n y circumstances, prove to be a serious i m p e d i m e n t to n o r m a l a n t i b a c t e r i a l t h e r a p y Y T h e m e c h a n i s m s of r e s i s t a n c e to m a n y of the c o m m o n l y used a n t i m i c r o b i a l agents have 1Unfortunately the bacterial strains and bacteriophage stocks referred to in this article "are not available from any central source. Many E. coli strains may be obtained from the Coli Genetic Stock Center, Yale University (Curator, Dr. Barbara Bachman), and S. typhimurium can be obtained from the Salmonella Stock Center, University of Alberta, Calgary (Curator, Dr. Kenneth Sanderson). The authors of referenced manuscripts are the best source of strains which have been described in publication. 2S. Mitsuhashi, "Transferable Drug Resistance Factor R." University Park Press, Baltimore, Maryland, 1971. "The Problems of Drug-Resistant Pathogenic Bacteria" (E. L. Dulaney and A. L Laskin, eds.), Ann. N.Y. Acad. Sci. Vol. 182, 1971.
[3]
METHODS FOR STUDY OF ANTIBIOTIC RESISTANCE PLASMIDS
41
years that industrial laboratories seem to have been reluctant to investigate the potential benefits to be derived from the application of more sophisticated genetic studies.
[3] Genetic Methods for the Study of Antibiotic Resistance Plasmids B y JULIAN DAVIES I. Introduction . . . . . . . . . . . . . . . . II. Genetic Transfer of Resistance Plasmids . . . . . . . . A. Conjugation . . . . . . . . . . . . . . . B. Transduction . . . . . . . . . . . . . . . C. Transformation . . . . . . . . . . . . . . III. Enhanced Segregation of Resistance Plasmids by Curing Agents A. Acridine Orange, Quinacrine, and Ethidium Bromide Curing B. Sodium Dodecyl Sulfate . . . . . . . . . . . . C. Mitomycin . . . . . . . . . . . . . . . . IV. Compatibility Properties of R Plasmids . . . . . . . . A. Determination of fi Character . . . . . . . . . . B. Testing for Compatibility of R Factors . . . . . . . .
. . . . .
. . . . .
. . . . . . . . . .
41 43 43 45 48 49 51 51 52 52 54 54
I. I n t r o d u c t i o n T h e extensive use of c h e m o t h e r a p e u t i c agents has p r o v i d e d a n e n v i r o n m e n t conducive to the selection of b a c t e r i a l s t r a i n s I r e s i s t a n t to these agents. Since t h e i r discovery in J a p a n in the 1950's, the occurrence of b a c t e r i a r e s i s t a n t to a n t i m i c r o b i a l agents in clinical s i t u a t i o n s has been reported t h r o u g h o u t the world, a n d it is n o w clear t h a t the presence of such r e s i s t a n t b a c t e r i a can, u n d e r m a n y circumstances, prove to be a serious i m p e d i m e n t to n o r m a l a n t i b a c t e r i a l t h e r a p y Y T h e m e c h a n i s m s of r e s i s t a n c e to m a n y of the c o m m o n l y used a n t i m i c r o b i a l agents have 1Unfortunately the bacterial strains and bacteriophage stocks referred to in this article "are not available from any central source. Many E. coli strains may be obtained from the Coli Genetic Stock Center, Yale University (Curator, Dr. Barbara Bachman), and S. typhimurium can be obtained from the Salmonella Stock Center, University of Alberta, Calgary (Curator, Dr. Kenneth Sanderson). The authors of referenced manuscripts are the best source of strains which have been described in publication. 2S. Mitsuhashi, "Transferable Drug Resistance Factor R." University Park Press, Baltimore, Maryland, 1971. "The Problems of Drug-Resistant Pathogenic Bacteria" (E. L. Dulaney and A. L Laskin, eds.), Ann. N.Y. Acad. Sci. Vol. 182, 1971.
42
METHODS FOR T H E S T U D Y OF ANTIBIOTICS
[3]
TABLE I RESISTANCE CHARACTERS ASSOCIATED WITH RESISTANCE PLASMIDS a
~-Lactam antibiotics (penicillin, cephalosporins) Streptomycin Spectinomycin Gentamiein Tobramycin Neomycin Kanamycin Lividomycin Butirosin Chloramphenicol
Tetracycline Sulfonamides Trimethoprim Erythromycin Lincomycin Fusidic acid Bacteriophages (restriction) Metals (cadmium, nickel, cobalt, bismuth, lead, antimony, mercury, arsenate, arsenite) Ultraviolet irradiation
a This list includes characters on conjugative plasmids (gram-negative organisms) and nonconjugative plasmids (gram-positive bacteria). been characterized, and there are only few antibiotics which are refract o r y to the resistance mechanisms of these strains. Covalent changes in antibiotics are the most common forms of resistance mechanism, but alterations in permeability and other mechanisms are possible. The discovery of this form of resistance, in which genes coding for resistance mechanisms to several antibiotics (see Table I) are linked on an extrachromosomal genetic element or plasmid which replicates autonomously and in many cases codes for its own conjugal transfer, required the application of a number of genetic techniques which are summarized here. In addition, a variety of physical techniques, 4,5 such as dye-buoyant density centifugation, 6 equilibrium density centrifugation, 6 nitrocellulose fractionation, ~ and heteroduplex mapping, s have been applied to the study of resistance plasmids. The term "resistance plasmid" is used to describe extrachromosomal elements in general; these include those plasmids capable of transfer by conjugation (conjugative plasmids, also known as R factors) and those not capable of self-transmission (nonconjugative plasmids). 5,9,1° The latter are found in many gram-positive bacteria and some gram-negative bacteria; the former are found exclusively in gram-negative organisms (see Table II). D. Freifelder, this series, Vol. 21 [6]. R. C. Clowes, Bacteriol. Rev. 36~ 361 (1972). e W. Szybalski, this series, Vol. 12B [124]. Tj. A. Boezi and R. L. Armstrong, this series, Vol. 12A [90b]. 8R. W. Davis, M. Simon, and N. Davidson, this series, Vol. 21 [31]. 9j. Davies and R. Rownd, Science 176, 758 (1972). 1oG. G. Meynell, "Bacterial Plasmids." M.I.T. Press, Cambridge, Massachusetts, 1973.
[3]
METHODS
F O R S T U D Y OF A N T I B I O T I C
RESISTANCE
PLASMIDS
43
TABLE II BACTERIAL SPECIES CAPABLE OF HARBORING
Acinetobacter calcoaceticus A eromonas spp. A lcaligenes faecalis Arizona Citrobacter spp. Enterobacter spp. Erwinia spp. Escherichia coli KlebsieUa pneumoniae Neisseria perflava Proteus spp.
R FACTORS a
Providence spp. Providencia stuartii Pseudomonas spp. Rhizobium japonicum Rhodopseudomonas spheroides RhodospiriUum rubrum Salmonella spp. Serratia marcescens Shigella spp. Vibrio cholerae Yersinia spp.
" Not all R factors have a host range that includes all these strains; in addition many resistance plasmids are not self-transmissible in these species. Among gram-positive strains of bacteria, Staphylococcus aureus is known to harbor nontransmissible resistance plasmids. There have been extensive studies of the structure of plasmids both from a molecular and genetic standpoint. I t is clear t h a t most plasmids consist of covalently closed circular D N A duplexes with molecular weights in a range from about 20 X 106 to 70-80 X 106 (larger composite structures can occur under certain circumstances). Plasmids studied in Escherichia coli, Proteus mirabilis, and Salmonella t y p h i m u r i u m have been found to consist of two separate parts which can dissociate in the bacterium. These two components are the resistance transfer factor, with genes for maintenance, replication, and conjugal transfer, and the resistance determinants, which are the genes coding for the various resistance characters of the particular R factor.
II. Genetic Transfer of Resistance Plasmids
I t is now possible to transfer resistance plasmids between gram-negative species by conjugation, transduction, or transformation. In grampositive species transduction is the most common technique although transformation has also been reported.
A. Conjugation 11 T h e demonstration of transfer and subsequent autonomous maintenance of resistance characters in a recipient strain provides the most con11D. Freifelder,this series,Vol. 21 [34].
44
METHODS FOR THE STUDY OF ANTIBIOTICS
IS]
vincing evidence for the presence of con]ugativc resistance plasmids (R factors) in strains such as E. coli. 1~ The recipient strain generally carries a genetic marker which can be used for counterseleetion against the donor strain. The commonly used markers are nalidixic acid resistance (nalr), sodium azide resistance (aziR), or rifamycin resistance (rif R) although other counterselective markers such as auxotrophy can be used. 1~ Cultures of donor and recipient cells are grown to saturation in a rich medium [for example, Penassay Broth (Antibiotic Medium No. 3)] and 0.1 ml of each is added to the same medium (5 ml) and allowed to stand at 37 ° for 1-2 h r - - t h e mixture is then incubated with shaking for 3-4 hr and samples (0.1 ml or, a sterile loopful) are streaked onto solid medium containing the counterselecting agent (depending on the strain, nalidixic acid 20 ~g/ml, sodium azide 200 ~g/ml, or rifampicin 50 ~g/ml) and one or more of the drugs for which resistance is coded by the plasmid, at appropriate concentrations (since there are often host effects on the levels of expression of resistance, it is advisable to use a low concentration of antibiotic (20-30 ~g/ml) in selecting for transfer. Often the frequency of transfer is low, and this is particularly true during intergeneric crosses, e.g., E. coli X P. aeruginosa, and even in P. aeruginosa X P. aeruginosa. 14 In such cases, methods involving more constant contact between the p u t a t i v e mating pairs is advisable, and thins can be accomplished by carrying out the coniugation on the surface of a nitrocellulose filter or in a thin layer in a medicine bottle. In crosses involving P. aeruginosa and E. coli, since P. aeruginosa is a strict aerobe, anaerobic conditions (anaerobic jar) can be used to counterselect against the P. aeruginosa strain although this is not as convenient as counterselection with an antimicrobial agent, such as rifampicin. I t is important to realize that failure to observe transfer does not mean that a resistance plasmid is not present and even less so that the resistance genes in question are chromosomal. I t is often necessary to examine a large number of bacterial isolates for the presence of con]ugative plasmids, and under these circumstances it is unwieldy to set up many tube crosses in liquid medium, as described above. I t is convenient to use "plate" crosses when m a n y colonies are to be screened. Using a sterile toothpick, single colonies of the desired recipi12H. Watanabe, Bacteriol. Rev. 27, 87 (1963). 13Suitable resistant mutants may be isolated by direct selection; a variety of resistance strains may be obtained from the Coli Genetic Stock Center, Yale University. ~' L. E. B~an, S. D. Semaka, H. M. Van den Elzen, J. E. Kinnear, and R. L. S. Whitehouse, Antimicrob. Ag. Chemother. 3, 625 (1973).
[3]
METHODS FOR STUDY OF ANTIBIOTIC R E S I S T A N C E PLASMIDS
45
ent strain (a nalidixie acid resistant strain, for example) are gently stabbed into the surface of a rich agar plate (containing no selective agents). The colonies should be well separated, about 50 or so can be put on a standard sized petri dish. Colonies of putative R + strains are then stabbed into the recipient colonies, in turn, using sterile toothpicks. The colonies m a y be numbered on the back of the plate with a marker. Thus, each (master) agar plate will contain 50 or more mixed colonies of different R ÷ bacteria with recipient bacteria. After overnight incubation at 37 °, the master plate is replicated (using velvet pads) onto suitable agar plates containing the necessary nutrients and inhibitors that select only the recipients which have received resistance plasmids.
B. T r a n s d u c t i o n ~
Transduction of resistance plasmids was observed soon after they were discovered. Bacteriophage P1 kc has been used with E. coli, and this phage transduces all the resistances plus the resistance transfer factor of a conjugative resistance plasmid. On the other hand, bacteriophage P22, which is used with S. t y p h i m u r i u m is not capable of transducing the entire resistance plasmid and transduction by P22 often leads to loss of transferability and loss of one or more of the resistance characters. This difference is presumably due to the fact that P1 (73 X 106 daltons DNA) is much bigger than P22 (26 X 106 daltons DNA) and is thus capable of incorporating more D N A for transduction. 1~,17 In addition, transduction of resistance plasmids has been reported in Shigella flexneri Is and P r o t e u s mirabilis ~9 among gram-negative species, and transduction is the most convenient way of transferring resistance plasmids between strains of S t a p h y l o c o c c u s aureus. 2°-2~- Stable transduction into a recombination-deficient recipient provides a simple test for the existence of resistance characters on an autonomously replicating unit. Transductional studies have also proved to be useful in genetic mapping studies in both S a l m o n e l l a t y p h i m u r i u m (P22) and S t a p h y l o coccus aureus. Deletions are generated during transduction because of 1, L. Caro and C. M. Berg this series, Vol. 21 [35]. 1~T. Watanabe and T. Fukasawa, Y. Bacteriol. 82, 202 (1961). 1, Mitsuhashi,2 pp. 51-54. 18R. Nakaya, A. Nakarnura, and T. Murata, Biochem. Biophys. Res. Commun. 3, 654 (1960). 1~R. Nakaya and R. Rownd, ]. Bacteriol. 106, 773 (1971). 20R. P. Novick, Virology 33, 155 (1967); K. Smith and R. P. Novick, Y. Bacteriol. 112, 761 (1972). 21R. P. Novick, Bacteriol. Rev. 33, 210 (1969). ~2R. P. Novick and D. Bouanchaud, Ann. N.Y. Acad. Sci. 182, 279 (1971).
46
METHODS FOR THE STUDY OF ANTIBIOTICS
[3]
the limited capacity of the transducing bacteriophages for DNA; these deletions can then be used in the deletion-mapping of point mutations.
Transduction with P1 in E. coli Preparation o] Phage Stock. Two drops of a saturated culture of the donor bacterium (containing the resistance plasmid) and one drop of a suspension of bacteriophage P1 kc (or a clear mutant) (10 s PFU/ml) are added to 2 ml of soft (0.6% agar) nutrient-calcium agar (16 g of nutrient broth (Difco), 8 g of sodium chloride, and agar in 1000 ml of H~O; sterile calcium chloride added to 1 mM after autoclaving) at 45 °. The combination is mixed by agitation and immediately poured onto the top of a fresh (wet and warm, usually prepared the same day) agar (1% agar) plate of nutrient-calcium agar. The petri plate is then incubated at 37 ° for 8-12 hr (5 plates per phage stock are prepared). One milliliter of rich broth is added to each plate and the top agar and medium is scraped into a 50-ml centrifuge tube with a spatula or a thin glass rod which has been bent to form a scraper. Two drops of chloroform are added and the mixture is agitated on a Vortex mixer for 30 sec. The tube is then centrifuged in a table top centrifuge at room temperature (approximately 5000 rpm for 10 min) and the liquid layer of phage stock carefully poured off. This stock is then titered against a suitable indicator strain; many workers prefer Shigella, but a strain such as E. coli C600 is usually satisfactory. The titer of the stock should be 5 X 109/ml or greater, the above procedure is then repeated with a dilution of this phage stock, so that it is grown through the donor strain twice. Conditions ]or Transduction. For transduction of resistance characters, the recipient strain is grown in 5 ml of rich broth, centrifuged, and resuspended in 2 ml of MC solution (1 mM CaCl~, 10 mM MgCl~, pH 7.0), and to 0.9 ml of this suspension is added 0.1 ml of the P1 phage suspension (the multiplicity of infection should be 5:1 for P1 kc, if the clear-plaque mutants P1 vir or P1 clr are used, the multiplicity of infection should be 0.1-0.2 phage per bacterium). The bacteria/phage mixture is allowed to stand at room temperature for 20 min and then centrifuged, resuspended in rich broth (10 ml), and incubated with shaking at 37 °. At appropriate time intervals, 0.1-ml samples can be spread onto rich agar or minimal agar containing nutrients necessary for growth of the recipient strain and also the required selective antibiotic. The required linkage analyses may be performed on clones appearing after incubation by replicating. Propagation in liquid medium after transduction is necessary for phenotypic expression of the resistance characters. As an alterna-
[3]
METHODS FOR STUDY OF ANTIBIOTIC RESISTANCE PLASMIDS
47
tive to growth in liquid medium, the mixture, after transduction, may be spread onto the surface of rich agar plates (approximately 108 cells) and incubated at 37 ° for 4-6 hr. At this time the agar is rimmed with a sterile spatula and gently lifted up from one side of the plate. An appropriate concentration of antibiotic is then pipetted under the agar, the agar is replaced, and the petri plate is allowed to stand in a refrigerator overnight, to allow diffusion of the antibiotic through the agar. Incubation at 37 ° is then continued and the resistant clones picked and scored for necessary auxotrophic and resistance markers.
Transduction with Phage P22 in S. typhimurium Preparation o] Phage Stock. A saturated culture of the donor strain in rich broth is prepared by overnight incubation and 0.I ml of this culture is diluted into 1 ml of rich broth and incubated at 37 ° for 2 hr. To this 10 ml of culture (approximately l0 s cells/ml) is added 107 particles of phage P22 and vigorous incubation is continued for 5-8 hr until the broth begins to clear slightly. Centrifuge the cultures at low speed, remove the supernatant, and centrifuge the supernatant in a Spinco Model L ultracentrifuge at 30,000 g for 1 hr (40 rotor). Remove and discard the supernatant and resuspend the phage pellet in 2 ml of T2 buffer (T2 buffer contains 10 ml of 0.1 M MgS04, 10 ml of 0.01 M CaC1._,, 1.0 ml of a 1% solution of gelatin, 3 g of Na:HP04, 1.5 g KH~P04, 4 g of NaC1, 5 g of K2S04 and distilled water to 1000 ml; when the solution is autoclaved it becomes cloudy, but this clears on cooling). To ensure that the phage suspension is completely sterile, 0.5 ml of chloroform is added and the mixture is well shaken. The chloroform can be removed by bubbling sterile air through the suspension. The phage suspension is stored in a refrigerator; the titer should be approximately 1011 PFU/ml. Conditions ]or Transduction. For transduction, the recipient strain of S. typhimurium is grown overnight in rich broth and 1 ml is placed in a sterile tube and incubated at 37°; to this tube is added 0.1 ml of P22 phage stock, and incubation is continued for 10 rain to allow for phage adsorption. Phenotypic expression of resistance characters is then carried out by incubation in broth or on agar plates as described for P1 transduction in E. coli; following this period of growth, the bacteria are plated on media containing appropriate antibiotics to select for transductants which have received the resistance plasmid. The colonies are subsequently scored for other phenotypes of the plasmid; remember that in P22 transduction the transfer genes are not necessarily cotransduced with the resistance genes. Scoring is most conveniently carried out by
48
METHODS FOR THE STUDY OF ANTIBIOTICS
[3]
transferring colonies to a grid on a "master" plate (containing no antibiotic), incubating at 37% and replica plating onto antibiotic plates. C. Transformation
The discovery of transformation in E. coli23,24has provided an important tool for the study of R plasmid structure and function; it seems likely that further work will provide the technical details whereby other bacterial strains and their plasmids may be studied in similar fashion. Preparation of R PMsmid DNA. It is advisable to use R plasmid DNA in the covalently closed circular form, which can be obtained by ethidium bromide-cesium chloride equilibrium centrifugation or nitrocellulose chromatography. Both these methods provide convenient ways of obtaining active transforming DNA; such DNA preparations have been adequately described in a recent review by Clowes2 The DNA is kept in TEN buffer (20 mM Tris, 1 mM EDTA, 20 mM NaC1, pH 8.0). Conditions ]or Trans]ormation in E. coli. The calcium chloride pretreatment, as described below, is satisfactory for converting most E. coli strains into efficient recipients for transformation. However, certain substrains and mutants lose viability when exposed to calcium chloride. It is important to perform adequate controls and modify the treatment (exposure to calcium chloride, stage of growth, length of 42 ° heat pulse) where necessary, so as to maintain viability of the recipient. The recipient E. coli strain is grown in 10 ml of phosphate-buffered minimal medium to a cell density of 2-3 X 108/ml and chilled quickly by plunging the culture tube in ice water. The cells are collected by centrifugation (5000 rpm X 10 rain) and washed in 5 ml of 10 mM NaC1. The cells are then resuspended in 5 ml of cold 30 mM CaC12 and allowed to stand in ice for 20 min, pelleted by centrifugation and resuspended in 0.5 ml of 30 mM CaC12. To 0.2 ml of cells in CaCi~ is added 0.1 ml of DNA solution (in T E N buffer adjusted to 30 mM CaC12 ; various concentrations should be tested, but 1 ~g is suitable for most experiments), and the mixture is allowed to stand in ice for 1 hr, placed in a 42 ° water bath for 2 min, and chilled to room temperature. The transformants can be detected by one of the following methods: (a) Samples of the transformation mixture are plated directly onto rich medium containing the appropriate selective antibiotics and incubated at 37 °. (b) Samples of the transformation mixture are diluted into rich broth containing no antibiotic, incubated at 37 ° for at least 1 hr, and then plated on solid medium containing antibiS. N. Cohen, A. C. Y. Chang, and L. Hsu, Proc. Nat. Acad. Sci. U.S. 69, 2110 (1972). S. D. Cosloy and M. Oishi, Proc. Nat. Acad. Sci. U~. 70, 84 (1973).
[3]
METHODS FOR STUDY OF ANTIBIOTIC RESISTANCE PLASMIDS 1
I
I
1
1
I
I
1
|
49
12
4O 0 ~c
8
~, 6 E ~4 I--
0.40 0.80
I
1.20
t.60 2.00 2 . 4 0
A m o u n t of DNA ( p . g / m l )
FIG. 1. Effect of concentration of R factor DNA on transformation frequency. Various concentrations of covalently closed R6 DNA were assayed for their ability to transform CaCI2-treated Escherichia coli to kanamycin resistance. Transformation frequency was determined after a 120-min incubation in antibiotic-free medium to allow complete expression of kanamycin resistance. [From S. N. Cohen, A. C. Y. Chang, and L. Hsu, Proc. Nat. Acad. Sci. U.S. 69, 2110 (1972).]
otics. (c) Samples of the transformation mixture are spread onto rich agar medium lacking antibiotic, incubated at 37 ° and subsequently replica-plated onto medium containing antibiotic. In general, method (b) is the most convenient since it allows ample time for phenotypic expression of the antibiotic resistance characters. Method (a) is unsatisfactory for certain antibiotic resistances such as streptomycin, which have a long phenotypic lag. Linkage relationships and other properties may be examined by replica plating. Figures 1 and 2 show a typical DNA requirement for transformation, and the kinetics of appearance of resistance phenotypes after transformation. Transformation of resistance plasmids by similar methods has also been reported for S. aureus. T M
III. Enhanced Segregation of Resistance Plasmids by Curing Agents Evidence for the presence of a resistance plasmid in a bacterial strain can be obtained by demonstrating the loss of the resistance characters 24aL. Rudin, J. E. SjSstrSm, M. Lindberg, and L. Philipson, J. Bacteriol. 118, 155
(1974).
50
METHODS FOR THE STUDY OF ANTIBIOTICS I
I
I
I
|
I
I 20
I 40
J 60
I 80
I t00
I ~20
[3]
I0-6
~:
40 - ~
c ~r
~0 -~
h
10-9 0
(rain)
Fla. 2. Kinetics of expression of kanamycin resistance in transformed Escherichia coli. After incubation of CaC12-treated cells with R6 D N A (0.6 ~g/ml), bacteria were diluted 10-fold into antibiotic-free L broth. At the times shown, 0.1-ml samples of the bacterial culture were spread onto nutrient agar plates containing kanamycin and incubated overnight at 37 ° for determination of number of transformants. An identical sample was diluted appropriately, and plated on antibiotic-free nutrient agar to determine the total number of viable cells. Transformation frequency is expressed in terms of the number of kanamycin-resistant bacteria relative to the total number of viable cells. [From S. N. Cohen, A. C. Y. Chang, and L. Hsu, Proc. Nat. Acad. Sci. U.S. 69, 2110 (1972).]
following loss of the plasmid. In some strains (notably Salmonella typhimurium) and with certain plasmids a relatively high spontaneous segregation of the resistance plasmid ( > 5 % ) is common. ~,5,l° For this reason it is recommended that all stock cultures of resistant strains be mainrained on agar containing a low concentration of one or more of the antibioties to which resistance is determined by the plasmid. However, in most bacterial strains carrying resistance plasmids, spontaneous segregation of the plasmid is not detectable and "curing" agents must be used to enhance this. These curing agents consist of compounds such as acridine orange, quinacrine, ethidium bromide, mitomycin, or
[3]
METHODS FOR STUDY OF ANTIBIOTIC RESISTANCE PLASMIDS
51
sodium dodecyl sulfate. 2,5,1° A variety of other methods are also effective. Some typical protocols are described below. Again, it should be emphasized that failure to remove resistance characters by curing agents is not strong evidence for the lack of a plasmid, neither is it evidence for chromosomal resistance genes.
A. Acridine Orange, Quinacrine, and Ethidium Bromide Curing 25-27 An overnight culture of the resistant strain is diluted into five different 5-ml cultures of Penassay broth or nutrient broth, previously adjusted to pH 7.5, to give a cell concentration of 10~ to 105 per milliliter. Increasing concentrations of the curing agent added to the five tubes cover the range from 10 to 200 ~g/ml. The cultures are then incubated overnight at 37 ° and observed for growth. The cells from the culture tube that contains the highest concentration of curing agent permitting visible growth (usually 50-75 ~g/ml) are diluted and plated onto rich agar plates and grown up to single clones. These clones are tested for the absence of resistance characters by replica-plating onto plates containing antibiotics. It is essential that several of the resistance characters be examined since, in certain cases, resistance plasmids show a tendency to segregate single resistance characters without loss of the plasmid.
B. Sodium Dodecyl Sulfate 2s Sterile Penassay broth containing 10% sodium dodecyl sulfate is prepared and is inoculated with a low dilution of cells of the resistant strain (to 103 to 104 cells/ml). The culture is then incubated for 48-72 hr at 37°; at this time the culture medium should be (apparently) free of bacteria and very viscous. Most of the cells have lysed at this stage, but the survivors are usually enriched for bacteria which have lost the plasmid. It appears that cell surface characters associated with plasmids (pill, etc.) make these cells more sensitive to lysis by the detergent than the corresponding R- strain. Recovery is low, and this cannot be used as a quantitative measure of induced plasmid loss. 2: T. Watanabe and T. Fukasawa, J. Bacteriol. 81, 679 (1961). 28D. Bouanchaud, M. R. Scavizzi, and Y. A. Chabbert, J. Gen. Microbiol. 54, 417 (1969). 27F. E. Hahn and J. Ciak, Ann. N . Y . Acad. Sci. 182, 295 (1971). :s M. Tomoeda, M. Inuzuka, N. Kubo, and S. Nakamura, J. Bacteriol. 95, 1078 (1968).
52
METHODS FOR THE STUDY OF ANTIBIOTICS
[3]
C. M i t o m y c i n 29
This drug has been used as a means of curing plasmids from strains of P. aeruginosa. An overnight culture of the resistant strain is diluted to 105 cells/ml in tubes of rich medium containing successively higher concentrations of mitomycin C (a maximum of 100/~g/ml is usually sufficient). The tubes are incubated overnight with shaking and organisms from the tube containing the highest concentration of mitomycin which permits growth are plated onto rich agar. Colonies are then picked onto rich agar plates and replica-plated to score for antibiotic resistance characters. A relatively high frequency of curing induced by any of these agents is good evidence for the presence of a resistance plasmid, although lack of curing cannot be taken as evidence for lack of a plasmid, or the existence of chromosomal resistance genes since many resistance plasmids are quite refractory to curing. Demonstration of concomitant loss of resistance phenotype and plasmid DNA (by methods such as dye-buoyant density analysis) provides strong support for plasmid-mediated resistance. In those instances where it is simply desired to obtain the host bacterium free of resistance plasmid, any of the above methods can be combined with B-lactam antibiotic screening (penicillin selection) for the antibiotic strains. Thus if a tetracycline-sensitive strain is required, curing treatment followed by two cycles of treatment with a fl-lactam antibiotic in the presence of tetracycline can be used to enrich for rare tetracycline-sensitive plasmid-negative strains, or mutants. The survivors of this treatment should be screened against other antibiotics for which resistance is determined by the R plasmid to determine whether some or all of the resistance genes have been lost or mutated. The choice of /3-1actam antibiotic is important; for most E. coli or S. typhimurium strains ampicillin selection is sufficient, although cefotoxin or another/3lactamase resistant penicillin or cephalosporin is convenient when the strain is a/3-1actamase producer. For P. aeruginosa carbenicillin selection can be used, but resistance to this drug is common in this organism. Penicillin G or methicillin (for/3-1actamase producers) are used for screening of S. aureus strains. IV. Compatibility Properties of R Pla'smids The most common way of characterizing R plasmids is on the basis of their property to coexist or not to coexist in the same bacterium. The original observation of incompatibility was by Watanabe, 12 who found ~oj. G. Rheinwald, A. M. Chakrabarty, and I. C. Gunsalus, Proc. Nat. Acad. Sci. U.S. 70, 855 (1973).
[3]
METHODSFOR STUDY OF ANTIBIOTIC RESISTANCE PLASMIDS
53
TABLE I I I R FACTOF~COMPATIBILITYGROUPSa Representative plasmid RA1 R57b R386b Rldrdl6
ColB.K98 R124 Flac
R811 R27 R144 R483 R621a JR66a R391 R387 R446b N3T R724 RP4 R751 Rtsl R388 RCK
Compatibility group
Resistances b
A
T,Su
C
A,C,Gk, Su
Ft FII FII Fiv Fv G H I I I I J K M N O P P T W X
T K T A T T,K S,Tp T S,K K S,C S,T T S,T,C,Su A,T,K Tp K Su,Tp A,S
See J. N. Coatzee, N. Datta, and R. W. Hedges, J. Gen. Microbiol. 72, 543 (1972); Y. A. Chabbert, M. R. Scavizzi, J. L. Witchitz, G. R. Gerband, and D. H. Bouanchaud, J. Bacteriol. 112, 666 (1972); N. D. F. Grindley, J. N. Grindley, and E. S. Anderson, Mol. Gen. Genet. 119, 287 (1972) ; R. W. Hedges and N. l)atta, J. Gen. Microbiol. 77, 19 (1973). bT, tetracycline; Su, sulfonamide; A, ampicillin; C, chloramphenicol; GK, gentamicin/kanamycin; K, kanamycin; Tp, trimethoprim, S, streptomycin that the R plasmids which he was studying could be divided into two classes, one that would allow a sex factor to coexist in the same host (fi-), and the other which led to the expulsion of the sex factor (fi÷), presumably by segregation during replication and division. The molecular basis for this expulsion is still not understood, but by examining the abilities of various R plasmids to coexist in E . coli it has been possible to assign more than 20 different compatibility types~°; a similar phenomenon has been described in S t a p h y l o c o c c u s aureus. '~1 The prototype for studies of this kind is the work of Coatzee et al., 3° and Table I I I shows the currently known compatibility types among a group of plasmids. ~J. N. Coatzee, N. Datta, and R. W. Hedges, J. Gen. Microbiol. 72, 543 (1972).
54
METHODS FOR THE STUDY OF ANTIBIOTICS
[3]
A. D e t e r m i n a t i o n o f fi C h a r a c t e r 3°
The R factors to be tested are transferred by conjugation or transduetion to an E. coli Hfr donor such as Hfr Hayes or Hfr Cavalli. To facilitate transfer it is convenient to use a nalidixic acid- or rifampicinresistant mutant of the recipient strain. Conjugation is carried out as described previously. A culture of the R÷/Hfr strain is then grown in rich medium and 2-3 drops spread onto a rich agar plate containing CaC12 (1 raM). Samples of a stock lysate of the male-specific phages MS2, f2, or R17 are dropped (spotted) onto the bacterial lawn with a Pasteur pipette or spread on the surface of the lawn with a sterile loop or paper strip. Visible lysis after overnight incubation at 37 ° indicates that the plasmid is fi-; no lysis indicates that the plasmid is fi+ and is repressing the synthesis of sex pili necessary for attachment and infection by the male-specific bacteriophages.
B. Testing for Compatibility of R Factors 3°-~3 This is determined by testing the ability of two different R factors of a particular group to coexist stably in the same host. For this to be done, the two R plasmids must differ in resistance characters in order that a combination of resistance characters from the two may be selected. For ease of selection, it is convenient to have one of the R plasmids present in a standard nalidixic acid, or rifampicin-resistant recipient to allow selection against growth of the donor host harboring the entering R plasmid. The frequency of transfer is compared against that into the same recipient which does not carry an R plasmid. Recipient colonies are purified on nonselective medium and tested, by replica plating, for the presence of characters of each plasmid to give a measure of compatibility. In this way it can be easily seen whether the presence of one R plasmid excludes (or is incompatible) with another, and they can be assigned to incompatibility groups (Table III). If on the basis of resistance phenotypes, two R plasmids apparently coexist in the same host it is important to determine whether they remain as independent R plasmids, since recombination could have occurred. To test for this, the host carrying the two R factors is submitted to a series of conjugations with a suitable recipient (as previously de~1y. A. Chabbert, M. R. Scavizzi, J. L. Witchitz, G. R. Gerband, and D. H. Bouanchaud, J. Bacteriol. 112, 666 (1972). s2N. D. F. Grindley, J. N. Grindley, and E. S. Anderson, Mol. Gen. Genet, 119, 287 (1972). 3~R. W. Hedges and N. Datta, J. Gen. Microbiol. 77, 19 (1973).
[4]
ANTIBIOTIC ASSAYS
55
scribed). Each resistance character of the two coexisting plasmids is selected in turn and the transcipients are analyzed separately for the resistance characters of both plasmids, by replica plating. In this w a y it can be seen whether the characters of the two plasmids remained intact in the host, or any assortment occurred. In like manner incompatibility groups have been assigned for resistance plasmids in P. aeruginosa. 14 In S. aureus the stable coexistence of R plasmids in the same host can be tested by transduction. 21
[4] A n t i b i o t i c Principles
Assays--
and Precautions
B y FREDERICK KAVANAGH I. Introduction . . . . . . . . . . . . . . . . . . II. General Considerations . . . . . . . . . . . . . . . A. Organisms Used in Assays . . . . . . . . . . . . . B. Preliminary Screening Methods . . . . . . . . . . . . C. Quantitative Aspects of Relatively Pure Samples . . . . . . . III. Kinds of Assays . . . . . . . . . . . . . . . . . A. General Comments . . . . . . . . . . . . . . . B. Diffusion Methods . . . . . . . . . . . . . . . C. Dilution Methods . . . . . . . . . . . . . . . . IV. Assays of Diverse Biological Samples . . . . . . . . . . . V. Relative Inhibitory Coefficient of Antibiotics . . . . . . . . .
55 57 57 58 58 59 59 60 62 65 66
I. I n t r o d u c t i o n Anyone who works with antibiotics needs to understand the principles of the several kinds of assays, their applicabilities, and their deficiencies. In laboratories t h a t have assay groups, the researcher should always discuss the reasons for the assays and a n y anticipated difficulties with this group. Otherwise the preparations will be processed in the routine assay. The numbers obtained in such assays m a y have little intrinsic meaning, but neither the researcher nor assay group will realize it. Without a common understanding of principles, the two groups cannot hold meaningful discussions of their common problems. Analytical microbiology is a p a r t of the general subject of analysis, not simply a minor branch of bacteriology. The bacteria are employed as reagents and usually are the most dependable p a r t of the methods.
[4]
ANTIBIOTIC ASSAYS
55
scribed). Each resistance character of the two coexisting plasmids is selected in turn and the transcipients are analyzed separately for the resistance characters of both plasmids, by replica plating. In this w a y it can be seen whether the characters of the two plasmids remained intact in the host, or any assortment occurred. In like manner incompatibility groups have been assigned for resistance plasmids in P. aeruginosa. 14 In S. aureus the stable coexistence of R plasmids in the same host can be tested by transduction. 21
[4] A n t i b i o t i c Principles
Assays--
and Precautions
B y FREDERICK KAVANAGH I. Introduction . . . . . . . . . . . . . . . . . . II. General Considerations . . . . . . . . . . . . . . . A. Organisms Used in Assays . . . . . . . . . . . . . B. Preliminary Screening Methods . . . . . . . . . . . . C. Quantitative Aspects of Relatively Pure Samples . . . . . . . III. Kinds of Assays . . . . . . . . . . . . . . . . . A. General Comments . . . . . . . . . . . . . . . B. Diffusion Methods . . . . . . . . . . . . . . . C. Dilution Methods . . . . . . . . . . . . . . . . IV. Assays of Diverse Biological Samples . . . . . . . . . . . V. Relative Inhibitory Coefficient of Antibiotics . . . . . . . . .
55 57 57 58 58 59 59 60 62 65 66
I. I n t r o d u c t i o n Anyone who works with antibiotics needs to understand the principles of the several kinds of assays, their applicabilities, and their deficiencies. In laboratories t h a t have assay groups, the researcher should always discuss the reasons for the assays and a n y anticipated difficulties with this group. Otherwise the preparations will be processed in the routine assay. The numbers obtained in such assays m a y have little intrinsic meaning, but neither the researcher nor assay group will realize it. Without a common understanding of principles, the two groups cannot hold meaningful discussions of their common problems. Analytical microbiology is a p a r t of the general subject of analysis, not simply a minor branch of bacteriology. The bacteria are employed as reagents and usually are the most dependable p a r t of the methods.
56
METHODS FOR THE STUDY OF ANTIBIOTICS
[4]
Basic knowledge of bacteriology, especially bacterial physiology, is necessary to the proper practice of microbiological assaying. Most of the causes of the low quality of assays come from lack of both analytical knowledge and of analytical sense by practitioners of the art. Microbiological assaying should be considered to be an exacting profession. No one would expect to become an analytical chemist or an expert at synthesizing new antibiotics by reading a brief chapter in a book; he would expect to spend years learning the trade. The purpose of this brief article is to give principles of the several kinds of assays for antibiotics, to discuss problems of application, to indicate reasons for assaying, and to consider the general topic of relation between structure and activity. Details of assay methods will not be given because to do so would fill the volume and would duplicate other publications. The general references, where details may be found, are the two volumes of "Analytical Microbiology," edited by F. Kavanagh. 1,2 Microbiological assays are performed for two reasons. The first is that antimicrobial activity is the only property the rather diverse group of compounds known as antibiotics have in common, and the appropriate method for measuring their quantity is by means of that activity. The second reason is that the alternatives to microbiological methods, namely, chemical methods, may take too much time to develop and may lack specificity for only those compounds with antimicrobial activity. Antibiotic assays are relative assays in which responses to a sample are compared to responses to a standard. This relativity places certain constraints upon the system if the assays are to be valid. The most important constraint is the requirement of qualitative identity of standard and sample solutions and of their treatments. To meet these requirements of validity, standard and sample must have the same chemical compositions, must be dissolved in identical solvents (usually aqueous solutions that have the same pH and buffer capacity), and the solutions must be processed in the assay in an indistinguishable manner. Usually the first requirement, that of chemical identity, is not established. The chemical steps are under the control of the analyst. The final requirement, that of identity of processing, is provided by the AUTOTURB ® System2 ,4 Close approximation to these requirements can be achieved by any analyst without extensive mechanical aids and automation if he is skilled in the art and practice of microbiological assaying. 1 F. Kavanagh, ed., "Analytical Microbiology." Academic Press, New York, 1963. 2 F. Kavanagh, ed. "Analytical Microbiology," Vol. II. Academic Press, New York, 1972. s N. Kuzel and F. Kavanagh, J. Pharm. Sci. 60, 764 (197,1). 4 N. Kuzel and F. Kavanagh, J. Pharm. Sci. 60, 767 (1971).
[4]
ANTIBIOTIC ASSAYS
57
II. General Considerations A. Organisms Used in Assays There are several reasons for measuring the susceptibility of different microbial species to antibiotics. One is to obtain data to guide clinical applications. Another is an aid in selecting appropriate assay organisms. Certain bacteria are employed in diffusion assays and others in tube methods. The most popular test organisms for diffusion assays are Staphylococcus aureus, Sarcina lutea, and Bacillus subtilis. Occasionally Esch-
erichia coli, Klebsiella pneumoniae, Photobacterium fished, Salmonella gallinarium, and Pseudomonas sp. are used for specific antibiotics. Food and Drug Administration '~ uses 15 species and strains of bacteria and fungi to assay 56 different antibiotics by the plate method. They use four bacteria, S. aureus, K. pneumoniae, E. coli, and Streptococcus ]aecalis, and one yeast, Saccharomyces cerevisae, to assay 23 antibiotics by turbidimetric methods. Staphylococcus aureus is the most often used organism in tube assays. Streptococcus faecalis is employed when the pH of the assay medium is low as, for example, in the assay for monensin ~ in animal feeds. Salmonella gallinarium is sensitive enough to preservatives, such as thimerosal, 7"that it can be used in a turbidimetric assay for preservatives in biological preparations as well as for antibiotics. Escherichia coli, K. pneumoniae, and P. aeruginosa are used to assay antibiotics with special activity against gram-negative organisms. The test organism must be susceptible to the drug to be measured. It must grow reasonably fast in practical assay media. It should not be virulent to man for reasons of safety. It should be easy to maintain. Its susceptibility should not change with successive subcultivation. Old laboratory strains are much more likely to satisfy these criteria than freshly isolated strains. Most test organisms are old laboratory strains and have been in ~lse for 30 years or more. The population of cells produced in the inoculum broth should be of uniform susceptibility. In certain types of assays, e.g., overnight serial dilution, resistant forms could grow out and give confusing answers. The first example of this was a tube assay for streptothricin by means of S. aureus (Heatly strain, ATCC 9144). A strain of Klebsiella pneumoniae, ATCC 9997, did not have the resistant forms and was suitable 5B. Arret, D. P. Johnson, and A. Kirshbaum, Y. Pharm. Sci. 60, 1689 (1971). 6 F. Kavanagh and M. Willis, J, Ass. Offic. Anal. Chem. SS, 114 (1972). 7F. Kavanagh, in "Analytical Microbiology" (F. Kavanagh, ed)., Vol. 2, p. 343. Academic Press, New York, 1972.
58
METHODS FOR THE STUDY OF ANTIBIOTICS
[4]
for streptothricin and streptomycin assays. This experience with streptomycin assay indicates the necessity for ascertaining the suitability of test organisms. More details and method of testing the organism are given by Kavanagh. 8,9 If a preparation of a single component antibiotic, for example, penicillin G, is to be assayed in terms of a single component standard of the same chemical structure, any susceptible test organism could be used under any suitable condition. When both standard and sample are mixtures, as is usual, the organism and operating conditions must be specified if others are to obtain comparable answers. Interpretation of answers in terms of weight of active drug must be done with caution when standard and samples are mixtures.
B. Preliminary Screening Methods The usual way of detecting antibacterial activity in preparations of unknown activity is by means of a simple diffusion test. Concentrations of about 1, 10, and 100 ~g/ml in water, buffer, or dilute alcohol are prepared. Filter paper disks saturated with the solutions are placed on agar plates seeded with B. subtilis, S. lutea, S. aureus, or E. coli. The plates are then incubated at 33-35 ° overnight to permit growth of the test bacteria. Clear zones of inhibition will be found around certain of the disks if the compound has antibacterial activity. A zone around only the 100 ~g/ml disk on S. lutea plates usually indicates a very weak antibiotic. If zones at the l~g/ml concentrations occur on the S. lutea, S. aureus, and B. subtilis, but not on the E. coli, plates at 100 ~g/ml, then the compound has good activity against gram-positive bacteria but little against gram-negative bacteria. If activity is mainly on the E. coli plates, the compound should be tested against other gram-negative bacteria. Activities in this crude screening test are used as a guide in designing assay methods for the compound. The character of the zone edge as well as sensitivity needs to be considered in selecting a plate method. Usually zone edges are better defined in the B. subtilis assay than in the others. If activity against S. aureus, or E. coli is indicated, then one of them could also be used in a tube method.
C. Quantitative Aspects of Relatively Pure Samples More antibiotic assays are done to measure the quantity of an active antibiotic than for all other purposes. Samples may be pharmaceutical s F. Kavanagh, Bull. Torrey Bot. Club 74, 303 (1947). 9F. Kavanagh, in "Analytical Microbiology" (F. Kavanagh, ed.), Vol. 1, p. 125. Academic Press, New York, 1963.
[41
ANTIBIOTIC ASSAYS
59
dosage forms of high purity and precisely known composition, agriculture products such as premixes and animal feeds in which the active drug may be of high purity, and process samples of lessened purity but known identity. The first two classes of products must meet a label claim of quantity. The third class of samples are of great interest to the chemists who must supervise production of the product. The assays enable them to monitor their operations and to determine yields across purification steps. All samples discussed in this section are of known identity and of high purity so that measurement of quantity is possible. This is a good place to make a point crucial to microbiological assays as well as to many chemical assays in which a sample is measured in terms of a standard. It is that the quantity of sample can be exactly determined only when sample and standard have identical composition. When the composition of sample is unknown or is known to differ from the standard, then the sample is said to have an activity equivalent to a certain quantity of standard when assayed in a particular assay under specified conditions. The exact quantity of active product as a weight of product compound cannot be obtained from such assays. The degree of purity of a preparation can be determined if the active drugs in the samples have the same relative concentrations as the active materials in the standard. The sample under these conditions may be considered to be a diluted standard so long as the inert materials do not interfere with the assay. Preparations obtained during purification of an antibiotic being manufactured usually meet the criteria. The safest practice is to consider the figures obtained on impure samples only as approximate values until the preparations become nearly pure or chromatography indicates no unexpected active substances.
III. Kinds of Assays A. General Comments The two kinds of assays are those usually referred to as dilution and diffusion. In the former, the antibiotic is presented to the test organism at one concentration. The concentration is uniform throughout the medimn. The medium may be liquid as in tube methods or solidified by agar as in a plate dilution method. Activity may be measured by an end point, as in serial dilution in tubes s,%1° or agar dilution in plates, 1° or as a graded response, as in the diffusion and turbidimetric methods. 1,~ lo H. M. Ericsson and J. (3. Sherris, "Antibiotic Sensitivity Testing." Acta Pathol. Microbiol. Stand., Suppl. 217 (1971).
60
METHODS FOR THE STUDY OF ANTIBIOTICS
[4]
Three kinds of dilution methods are in use; namely, agar dilution, serial dilution in tubes, and photometric assays. Some sort of all-or-none response is obtained with the first two types. The third represents the high precision turbidimetric assays in which graded response is obtained to graded concentrations of drug. B. Diffusion Methods
In diffusion assays, the concentrations of antibiotic vary between the point of application and the point of inhibition. The response is observed as a clear zone of no growth in which the diameter of the zone is approximately proportional to the logarithm of concentrations of antibiotic for small zones. Theory of the method was reviewed by Cooper. 1,2 The two general methods and their applications will be considered in detail in the following sections. Diffusion assays may be performed in tubes or in dishes. The tube method is used in Japan where it is called the superposition method. The test organism is in nutrient agar in a layer about 2 cm deep in small tubes. The sample is added to the tube to a depth of about 1 cm. The tubes are incubated to permit development of the uninhibited bacteria. The length of the zone of no growth is measured. Zone length is proportional to logarithm of concentration of antibiotic. 2 The tube method is a simple method that does not require special equipment. Incubation can be done in a water bath. Most diffusion assays are done in petri dishes or in large plates. The samples may be applied in disks, in cylinders, or in holes in the agar layer. Inoculation of the agar may be uniform or as a thin layer on the surface of the bulk agar for aerobic organisms or next to the glass for microaerophilic organisms. Details of specific methods are given in the two general references. 1,2 When a new substance is to be assayed, the general procedure is to select a test organism that has requisite susceptibility for plate assaying. The assay design can be selected from those given for penicillin or for a substance of the same general class as that of the new substance. All designs are derivatives of those devised for assay of penicillins. Advantages and disadvantages need to be considered before plate methods are applied. The method is deceptively simple but the theory is complex. Details must be carefully observed if precision is to be high. Precautions to be observed to obtain high precision assays are outlined by Kavanagh. 11 The diffusion assay is a physicochemical method in which a bacterium 11F. Kavanaugh, in "Analytical MicrobiOlogy" (F. Kavanaugh, ed.), Vol. 2, p. 31. Academic Press, New York.
[4]
ANTIBIOTIC ASSAYS
61
is used as indicator of concentration of active compound. Diffusion of the drug is determined by diffusion constant, temperature, and to a minor extent by pH, salt concentration, concentration of agar, etc. The diffusion constant is related to molecular weight of the diffusing species. The smaller the molecular weight, the farther the drug diffuses in a given time. Location of the edge of the inhibition zone is determined by arrival of a certain concentration (critical concentration) of the drug before the bacteria have grown to a critical concentration of cells. 1-~ Because zone size is dependent upon several physical and biological factors, size per se is not an indication of concentration of drug or of sensitivity of test organism. Diffusion assays are relative; a standard is required to calibrate the system. Standard and sample must be of the same chemical species for the assays to have meaning. When the dose-response line relating zone diameter to logarithm of concentration of drug covers a relatively short range of concentrations, graphs of the lines on semilog paper are straight. A range of less than 8 times the least concentration showing a zone of inhibition usually will give straight or nearly straight lines. Closely related antibiotics will plot into families of approximately parallel lines. Dissimilar substances may give lines that are not parallel. Thus two factors govern the appearance of dose-response lines. One is the sensitivity of the system which locates the starting point. The other factor is the slope of the line which is determined by the product of the diffusion coefficient and critical time (DTo). Influence of the several factors affecting zone size was considered in detail by Cooper 12,13 and Kavanagh. TM Conclusions from this discussion of diffusion assays are that the method is suitable for obtaining a quick indication of activity, and, if care is exercised, for obtaining a precise measure of a sample in terms of a standard. It cannot be used to obtain an absolute measure of susceptibility of bacteria or of activity of a drug. As ordinarily done, diffusion assays may determine concentration of a sample with errors varying from 1 to 10%. The former errors are for very carefully performed large plate assays. Because of poor control of operating factors, petri dish assays of drugs in nearly pure solution may have errors ranging from 5 to 10%. High accuracy and precision are much more difficult to achieve with diffusion than with turbidimetric methods. 1: K. E. Cooper, in "Analytical Microbiology" (F. Kavanagh, ed.), Vol. 1, p. 1. Academic Press, New York, 1963. 1~K. E. Cooper, in "Analytical Microbiology" (F. Kavanagh, ed.), Vol. 2, p. 13. Academic Press, New York, 1972. 14F. Kavanagh, J. Pharm. Sci. in press.
62
METHODS FOR THE STUDY OF ANTIBIOTICS
[4]
C. Dilution Methods
1. Agar Dilution The active substance is diluted in agar contained in petri dishes. The surface is streaked with several species of bacteria or small areas are inoculated with a large number of strains of bacteria by means of a replicator. The plates are incubated overnight at suitable temperatures and observed for growth of test bacteria. The lowest concentration of drug that shows no growth of a particular test organism is taken as the minimum inhibitory concentration (MIC). This very simple method is suitable for obtaining approximate MIC's for a number of bacteria simultaneously. The error in the determinations comes from both the design and the difficulty in determining the end point, which is not always sharp. Sharpness of end point depends, in part, upon the size of the step in concentration. Usually concentration changes by 2-fold steps. In other words, the concentrations form a geometric series, such as 1, 2, 4, 8, etc., just as in diffusion assays. In a 2-fold dilution test as described, there is an inherent uncertainty of 50% (1 step in the concentration scale) in determining the end point in addition to the subjective one mentioned above. Agar dilution is a widely used test of severely restricted accuracy.
2. Serial Dilution in Tubes Dilution in tubes was used early in the penicillin program to measure penicillin in experimental samples. It had two forms. One was a 2-fold serial dilution method and the other was one in which concentrations formed an arithmetic series of closely spaced values. The latter was considerably more accurate and precise than the serial dilution method. The end point in these tube assays is indicated by the absence of perceptible growth after 16 hr of incubation, by absence of hemolysis of red cells in the short-term test, 15 or by absence of luminescence of photobacteria. TM The last-named test could provide independent information on both suppression of luminescence and of growth of the test photobacterium. Details of the methods and their inherent errors are given by Kavanagh2 '17 One advantage of the tube dilution methods is the ease of performing tests with a large variety of organisms in many media. Results from such tests may be adequate for survey work early in a program. The dilution of the test substance causing inhibition may provide useful information 1~G. Rake and H. Jones, Proc. Soc. Expl. Biol. Med. 54, 189 (1943). ~eG. Rake, C. M. McKee, and H. Jones, Proc. Soc. Exp. Biol. Med. 51, 273 (1942) 1TF. Kavanagh, Bull. Torrey Bot. Club 74, 414 (1947).
[4]
ANTIBIOTIC ASSAYS
63
in the absence of a standard. A serial dilution test may be used for measuring MIC of clinical isolates but not for quantitation of active drugs when accuracy is important. 3. Photometric Methods
Serial dilution methods soon were found to be inadequate for the penicillin program and were supplanted by the method of measuring response of bacteria to graded concentrations of antibiotics. The first successful turbidimetric assay was that of McMahan. TM It was used essentially unchanged for nearly 25 years by laboratories in pharmaceutical companies, official bodies, and regulatory agencies. The method was derived from microbiological vitamin assay methods. It is very simple in principle. Graded quantities of drug are added to test tubes, inoculated broth added, tubes incubated until substantial growth has occurred in the control tubes, growth stopped, and concentration of bacteria in the tubes determined by a photometric method. Potency of samples is obtained by interpolation from a calibration curve prepared from responses of the standard tubes in each test. The assays can be of high accuracy. Both manual and partially automated methods are used. Details of both will be found in the appropriate chapters of the two volumes of "Analytical Microbiology. ''1,2 A turbidimetric method can be designed for nearly any antibiotic or antibacterial substance. A wide variety of gram-positive and gram-negative bacteria, yeasts, protozoa, and algae may be used as the test organism. Quite often the test organism is selected from the dominant group inhibited by the drug. The requirement is that growth of the test organism be inhibited by the drug and that the generation time be less than 60 rain, otherwise incubation time becomes excessive. The assay medium must support growth of the test organism and not interfere to any great extent with the action of the drug. The test organism should not be appreciably pathogenic. It must grow uniformly suspended, and neither clump, form strings, nor form a surface pellicle. In the range of concentrations of drug of interest in assaying, most. drugs interact with the test organism to reduce the growth rate. Therefore, the assay really is a rate method in which the integrated effect of reduction of growth rate is measured. Since a lag period also is involved, the total incubation time is longer than the log phase growth period. The length of the incubation is unimportant so long as it is sufficient and is exactly the same for standards and samples. Since the assays are growth rate methods and the medium has an upper limit to the maximum 18j. R. McMahan, J, Biol. Chem. 153, 249 (1944).
64
METHODS FOR THE STUDY OF ANTIBIOTICS
[4]
concentration of bacteria possible, unduly prolonging incubation time will eliminate the effect of the drug. Anything affecting growth rate other than drug will cause a bias in the assay. 19 The analyst must always be alert to such interferences. They are very common in feed assays but may be rare in pharmaceutical dosage forms and relatively pure preparations from chemical syntheses. An important interfering substance is a slightly different relative of the principle drug. An example would be deacetyl cephalothin in a sample of cephalothin. A theory of the relation between drug and organism for the simple case described above was given by Kavanagh. 19,2° The following theoretical equation is based on the work of Garrett and Miller 2~ N = No exp (ko - k~C)(t - no)
(1)
where N is the concentration of bacteria as number of organisms per milliliter after an incubation time of t, No is the concentration of bacteria at time zero, ko is the growth rate constant in the absence of antibiotic, k~ is the inhibitory coefficient characteristic of antibiotic and organism, C is the concentration of antibiotic, and Lo is the lag time from inoculation to the beginning of exponential growth. In any particular turbidimetric assay No, ko, ka, t, and Lo are constant for all values of C of both standard and sample; otherwise, the assay is invalid. Therefore, Eq. (1) may be reduced to the form logN = A-BC
(2)
A graph of N against C on semilogarithmic paper would be a straight line of slope B3 ° This equation has been a valuable dose-response line for obtaining potencies of unknowns. It has been applied to assay of penicillins, cephalosporins, and erythromycin in pharmaceutical dosage forms and of tylosin, monensin, hygromycin B, and tetracyclines in animal feeds. The equation does not apply to assays employing Klebsiella pneumoniae (ATCC 10031) for unknown reasons. The value of N in Eq. (2) can be obtained from photometric measurements with a calibrated instrument.19, 22 In preparing calibration lines for assays, absorbance is used in place of N to avoid the errors and inconvenience of converting absorbance into N. The line will be somewhat more curved than the log N vs. C line. 1~F. Kavanagh, in "Analytical Microbiology" (F. Kavanagh, ed.), Vol. 2, p. 44. Academic Press, New York, 1972. ~oF. Kavanagh, Appl. Microbiol. 16, 777 (1968). 21E. R. Garrett and G. I-I. Miller, J. Pharm. Sci. 54, 427 (1965). ~F. Kavanagh, in "Analytical Microbiology" (F. Kavanagh, ed.), Vol. 1, p. 141. Academic Press, New York, 1963.
[4]
ANTIBIOTIC ASSAYS
65
The dose-response line is drawn point-to-point. An important point is that the concentrations of standards must be on a linear scale and not form a geometric series as in diffusion assays.
IV. Assays of Diverse Biological Samples The implicit assumption made to this point ~in the discussion is that the samples are reasonably pure and derived from chemical operations. Such samples usually are available in adequate quantity for any kind of test and do not contain substances of biological origin that interfere with assays. The situation changes drastically when the antibiotics are tested in various biological systems because the samples are small and may contain interfering substances from the system. Usually the most difficult to assay accurately are blood samples. Urine samples may present fewer problems because the quantity is larger and, quite often, the concentration is higher than in blood. Diffusion assays have been the method of choice because the assays can be done with small samples and the diffusion process reduces interferences. Diffusion assays usually are more sensitive than tube methods because the most susceptible test bacteria are suitable for plate methods, but not for tube methods. Usually, tube methods are considered to be too insensitive for assay of blood samples. Unless the sample can be diluted substantially, the proteins and other materials in the plasma or serum may affect a tube assay. A problem more likely to occur in samples of biological origin is the presence of active metabolic products of the drug. An example would be deacetyl cephalothin 23 in blood or urine from animals given cephalothin. The metabolic product has considerably less activity than the parent compound when assayed by S. aureus. The derivative may be as active as the parent in certain plate assay systems. When this is so, the assay is a measure of activity, not of a quantity of a particular substance. Quite often the derivative is less active than the parent and may not be or may be only incompletely measured in the assay, which then becomes a method for the parent compound. However, in a turbidimetrie method, all active compounds will be measured because the test organism is exposed to all. There may be pharmacological reasons to use derivatives of the active drug to increase stability, to improve rate of absorption, to decrease toxicity, or to increase activity. Derivatives of several antibiotics have no activity until hydrolyzed to release the parent compound. When as~ R. J. Simmons, in "Analytical Microbiology" (F. Kavanagh, ed.), Vol. 2, p. 193. Academic Press, New York, 1972.
66
METHODS FOR THE STUDY OF ANTIBIOTICS
[4]
sayed without preliminary hydrolysis, apparent potency of the derivatives will depend on the kind of assay and extent of incubation. V. Relative Inhibitory Coefficient of Antibiotic8 The scientist who works with a series of related antibiotics, such as the penicillins or cephalosporins, needs a meaningful method for comparing antibacterial activities. The activity of each new compound must be compared with the parent or a model compound to learn whether the new molecular change has altered the antibacterial activities. The search is for products with higher activities and activities against a broader range of bacteria than the reference compound. Changes in molecular structure may cause an increase in activity, a decrease in activity, or no change, and it is necessary to know how much the change in structure has changed activity. The more accurately changes can be measured, the more reliably can the decision be made concerning the effect of molecular changes. To 1973, the great advances in preparing semisynthetic penicillins and cephalosporins were all made using a very crude measure of activities. How many superior compounds were missed and how much needless work was done because of inadequate measurements of antibacterial activities can not be estimated. In the work published, 24-26 activities were all reported as MIC (minimum inhibitory concentration) ascertained by either a tube or agar dilution method. The minimum inherent error 8,1° in MIC tube methods is 50% and usually is no less in agar dilution assays. These assays could not distinguish between compounds differing in activity by 20%. MIC is a quick way to ascertain influence of changes in structure upon activity against many organisms. The resulting spectrum of activities can be a valuable guide for selecting candidates for the more precise characterization of activities to be described next. Nonetheless, the assays are a great improvement over pure chance. A much more precise measure of activity of a new compound can be obtained by relating its activity to a parent compound or other reference substance of closely related structure. The factor that characterizes antibacterial activity of a compound in a particular turbidimetric assay is the specific inhibitory coefficient, ka, of Garrett and Miller. 21 The theoretical equation, Eq. (1), applicable to many assay systems relates inoculum size, growth constant (ko), specific inhibitory coefficient (ka), and concentration of antibiotic. 19,~° Because inoeulum size and 24j. p. Hou and J. W. Poole, J. Pharm. Sci. 60, 503 (1971). ~5 M. Misiek, T. A. Pursiano, L. B. Crast, :F. Leitner, and K. E. Price, Antimlcrob. Ag. Chemother. 1, 54 (1972). ~e M. Gorman and C. W. Ryan, in "Cephalosporins and Penicillins" (E. Flynn, ed.), Chapter 12, pp. 532-582. Academic Press, New York, 1972.
[4]
ANTIBIOTIC ASSAYS
67
growth constant vary from assay to assay and are inconvenient to measure, absolute values of ka are not determined. Inoculum size and growth constant of the test organism are the same for all antibiotics in one assay. The necessity for knowing them is obviated by obtaining the ratio of /ca of new compounds to k~ of reference compound. The ratio is called the relative intrinsic activity of the new substance relative to reference substance. The value is, of course, a constant only for a particular assay organism and changes with test bacteria. The value also changes with activities of the reference compound. The relative inhibitory coefficient can be easily measured in modern automated turbidimetric assays. Should absolute values of ka be needed, the method of Garrett and Miller 21 or a photometric equivalent ~9 can be used. A procedure was developed for determining the relative inhibitory coefficient for those antibiotics and test organisms that follow Eq. (1). By referring the inhibitory coefficients of several drugs to that of a reference compound, the influence of unavoidable variables in the test system will be reduced to second-order importance. The following assumptions are made: 1. The compound reduces growth rate of the test bacteria. 2. The compound does not affect lag time in a manner different from the standard. 3. The bacteria in all test solutions are still in the log phase of growth when growth is terminated. 4. The apparent growth rate constant is a linear function of concentration of antibiotic. 5. The apparent growth rate ( k o - k.~C) is constant throughout the incubation period for each concentration of each test substance. 6. Drug is not consumed by the test bacteria. 7. Organisms grown in the presence of the test compounds possess optical properties identical with those grown in the absence of the compounds, Given the above assumptions, the growth of test organisms in the presence of a growth-rate inhibitory compound may be expressed by Eq. 1. In an assay, ko, t - - L o , and No are the same for all tubes, are unknown and are not easily measured. However, the value of k , ~ ( t - L~,) can be obtained from measurements of N for two values of C for each antibiotic as follows:
ka(t -- L0) = [2,3 log (NI/N2)]/(C2 - CI)
(3)
where N1 is the concentration of bacteria at antibiotic concentration C1 and N2 the concentration at C2. Although total incubation time can be measured accurately, t - Lo is unknown because the value of L0 is unknown. The values of (t - L0),
6S
M~.T~ODS ~Oa TI~. STVOY Or ANTIBIOTICS
[4]
and N o , are different in different tests even though the tests are prepared from the same inoculated broth. A way around the obvious difficulties is to refer all activities to a standard compound in the test and to treat each test as a unit. Represent the k~ of the reference substance by t l! t! t k,, and the k, of samples by k~. The ratio k~/k~ should be independent of No, t, L0, and k0 because kT(t -
Lo)/k'~(t - Lo) = kT/k'~ = R
(4)
where R is the relative inhibitory coefficient. R is a function of the activity of the reference substance as well as of the sample. If the same reference compound is used in testing a series of derivatives, the several values of the R may be used to measure the influence of a change in molecular structure upon activity. As a practical matter, medium, buffer, size of inoculum, and condition of inoculum are controlled. Restrictions are placed on the assay systems by choosing concentration of antibiotics so that at least one permits growths between 50 and 70% of uninhibited growth and at least one permits growths between 50 and 30%. In addition, a plot of log N vs C on semilog paper of N obtained for at least three concentrations of drug including the above two concentrations should approximate a straight line. The inhibitory coefficients are computed from activities around the 50% growth point of the inhibition curve and gives no indication of activity near the ends of the curve. The full range of activities are better shown in a log-probability plot. 19,22 In these graphs, bacterial concentration relative to uninhibited growth (C = 0) for a number of concentrations of antibiotic are plotted against corresponding C on log-probability paper. The lines should be slightly curved toward the C axis. A family of compounds should form a series of approximately parallel lines. Only for such families can the relative inhibitory coefficients have meaning. The compounds may be expected to have different MR (median response), which is defined as the concentration of antibiotic permitting 50% growth. A procedure for computing relative inhibitory coefficients from the several median responses can be derived from Eq. (1). Although the two procedures have equal theoretical validity, the one given is preferred because no assumptions must be made about linearity of the line representing uninhibited growth. Many measurements of numerous systems showed inhibited growth following the theoretical growth equation much more accurately than uninhibited growth. Lack of linearity of the uninhibited growth causes the computed median responses to be larger than the true value.
[5]
f~-LACTAMASE ASSAYS
69
Nothing can be learned from the dose-response line about resistance to enzymic attack, animal toxicity, absorption and excretion, and effectiveness in treating an infected animal. The dose-response line is the quickest way to obtain an idea of the quantitative response of the test organisms to graded concentrations of the antibiotic. A high accuracy turbidimetric assay such as that provided by an AUTOTURB ® System should be used to obtain the basic data. A reference standard and five samples at three concentrations would constitute one test in the AUTOTURB ® System. The important parameter in Eq. (3) are the values of N which are obtained from a calibration curve relating transmittance or absorbance of the suspension to concentration of bacteria.19, 22 In a study of one series of commercially important antibiotics using S. aureus as the test organism, R ranged from 0.03 to 1.56. The value of R, obviously, can have nearly any value. If the molecular weights of the compounds vary significantly within a series, C should be in molar and not in weight concentrations of active compounds.
[5] f~-Lactamase Assays B y GORDON W. Ross and CYNTHIA H. O'CALLAGHAN I. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . II. D e f i n i t i o n of U n i t . . . . . . . . . . . . . . . . . III. Assay Methods . . . . . . . . . . . . . . . . . A. M e t h o d s in W h i c h Penicillin a n d C e p h a l o s p o r i n B r e a k d o w n P r o d u c t s Are M e a s u r e d . . . . . . . . . . . . . . B. M e t h o d s in W h i c h t h e C o n c e n t r a t i o n of R e s i d u a l Penicillin or C e p h a l o s p o r i n Is M e a s u r e d . . . . . . . . . . . . . IV. D i s c u s s i o n of A s s a y M e t h o d s . . . . . . . . . . . . . A. I o d o m e t r i c A s s a y . . . . . . . . . . . . . . . . B. A c i d i m e t r i c A s s a y . . . . . . . . . . . . . . . . C. S p e c t r o p h o t o m e t r i c (UV) A s s a y . . . . . . . . . . . D. H y d r o x y l a m i n e A s s a y . . . . . . . . . . . . . . .
I. H R--CO--N~.H
C--C [
r
H
A penicillin
C
J
CH
oo"
74 80 82 83 84 84 85
Introduction
CHs H ~ . ~ [/CH s
o~C--N
69 74 74
÷ ..o
CH~
R-- CON..H HIS~ . 11CHs C--C C I i J
o ,C
OH
CO0"
A penicilloic acid
(I)
[5]
f~-LACTAMASE ASSAYS
69
Nothing can be learned from the dose-response line about resistance to enzymic attack, animal toxicity, absorption and excretion, and effectiveness in treating an infected animal. The dose-response line is the quickest way to obtain an idea of the quantitative response of the test organisms to graded concentrations of the antibiotic. A high accuracy turbidimetric assay such as that provided by an AUTOTURB ® System should be used to obtain the basic data. A reference standard and five samples at three concentrations would constitute one test in the AUTOTURB ® System. The important parameter in Eq. (3) are the values of N which are obtained from a calibration curve relating transmittance or absorbance of the suspension to concentration of bacteria.19, 22 In a study of one series of commercially important antibiotics using S. aureus as the test organism, R ranged from 0.03 to 1.56. The value of R, obviously, can have nearly any value. If the molecular weights of the compounds vary significantly within a series, C should be in molar and not in weight concentrations of active compounds.
[5] f~-Lactamase Assays B y GORDON W. Ross and CYNTHIA H. O'CALLAGHAN I. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . II. D e f i n i t i o n of U n i t . . . . . . . . . . . . . . . . . III. Assay Methods . . . . . . . . . . . . . . . . . A. M e t h o d s in W h i c h Penicillin a n d C e p h a l o s p o r i n B r e a k d o w n P r o d u c t s Are M e a s u r e d . . . . . . . . . . . . . . B. M e t h o d s in W h i c h t h e C o n c e n t r a t i o n of R e s i d u a l Penicillin or C e p h a l o s p o r i n Is M e a s u r e d . . . . . . . . . . . . . IV. D i s c u s s i o n of A s s a y M e t h o d s . . . . . . . . . . . . . A. I o d o m e t r i c A s s a y . . . . . . . . . . . . . . . . B. A c i d i m e t r i c A s s a y . . . . . . . . . . . . . . . . C. S p e c t r o p h o t o m e t r i c (UV) A s s a y . . . . . . . . . . . D. H y d r o x y l a m i n e A s s a y . . . . . . . . . . . . . . .
I. H R--CO--N~.H
C--C [
r
H
A penicillin
C
J
CH
oo"
74 80 82 83 84 84 85
Introduction
CHs H ~ . ~ [/CH s
o~C--N
69 74 74
÷ ..o
CH~
R-- CON..H HIS~ . 11CHs C--C C I i J
o ,C
OH
CO0"
A penicilloic acid
(I)
70
METHODS FOR THE STUDY OF ANTIBIOTICS
,I
i
I
CO0A cephalosporin
+
H20
~-
C~ C I
o.
I
[5]
CHi I
oo-
7/ I
]I
(2)
Unstable intermediate
Breakdown products
fl-Lactamases [penicillin (cephalosporin) fl-lactam amidohydrolases, EC 3.5.2.6] are enzymes of bacterial origin which hydrolyze the C - - N bond in the fl-lactam ring of a penicillin or a cephalosporin [Eqs. (1) and (2), respectively]. The term fl-laetamase covers a variety of enzymes from a variety of different organisms, fl-Lactamase activity has been detected in all organisms examined to date when an isoelectric focusing technique has been used. 1 The effect of fl-lactamases was first noted with benzylpenicillin (penicillin G), and this resulted in the term penicillinase being applied to any biological entity that rapidly inactivated benzylpenicillin. 2 With the discovery of the cephalosporins, it became evident that the term penicillinase was too narrow to encompass the many different enzymes capable of catalyzing the hydrolysis of the fl-lactam ring of cephalosporins and penicillins, and the general name fl-lactamase is now used. fl-Lactamases from at least 25 different strains of bacteria have been purified, and properties such as substrate profile, molecular weight, effect of pH and temperature, electrical charge, and susceptibility to inhibition have been studied. M a n y other fl-lactamases have been observed but not studied in detail. In a recent review, a classification of fl-lactamases from gram-negative bacteria into five main classes containing 15 different types of enzyme was proposed2 Some fl-lactamases are highly species specific, such as the Sabath and Abraham enzyme found in all strains of P s e u d o m o n a s aeruginosa, 4 whereas others, being mediated by an R factor such as RTEM,5 m a y be found in several different species. Some enzymes inactivate cephalosporins very much more rapidly than they inactivate penicillins, but even so their activity can usually be expressed in terms of benzylpen!cillin. If the main interest is in cephalosporins, 1M. Matthew, A. M. Harris, M. J. Marshall, and G. W. Ross, J. Gen. Microbiol., in press (1975). 2E. P. Abraham and E. Chain, Nature (London) 146, 837 (1940). s M. H. Richmond and R. B. Sykes, Advan. Microb. Physiol. 9, 31 (1973). 4L. Sabath, M. Jago, and E. P. Abraham, Biochem. J. 96, 739 (1965). N. D a t t a and M. H. Richmond, Biochem. J. 98, 204 (1966).
[5]
~-LACTAMASIg ASSAYS
71
then often the activity is related to that against cephaloridine, rather than benzylpenicillin. In the course of the past 30 years, many penieillins and cephalosporins have been prepared and tested as substrates for many fl-lactamases. Most of the compounds tested have been found to be susceptible to at least one fl-lactamase; recently, a new type of cephalosporin known generically as cephamyeins, has been described as highly resistant to most fllaetamases. As yet, little is known about the behavior of these compounds in the assay methods given here. Whatever the preparation of enzyme, or the purpose of the assay, the concentration of the fl-lactamase is primarily estimated from its effect on a known concentration of benzylpenicillin and/or cephaloridine, although its activity on other penicillins or cephalosporins may be tested in parallel at the same time. Methods for assaying fl-laetamase activity can be divided into two main groups. In one, the concentration of the unchanged substrate remaining is measured; in the other, the assay determines the concentration of breakdown products. In general, benzylpenieillin is comparatively simple to assay by a variety of methods. However, if these methods are extrapolated to other penieillins, care must be taken that the method chosen is still applicable. This is even more important with eephalosporins, and it cannot be assumed that the assay method selected as the most convenient for the estimation of benzylpenieillin will work in the same way, or even work at all for a particular cephalosporin. The fl-laetamase preparation may also affect the assay. A highly purified enzyme is tedious and time consuming to make but usually gives good results with most assays. However, if the preparation is very impure and has, for example, strongly reducing tendencies, then this can seriously interfere with several assay methods. When the fl-laetam ring of a penicillin is hydrolyzed by a fl-laetamase IEq. (1) 1, the corresponding penicilloic acid is produced in stoichiometrie proportions. A penieilloic acid has two acidic groups where the parent compound had only one [Eq. (1) ], and it is also a stable substance which can be readily assayed. The situation with eephalosporins is more complicated owing to the presence of another substituent on the nucleus at position 3. The first product of fl-laetamase attack on a eephalosporin is hypothetically a eephalosporoie acid, analogous to penieilloie acid [Eq. (2)]. However, unlike penieilloie acids, these compounds are usually very unstable and cannot be isolated because they undergo rapid decomposition. Only two exceptions are known to this, one being eephalosporins, where the 3-sub-
72
METHODS H
R--CO--N
F O R T H E S T U D Y OF ANTIBIOTICS
[5]
H
S-.
R--CO--N~_~S.~
0~ ~0
COO-
(a)
/---" NO~
(b)
FIG. 1. Stable products of hydrolysis of #-lactam ring of cephalosporins formed when 3-substituent is (a) a ~/-lactone or (b) 2:4-dinitrostyrene.
O ~ 9 " - - - - N ~ _ CH2OCOCHs COO
L
coo~
L
j
(Hypothetical, transient, intermediates)
coo-
/
Further fragmentation FIG. 2. Production of a second acidic group from cephalothin on fl-lactam hydrolysis. s t i t u e n t is a ,/-lactone 6 (Fig. la) ; the other comprises compounds where the 3-substituent is the highly conjugated s y s t e m of 2 : 4 - d i n i t r o s t y r e n e , 7 (Fig. l b ) . 6 E. P. Abraham and G. G. F. Newton, Biochem. J. 79, 377 (1961). 7 C. H. O'Callaghan, A. Morris, S. M. Kirby, and A. H. Shingler, Antimicrob. Ag. Chemother. 1, 283 (1972).
[5]
/~-LACTAMASE ASSAYS
73
CH~--CO--N..~_~S-~ 0
l_
CH2---N
~oo--
coo-
/}
1
Fia. 3. Decomposition products of eephaloridine formed on rupture of ~-laetam ring.
Cephalothin gives the expected acidic group from the fl-lactam ring but, in addition to this, the acetoxy group at position 3 is simultaneously expelled from the molecule, producing another acidic group 4 (Fig. 2). Thus, unlike the penicillins, cephalothin gives two additional acidic groups. Other cephalosporins with an acetoxy group at position 3, such as cephaloglycin, cephapirin, and cephacetrile, also give extra acid groups on fl-lactamase hydrolysis. This does not apply to all cephalosporins. Cephaloridine, for example (Fig. 3), also loses its 3-substituent simultaneously with rupture of the fl-lactam ringS; this 3-substituent is pyridine--a base, not an acid. Therefore, while the intact compound is a betaine with no net charge, the initial decomposition products have two acidic groups and one basic group. For each different cephalosporin, the overall effect will depend on the relative strengths of the acidic and basic groups. Not all cephalosporins lose their 3-substituent when the fl-lactam ring is hydrolyzed. Cephalexin retains its methyl group at position 3. Compounds such as cefazolin and cephamandole probably lose their 3-substituent, but this reaction may not proceed as rapidly as the decomposition of the fl-lactam ring, and the nature of the decomposition products is not yet firmly established. It is thus evident that while the determination of benzylpenicillin in a fl-lactamase reaction mixture is a.relatively simple matter, the situation with cephalosporins is much more complex. 8 C. H. O'Callaghan, S. M. Kirby, A. Morris, R. E. Waller, and R. E. Duneombe, J. Baeteriol. 110, 988 (1972).
74
METHODS FOR T H E STUDY OF ANTIBIOTICS
[5]
II. Definition of Unit A unit of fl-lactamase activity is commonly defined as the amount of enzyme which hydrolyzes 1 ~mole of substrate per minute at 37 ° and optimum pH. Measurement of enzyme activity at 37 ° rather than the 25 ° or 30 ° often used is preferred for these bacterial enzymes, which will usually be acting in vivo at 37 °. A standard pH of 7.0 has often been used for the same reason. The unit defined by Pollock and Torriani 9 (one unit hydrolyzes 1 ~mole of benzylpenicillin per hour at 30 ° and pH 7.0) has also been frequently used in fl-lactamase studies. Because of the diversity of the fl-lactamases and the many independent groups of workers investigating various aspects of the problem, there has been a notable lack of correlation between methods and conditions used. This has made comparisons between various sets of results ~lmost impossible in many cases. There is a great need to standardize methods and conditions to make comparisons possible. The obvious conditions to use should be those most closely related to the bacterial environment in which the fl-lactamases will do the most damage, i.e., pH 7 and 37%
III. Assay Methods
A. Methods in Which Penicillin and Cephalosporin Breakdown Products Are Measured 1. Iodometric Assay
This method is the most widely used at the present time, and several variations have been reported. Perret's macroiodometric method 1° is relatively straightforward, gives reproducible results, and does not require any specialized or expensive equipment. An excess of iodine, buffered at pH 4, is used to stop the enzyme reaction. The hydrolysis products of the fl-lactam antibiotic react with the iodine and the remaining iodine is measured by titration with sodium thiosulfate solution. Full details of this method are given below. Sargent 11 has described a variation of the Perret method in which the decrease in optical density at 490 nm is used instead of thiosulfate ' M. R. Pollock and A. M. Torriani. C. R. Acad. Sci. Paris 237, 276 (1953). soC. J. Perret, Nature (London) 174, 1012 (1954). 11M. G. Sargent, J. Bacteriol. 95, 1493 (1968)
[5]
/~-LACTAMASE ASSAYS
75
titration as a measure of iodine uptake by the products of enzyme action. Ferrari et al. 12 have described an Autoanalyzer method in which iodine uptake is followed spectrophotometrically at 420 nm. Zyk 13 has suggested that the enzyme reaction should be stopped by addition of iodine-tungstate solution before measuring the decrease in optical density at 620 nm due to the reaction of iodine with the products of fl-lactam hydrolysis. The macroiodometric methods, although easy to use, are relatively insensitive and cannot be used, for example, for studies on reaction kinetics. Several microiodometric techniques have been described in which a 1000-fold increase in sensitivity can be obtained by following spectrophotometrically the rate of decolorization of the starch-iodine complex by the penicilloic acid. Novick" followed the decolorization at 620 nm, but his method gave results that were 40% lower than those obtained by Perret's method. Sykes and Nordstrom 1~ showed that this discrepancy could be eliminated if sufficient time was allowed for a steady state to be reached between formation of penicilloic acid and its reaction with the starch-iodine complex; their method is given in detail below. None of the microiodometric assays has been applied successfully to cephalosporin substrates. Lindstrom and Nordstrom 16 adapted Novick's method for use on an Autoanalyzer. Goodall and Davies 1~ had previously described an automated procedure in which excess iodine remaining after reaction with the penicilloic acid was mixed with a starch-potassium iodide reagent and the resulting starch-iodine complex was measured at 526 nm. Cole et al. ~s described an automated procedure in which uptake of iodine was followed by measuring the reduction in height of the 288 nm absorption peak of the iodine/potassium iodide reagent. Mavroiodometric Determination of fl-Lactamase Activity Reagents
Phosphate buffer, 0.1 M, pH 7.0 Acetate buffer, 2 M, pH 4.0 Hydrolyzed starch, 2% Sodium thiosulfate, 0.0166 N (41.19 g/liter, dilute 1:10 v/v before use) 1,~A. Ferrari, F. M. Russo-Alesi, and J. M. Kelly, Anal. Chem. 31, 1710 (1959). 13N. Zyk, Antimcirob. Ag. Chemother. 2, 356 (1972). 1, R. P. Novick, Biochem. J. 83, 236 (1962). 15R. B. Sykes and K. Nordstrom, Antimicrob. Ag. Chemother. I, 94 (1972). I"E. B. Lindstrom and K. Nordstrom, A~timicrob. Ag. Chemother. 1, 100 (1972). 1~R. R. Goodall and R. Davies, Analyst 86, 326 (1961). ~8M. Cole, S. Elson, and P. D. Fullbrook, Biochem. J. 127, 295 (1972).
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METHODS FOR THE STUDY OF ANTIBIOTICS
[5]
Iodine reagent is 0.0166 N iodine, 60 mM potassium iodide and 2 M acetate buffer, pH 4.0. Dissolve 40.6 g of iodine + 200 g of KI in 1 liter of demineralized water. Dilute 1:20 with 2 M acetate buffer, pH 4.0, just before use. Substrate (5 mM) is prepared daily in phosphate buffer pH 7.0 Procedure. The reaction is carried out in a shaking water bath at the appropriate temperature.
50-ml flasks Addition
Control
Test
Substrate (ml) E n z y m e (ml)
5 --
5 1.0
Allow substrate and enzyme to reach operating temperature before addition of enzyme. Incubation in the shaking water bath is continued for 30 min before t h e reaction is stopped by addition of iodine reagent (10 ml) ; 10 ml of iodine reagent is also added to the control flask, followed by 1 ml of enzyme preparation. Ten minutes later (20 min for cephalosporins) the flasks are removed from the bath and titrated with 0.00166 N thiosulfate, using starch as indicator. If the titration is less than 4.5 ml, the assay is repeated with diluted enzyme. Under these conditions, 1 ml of 0.0166 N iodine is equivalent to 2 ~moles of a penicillin destroyed. The macroiodometric assay of cephalosporins is less reliable than with the penicillins, largely because the breakdown pattern of these molecules is less clear cut; 1 ml of 0.0166 N iodine is equivalent to about 4 t~moles of a cephalosporin destroyed, but the stoichiometry of the reaction varies somewhat with the nature of the cephalosporin. Calculation. C o n t r o l - test = x ml of iodine consumed; .'. x/15 = t~moles of penicillin substrate destroyed per minute, and x/7.5 = t~moles of cephalosporin substrate destroyed per minute. Microiodometric Determination o] fl-Lactamase Activity Reagents
Phosphate buffer, 0.1 M, pH 7.0 Hydrolyzed starch, 0.2% Iodine reagent 0.08 M in 3.2 M potassium iodide (2 g of I2 + 53.29 g of KI in 100 ml)
[5]
~-LACTAMASE ASSAYS
77
Starch iodine solution is prepared by adding 0.15 ml of iodine reagent to 100 ml of starch solution (giving a final iodine concentration of 120 t2Y/). Substrate (0.20 mM) in phosphate buffer, pH 7.0, is prepared daily and kept on ice for the period of the experiment. Procedure. The reaction is followed in a recording spectrophotometer with a heated cell carrier.
Cuvettes
Addition Starch-iodine (ml) Substrate (ml) Phosphate buffer (ml) E n z y m e (ml)
1
2
3
E n z y m e control
Substrate control
Test
1 -l. 9 0.1
1 1 1 --
1 1 0.9 0.1
Allow cuvettes to reach operating temperature in heated cell carrier before initiating reaction by addition of enzyme. Absorbance at 620 nm is then measured at various times. The initial absorbance of the normal assay mixture is 1.20. The reaction becomes linear after 15-20 minutes for enzyme activities up to 0.001 unit per assay mixture. At higher activities the starch-iodine is completely decolorized before the steady state is achieved. Calculation. The amount of iodine used in the assay corresponds to 30 nmoles of penicillin. At this concentration, the optical density (OD) at 620 nm decreases from 1.20 to 0 when a penicillin is completely destroyed by a fl-lactamase..'. ( 5 0 D / m i n / 1 . 2 0 ) X 0.03 X 10 = ~moles destroyed per minute per milliliter of enzyme. 2. Acidimetric Methods (or Alkalimetric Methods) These methods depend fundamentally on measurement of the rate of increase in acidic groups when the fl-lactam ring is ruptured. This rate can be measured directly by continuous titration with alkali in a pH stat, ~,19 or else by the change in color of an indicator which can be continuously recorded on a spectrophotometer. ~° It can be determined indil~j. p. Hou and J. W. Poole, J. Pharm. Sci. 61, 1594 (1972). 2oF..4.. Rubin and D. H. Smith, Antimicrob. Ag. Chemother. 3, 68 (1973).
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METHODS FOR THE STUDY OF ANTIBIOTICS
[5]
rectly by manometric measurement of carbon dioxide continuously released from a sodium bicarbonate solution by the increasing acidity.21 The techniques are relatively simple for penicillins but more difficult to apply to cephalosporins. a. D i r e c t M e t h o d s
Wise and Twigg 22 suggested a method for determining penicillinase activity in which the acid produced by the enzyme action was titrated with 0.01 N NaOH to a constant pH. (They chose pH 7.8, the optimum for their enzyme.) No indicator was used. Selzer and Wright ~3 used a similar method when studying inhibition of penicillinase by penicillinaseresistant penicillins. Sodium hydroxide (0.1 N) was added from a burette and the pH maintained at 7.0. Later workers used a pH star and different conditions. Sabath, Jago, and Abraham 4 hydrolyzed benzylpenicillin and cephalosporin C with a fl-lactamase from P. aeruginosa. They used Radiometer pH-stat equipment to measure uptake of 0.1 N sodium hydroxide solution, and hence the rate of fl-lactam hydrolysis when investigating pH activity curves and Michaelis constants. Hou and Poole 19 have recently used a similar method when investigating a variety of new penicillins. Details of this method are given below. An indicator method was first described by Saz et al., ~4 and the sensitivity of the method has recently been increased by Rubin and Smith 2° by decreasing the buffer concentration in the reaction mixture. They added enzyme solution (10 t~l, capable of hydrolyzing 2-10 nmoles of substrate per minute) to a reaction mixture (1.2 ml) containing substrate (0.2 raM), phenol red indicator (0.0013% w/v), and phosphate buffer (0.4 mM, pH 7.6). The absorption at 558 nm was followed with a recording spectrophotometer for 5 min at the temperature chosen for the assay. The assay should be standardized for each substrate by relating change in absorption at 558 nm to micromoles of substrate hydrolyzed. Imsande 2~ described an indicator method in which bromothymol blue was used instead of phenol red. The change in absorption at 620 nm of a reaction mixture containing substrate (6 mM), bromothymol blue indicator (0.0033% w/v), and cacodylate buffer (3 mM, pH 7.3) was followed for 12 min after addition of enzyme. 21R. J. Henry and R. D. Housewright,J. Biol. Chem. 167, 559 (1947). W. S. Wise and G. H. Twigg,Analyst 75, 106 (1950). ~8G. B. Selzer and W. W. Wright, in "Antimicrobial Agents and Chemotherapy" (G. Hobby, ed.), p. 311. AmericanSociety for Microbiology (1964). ~A. K. Saz, D. L. Lowry, and L. J. Jackson, J. Bacteriol. 82, 298 (1961). 25j. Imsande, J. Bacteriol. 69, 1322 (1965).
[5]
~-LACTAMASE ASSAYS
79
pH Stat Titration Method Reagents Potassium hydroxide solution, 0.04 N Substrate (5 mM) in dilute aqueous potassium chloride solution (ionic strength 0.025). Prepared daily and kept on ice before use Nitrogen gas washed with 10% sulfuric acid, 10% sodium hydroxide solution, and finally water. Procedure. Substrate solution (20 ml, 100 ~moles) is equilibrated to the operating temperature by stirring in a water-jacketed reaction vessel. A slow stream of the nitrogen gas is allowed to flow over the surface of the solution. B-Lactamase (less than 0.5 ml containing sufficient activity to hydrolyze 2-10 ~moles of substrate per minute) is added, together with enough dilute acid or base to set the initial pH. The rate of hydrolysis of 50% of the substrate is followed by the rate of addition of alkali required to maintain a constant pH, and the enzyme activity (~moles per minute) can be obtained from a calibration curve relating enzyme units to the time required for 100% hydrolysis of the 100 ~moles of substrate. b. Indirect Method Manometric measurement of released carbon dioxide was first described by Henry and Housewright21 in 1947 for the estimation of the rate of decomposition of benzylpenicillin by Bacillus cereus. The reaction is carried out in constant-volume Warburg respirometers. The enzyme solution and the sodium bicarbonate solution (total volume 4 ml) are placed in the main body of the vessel. The sidearm of the vessel contains 0.5 ml of penicillin solution, the penicillin being dissolved in the same concentration of bicarbonate solution as is contained in the main body of the vessel. The vessels and manometers are moved to and fro through a distance of 3 cm at a rate of at least 90 cycles per minute (to permit maximum C02 evolution); readings are taken every 2 rain until the reaction system is in equilibrium with the gas mixture. The penicillin solution in the sidearm is tipped into the main flask, and the evolution of CO~ begins. Readings are taken every minute, and often there will be a lag period of up to 4 rain before the rate of CO~ evolution becomes constant. The rate is measured in mm3/min of CO,.. evolved. The amount evolved is plotted against time, and the slope of the straight portions of the line is determined by the method of least squares. Concentrations of penicillin and enzyme must be adjusted so that the evolution of CO,, is fairly brisk. If the enzyme solution is excessively
80
METHODS FOR THE STUDY OF ANTIBIOTICS
[51
dilute, so that only a small amount of penicillin is hydrolyzed, then the amount of COz is too small to measure accurately. Similarly, if the penicillin solution is too dilute, complete decomposition occurs too quickly for the rate to be accurately measured, and the assay has a very low precision. The best results are obtained for a penicillin concentration of about i mM in 0.2% sodium bicarbonate solution. The preparations used must be in solutions of low buffering capacity, otherwise the C02 is not evolved in stoichiometric proportions. Iron, calcium, copper, and zinc ions interfere with the assay at levels as low as 5 t~g/ml for Fe 3÷. This method is not always readily extrapolated to cephalosporins, and, ideally, pilot experiments should be done first with new enzyme preparations to determine the best concentration of the enzyme preparation to use. It is a laborious and time-consuming method of penicillinase assay, and of poor reliability for cephalosporins; this method is now mainly obsolete. B. Methods in Which the Concentration of Residual Penicillin or Cephalosporin Is Measured
I. Spectrophotometric (UV) Assay The method described by O'Callaghan, Muggleton, and Ross 26 depends on the direct measurement of eephalosporin concentration and has proved to be particularly useful for comparison of the fl-lactamase resistanee of new eephalosporins and for/~-lactam inhibition studies. The rate of hydrolysis of the fl-laetam ring is followed by measuring the rate of decrease in optical density of a cephalosporin solution at the wavelengths of maximum absorption associated with the fl-lactam ring. This wavelength is 255 nm for cephaloridine; it must be determined for each analog assayed, together with the decrease in optical density at this wavelength for 100% hydrolysis. In a typical assay with cephaloridine as substrate, the concentration of the enzyme solution is adjusted so that a 10-15 ~1 sample mixed with 3 ml of a 0.1 mM (41.5 ~g/ml) solution of cephaloridine in 0.1 M phosphate buffer, pH 7.0, in a 1 cm euvette and incubated at 37°C will completely hydrolyze the cephaloridine in 5-10 min. The rate of decrease in optical density at 255 nm is followed for the first minute in a recording spectrophotometer (e.g., Unieam SP800). The change in optical density must be measured for each cephalosporin 2~C. H. O'Callaghan, P. W. Muggleton, and G. W. Ross, in "Antimicrobial Agents and Chemotherapy" (G. Hobby, ed.), p. 57. American Society for Microbiology, 1968.
[5]
~-LACTAMASE ASSAYS
81
being investigated. For example, the optical density of 0.1 mM solution of cephaloridine falls from 1.4 to 0.6 for complete hydrolysis; therefore, t~moles cephaloridine hydrolyzed/minute = (A OD/min/0.8)}( [(3 }( 41.5)/415] = A OD/min )< ~ . The range of the assay can be extended to cover other substrate concentrations by the use of optical cells of different light paths. 2. Hydroxylamine Assay This assay is based on that of Boxer and Everett :7 as modified by Hamilton-Miller et al. 2s Hydroxylamine reacts with the intact fl-lactam compound to give a hydroxamic acid which forms a chromogen with ferric ions. Reagents 1. Hydroxylamine hydrochloride (174 g in 500 ml of solution in water) 2. Buffer, pH 11.2 (173 g of sodium hydroxide + 20.6 g of anhydrous sodium acetate in 1 liter of solution) 3. Absolute alcohol + reagent 1 + reagent 2 + demineralized water (4:1 : 1 : 1.5 v/v) ; made freshly each day 4. Ferric ammonium sulfate (200 g) + concentrated sulfuric acid (95 ml) added to demineralized water to give 1 liter of solution 5. Substrate, 1 mM in 10 mM phosphate buffer, pH 7.0 Procedure. Reagent 3 (7.5 ml) is added to dilutions of substrate [1 ml in wide (diameter 1 inch) glass tubes] and mixed thoroughly (Vortex mixer). After 3..5 min (critical for cephalosporins) reagent 4 (2 ml) is added and mixed thoroughly. The mixture is immediately read in a spectrophotometer at 490 nm or in a colorimeter (623 filter) against a blank of reagent 3 (7.5 ml), phosphate buffer (1 ml), and reagent 4 (2 ml). Calibration curves are obtained for each substrate; they are usually straight lines passing through the origin (e.g., cephaloridine from 75 to 1500 ~g/ml). Substrate (1 mM) is incubated with enzyme at 37 ° for 5 min. Samples (1 ml) are assayed for residual substrate as described above. Five replicate samples are usually assayed. 3. Biological Assay Residual cephalosporin or penicillin can be estimated after fl-lactamase attack by biological plate assay against a suitable organism. Princi27G. E. Boxer and P. M. Everett, Anal. Chem. 21,670 (1949). J. M. T. Hamilton-Miller, J. T. Smith, and R. Knox, Nature (London) 208, 235 (1965).
82
METHODS FOR THE STUDY OF ANTIBIOTICS
[5]
ples and methods are given in detail in this volume [4]. Choice of the organism will depend on the particular substrate being assayed; for example, cephaloridine is usually assayed against Staphylococcus aureus, Bacillus subtilis, or Sarcina lutea. These organisms are all highly sensitive to ccphaloridine and will assay concentrations down to 0.1 ~g/ml. However, solutions of 12-500 ~g/ml can be assayed using Escherichia coll. This organism has the advantage that it is not sensitive to many other cephalosporins and penicillins, so that it will measure cephaloridine in the presence of a substance, such as cloxacillin, which may be present because it is being used as an enzyme inhibitor. It is very necessary, when using a biological method of estimating residual antibiotic, to stop the reaction immediately the sample for assay has been taken. This can be done by the addition of an iodine solution which is adequate to inactivate the enzyme without interfering with the growth of the assay organism. 29 Different enzymes require different amounts of iodine to inactivate them; the enzyme from Enterobacter cloacae P99 can be inhibited by 10 ~M iodine, but the one from E. coli TEM ÷ is not inactivated until the concentration of iodine is 0.7 mM. Method. Iodine reagent: 17.8 mg of iodine is dissolved in 100 ml of 0.06% potassium iodide solution. The chosen substrate at 1 mM is incubated with the enzyme preparation at 37 ° for 5 min. The reaction mixture is then diluted 100-fold with the iodine reagent (0.7 mM) to stop the enzyme reaction, and residual antibiotic assayed by a suitable plate diffusion assay. The concentration of iodine used does not interfere with the assay of antibiotic activity by either Staphylococcus aureus NCTC 7447 or Bacillus subtilis NCIB 8533. 59 IV. Discussion of Assay Methods The range of the different fl-lactamase assays described above and the large number of variations of these assays appearing in the literature, gives some indication of the widespread interest in fl-lactamases. The methods used to estimate their range and potency are numerous and modifications are constantly being made to the basic techniques. Consequently, it is very difficult to compare the results obtained by different groups of workers, although at least one attempt has been made to correlate the results obtained by running five methods in parallel, using 10 substrates and 3 fl-lactamases. ~9 At present, no single assay would seem to satisfy all requirements although there are two or three with definite advantages. aSG. W. Ross, K. V. Chanter, A. M. Harris, S. M. Kirby, M. J. Marshall, and C. H. O'Callaghan, Anal. Biochem. 54, 9 (1973).
[5]
~-LACTAMASE ASSAYS
83
For those workers requiring a rapid and sensitive assay for studying the fl-lactamase sensitivity and inhibitory activity of a large number of cephalosporin derivatives, the spectrophotometric assay is the method of choice. For similar studies on penicillin analogs, one of the recent microiodometric assays is suitable. If both cephalosporin and penicillin substrates are being compared, then microbiological assay is a very sensitive way of measuring residual substrate but is not well suited to enzyme kinetics. Some acidimetric assays offer rather less sensitive but more precise methods for measuring fl-lactamase hydrolysis of penicillins and cephalosporins, provided that the breakdown products of the cephalosporin analogs are sufficiently well characterized for the method to be properly calibrated. If the assay requirement is for a rapid, reliable but comparatively insensitive method of comparing the effect of fl-lactamase on a range of penicillins and cephalosporins, without the need for expensive equipment, then the macroiodomeric method of Perret is the obvious choice. If a method is used which requires intermittent sampling and reading, then the enzyme activity must be stopped at the time of sampling. In some assay methods, the reagents will themselves inhibit the action of the enzyme, for example, addition of hydroxylamine or iodine. This problem does not occur if a continuous recording device is employed.
A. Iodometric Assay Perret's macro assay is relatively straightforward, and reproducible results can be obtained quickly using inexpensive reagents and apparatus. The mechanism of action of iodine with breakdown products of fl-lactam antibiotics is uncertain and care has to be taken with tile interpretation of results for each new substrate studied. Fortunately, few intact substrates react with iodine, although examples are known. Grove and RandalP ° reported that intact allylmercaptomethylpenicillin reacts with iodine ; 3-dimethyldithioearbamoyloxymethyl-7fl-benzylthioacetan~idoceph-3-em-4-carboxylic acid also takes up iodine before/~-lactam hydrolysis, thus giving a high blank value in the assay, p-Hydroxybenzyl penicillin also gives a high blank value21 The enzyme preparation should be routinely checked for iodine uptake. The enzyme reaction can be stopped after 5 min instead of the usual 30 min if required. Care should be taken that the enzyme reaction is stopped by addition of the iodine reagent and Zyk '3 has followed the suggestion of Cshnyi '~'-' D. C. Grove and W. A. Randall, "Assay Methods of Antibiotics," p. 16. Medical Encyclopaedia Inc., Chicago. (1955). ~1p. H. A. Sneath and J. F. Collins, Biochem. J. 79, 512 (1961). 32V. Cs~nyi, Acta Physiol. Acad. Sci. Hung. 18, 261 (1961).
84
METHODS FOR THE STUDY OF ANTIBIOTICS
[5]
to include sodium tungstate in the iodine reagent to ensure inactivation of the enzyme. Perret's method is particularly useful for comparison of the substrate specificities of fl-lactamases, when a wide range of substrates has to be used. Its main disadvantage is lack of sensitivity; the usual substrate concentration is 5 mM. The microiodometric methods are extremely sensitive. Sykes and Nordstrom used a substrate concentration of 0.067 mM in the assay mixture and enzyme activities of below 0.001 ~mole of substrate per minute. At higher enzyme activities, the steady state is not reached before the starch-iodine complex is completely decolorized. Enzyme activities as low as 0.0004 unit per milligram dry weight of bacteria can be readily measured. 1~ The only equipment required is a relatively unsophisticated spectrophotometer. Microiodometric methods are particularly useful in studies of enzyme kinetics or in inhibition studies. A major disadvantage of the micro methods is that they cannot be used with cephalosporin substrafes. The effect of the starch-iodine complex on each new fl-lactamase must also be carefully examined.
B. Acidimetric Assay Although the original methods were "manual" assays, pH star equipment is required to obtain good precision and reproducibility in the direct assay. A substrate concentration of 5 mM is commonly used. Acidimetric assays have been applied principally to penicillin substrates rather than to the cephalosporins, where careful calibration is required owing to the possible production of more than one acidic group or even a basic group after hydrolysis of the fl-lactam ring (see introduction). Farrar and Krause have shown that the number of equivalents of acid released per mole of antibiotic hydrolyzed is 2 for cephalothin, 1.6 for cephaloglycin, and 0.6-1.0 for cephalexin. ~3 The indicator method is a useful alternative, especially for laboratories which have a spectrophotometer but not pH star equipment. This method is of intermediate sensitivity; a substrate concentration as low as 0.2 mM can be used. The manometric method is mainly of historical interest and is now seldom used.
C. Spectrophotometrie (UV) Assay This assay is easy to operate, is rapid, and gives valuable information on the enzyme-substrate interaction in the form of a reaction profile. 3a W. E. Farrer and J. M. Krause,
Inject. Immunity 2, 610 (1970).
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~-LACTAMASE ASSAYS
85
Up to four simultaneous enzyme reactions can be followed in a recording spectrophotometer, and this has been particularly useful for comparison of the fl-lactamase resistance of new cephalosporins and for inhibition studies. The products of fl-lactam hydrolysis may absorb in the UV range so that at any one time the absorption of a mixture of substrate and product(s) is being measured. The effect of UV absorption by the products is minimized by comparing only the initial rates of hydrolysis over the first minute. Reliable quantitative results are obtainable when a callbration curve is constructed relating fall in UV absorption to hydrolysis of the cephalosporin. The assay has been used for substrate concentrations from 0.05 to 1 mM. The main disadvantage of this technique is that it cannot be applied to penicillin substrates because they do not have the conjugated system associated with the fl-lactam ring in cephalosporins which gives rise to the absorption monitored in this method. 26 However, Jansson has reported 34 that the rate of hydrolysis of ampicillin by a B. cereus B-lactamase can be related to a fall in absorption at 244 nm. Spectrophotometric measurement has been used indirectly for assay of the products of B-lactamase hydrolysis of penicillins in the microiodometric and indicator methods. All fl-lactamases tested to date, including both penicillinase and cephalosporinase types, can be assayed by the chromogenic cephalosporin method and this is particularly useful for comparing several enzymesJ
D. Hydroxylamine Assay This assay has been found to be more difficult to operate than the other assay procedures described and it gave higher relative activities against penicillins and some cephalosporins in a study in which five assay methods were compared. 29 Mixing of the reagents is critical and unwanted precipitation can occur after addition of the ferric ammonium sulfate reagent or if the concentration of the phosphate buffer is increased. The chromogen is unstable, and gas bubbles formed on mixing the alcoholic and aqueous solutions can interfere with absorption measurements. When purified enzyme preparations are used, the assay measures residual intact B-lactam ring in the substrate, but, in less pure reaction mixtures, hydroxylamine can react with compounds containing a carbonyl group, or with esters, anhydrides, and amides25 The assay is relatively insensitive and has no apparent advantage. 34j. A. T. Jansson, Biochim. Biophys. Acta 99, 171 (1965). ~sj. :H. Ford, Ind. Eng. Chem. Anal. Ed. 19, 1004 (1947).
86
METHODS FOR THE STUDY OF ANTIBIOTICS
[6] I m m u n o l o g i c a l
Techniques
[6]
for
Studying/~-Lactamases
By M. H. :RICHMOND I. Introduction . . . . . . . . . . . . . . . . . . II. Preparation of Antisera . . . . . . . . . . . . . . . A. Using Purified Enzyme Preparations . . . . . . . . . . B. Using Crude Enzyme Preparations . . . . . . . . . . . C. Isolation of ~-Lactamase-Less Mutants . . . . . . . . . D. Inoculation Program for Crude Preparations . . . . . . . . III. Examination of ~-Lactamases with Specific Antisera . . . . . . . A. Neutralization Analysis . . . . . . . . . . . . . . B. Precipitation Analysis . . . . . . . . . . . . . . .
86 86 86 87 89 89 90 90 98
I. Introduction I n principle, fl-lactamases are no different from other enzymes in respect to their action with antisera, and m a n y of the techniques to be described below are capable of much wider application t h a n has occurred in the past. The reason for describing t h e m here, however, is t h a t they have a particular relevance to fl-lactamases since m a n y of the minor variants of this enzyme t h a t are found in naturally occurring strains have been characterized b y these immunological methods.
I I . P r e p a r a t i o n of Antisera
A. Using Purified Enzyme Preparations For the preparation of specific antisera, and those for reaction with enzymes are no exception, it is preferable to have the antigen as pure as possible. I n this w a y the sera are likely to have their highest titers and to be as free as possible of nonspecific side reactions. In practice, preparation of a pure antigen m a y be more exacting than normal protein purification since small amounts of impurity m a y be disproportionately immunogenic. On the other hand, these considerations do not often invalidate the use of sera for analytical purposes with fl-lactamases. Indeed all the studies on staphylococcal and Escherichia coli B-lactamases described in this section were carried out with enzymes purified by the methods described elsewhere in this volume. 1,2 1This volume [53c]. This volume [53d].
[6]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
27
A number of different animal species and dosage regimes have been used to raise fl-lactamase sera. All the most effective, however, use inoculation of rabbits with purified enzyme preparations treated in various ways2 ,4 For antistaphylococcal penicillinase,4 sandy lop rabbits are each injected in alternate thighs at 3-day intervals with a total of four 0.2-ml portions of a solution of purified staphylococcal penicillinase made by suspending about 5 mg of purified enzyme per milliliter of Freund's adjuvant2 After a further 2 weeks, each rabbit is injected intravenously through the ear vein with three successive 0.2-ml samples of an alum-precipitated preparation of purified enzyme (approximately 5 mg/ml) at 2-day intervals. Samples of blood are withdrawn from the rabbit's ear vein 6 days after the end of the second phase of treatment, the serum is separated by conventional techniques, and the antibody titer is determined. In practice, once a serum of suitable titer has been obtained, it is better to kill and exsanguinate the rabbit since this procedure provides maximum quantities of a serum of uniform properties. In a typical example with staphylococcal penicillinase, about 100 ml of a serum, with titer about 104 units per milliliter of undiluted serum, was obtained from a single sandy lop rabbit. After separation of the serum, the material is best stored by dividing into ampules in about 5-ml quantities and freezedrying. Ampules may then be reconstituted for use as required by adding 5 ml of distilled water. If diluted serum is needed, the reconstituted serum should be diluted for use in physiological saline.
B. Using Crude Enzyme Preparations The above method is the one that should be followed when reasonable supplies of purified enzyme are available. It is possible, however, to prepare adequate specific sera by inoculating relatively crude enzyme preparations in Freund's adjuvant, and then absorbing out the unwanted antibodies before use. For this procedure to be successful, it is vital to have a mutant of the producer strain that makes no fl-lactamase. Before going on to describe the actual techniques for preparation of fl-lactamase from crude ])reparations, the purification procedure for crude enzyme and the methods of isolating fl-lactamase-less mutants will be described. Preparations of fl-lactamase from most gram-negative bacteria may be prepared as follows. The method is based on the one described elsewhere in this volume for the purification of the fl-lactamase from Escherichia coli (Rr+~M).2 A culture of the producer strain growing exponena M. R. Pollock, J. Gen. Microbiol. 14, 90 (1956). 4 M. H. Richmond, Biochem. J. 88, 452 (1963). 5 Difco Bacto-adjuvant, Complete (Freund), 0638-60-7.
88
METHODS FOR THE STUDY OF ANTIBIOTICS
[5]
tially in 1% CY medium 1 is centrifuged and the bacteria are collected. These organisms are resuspended in 0.1 M Na2HPO4/KH2P04 buffer, pH 7.0, to a density of about 30 mg dry weight of bacteria per milliliter and disrupted in an ultrasonic disintegrator. ~ The broken bacteria are stored at 2 ° until disruption of the whole batch is complete; they are then centrifuged, first at 5000 g for 15 rain at 4 ° (to remove large cell debris), then at 40,000 g for 4 hr at 4 ° (to remove smaller fragments), and last at 105,000 g for 2 hr at 4 ° to sediment ribosomes and minute pieces of comminuted membrane. After centrifugation the supernatant containing the enzyme is dialyzed against 100 volumes of 0.1 M Na~ HPO4/KH2P04 buffer, pH 7.0, for 24 hr at 2 °. During this process a precipitate often forms, and this is removed by centrifugation at 5000 g for 15 min at 4 °. After dialysis and any necessary centrifugation the enzyme is run through a Sephadex column. Adequate preparations are obtained if a Sephadex G-75 column equilibrated against 0.1 M Na~HPO~/KH2P04 buffer, pH 7.0, is used, but cleaner enzyme preparations may be obtained by using either DEAE-Sephadex or CM-Sephadex, as appropriate. The choice of Sephadex depends on the nature of the enzyme, and to decide this it is useful to know something of the ionic properties of the protein. In general all fl-lactamases from gram-negative species with a predominant activity against cephalosporins are positively charged at neutral pH values 6 and therefore should be purified on CM-Sephadex. The remaining fl-lactamases are likely to be more acidic 6 and in this case DEAE-Sephadex may be more appropriate./~lthough a general tendency, however, this correlation between charge andsubstrate profile should not be taken as absolute, and it is always necessary to carry out some preliminary test absorptior~ experiments with the enzyme and various substituted Sephadexes when an enzyme is being examined for the first time. As a guide it is always helpful to know the electrophoretic properties of the enzyme before doing the experiments. When G-75 Sephadex is used, the column (150 X 1.5 cm) is equilibrated against 0.1 M Na2 HPO4/KH2P04 buffer, pH 7.0, and the material is eluted with similar buffer. After elution from the Sephadex column the enzyme preparation is dialyzed against distilled water to remove salts and then stored at 2 ° until required for use. In general it seems better not to freeze dry enzyme preparations destined for raising antisera since the freezing and thawing process is likely to denature some of the enzyme molecules, and this in turn is likely to broaden the specificity of the resulting antiserum. M. H. Richmond and R. B. Sykes, Recent Adv. Microb. Physiol. 9, 36 (1973).
[6]
IMMUNOLOGICALTECHNIQUES FOR STUDYING ~-LACTAMASES
89
C. Isolation of/~-Lactamase-Less Mutants The fl-lactamase producing culture is grown exponentially in any convenient growth medium and then treated with N-methyl-N-nitro-Nnitrosoguanidine (NMG) as described elsewhere in this volume. 1 After treatment and the necessary period of growth following removal of the mutagen, the bacteria are plated as single colony-forming units on 1% CY agar l and incubated overnight at 37 °. In the morning the colonies are flooded on the plate with a solution (5 mg/ml) of the chromogenic cephalosporin 87/312. 7 In this example, the great majority of the colonies will turn red (indicating production of fl-lactamase), but a few (probably not more than 1/50,000) will produce no color. It is therefore necessary to do this experiment on a large scale. About 100 plates with 1000 colonies per plate are commonly employed; but it may be necessary to work on an even larger scale. Any colorless colonies are picked onto fresh agar and purified by restreaking. The isolates are next checked for their production of fl-lactamase, and any producing no detectable enzyme by quantitative methods are retained for use. The process of isolating mutants in this way is tedious, since methods for selection of fl-lactamaseless mutants are not available, but fl-lactamase-less mutants of this type must be available for specific sera to be obtained when crude enzyme preparations are used as immunogens.
D. Inoculation Program for Crude Preparations As with the program for purified enzyme, rabbits are the most convenient animal for raising sera in the conventional laboratory. The larger the rabbit the better, since the final yield of serum will depend to some extent on the blood volume of the animal; and absorbed sera are likely to have lower titers than those prepared against purified antigens. A typical program for raising an antiserum using a crude enzyme preparation is as follows. Samples of crude enzyme, purified as described above, are injected after mixing with equal volumes of Freund's adjuvant into alternate thighs of a sandy lop rabbit. In all, a sequence of six injections at 2-day intervals is used. Two weeks after the last injection, the animal is injected through the ear vein with 0.2 ml of alum-precipitated crude enzyme. Four injections of this material are made at 2-day intervals. About 10-14 days after the end of this sequence of injections, the animal is test bled, and the antiserum titer is determined against the appropriate enzyme (see below). When two test bleeds 2 days apart show the same titer, the animal is killed and exsanguinated, and serum is prepared from TC. H. O'Callaghan, A. Morris, S. M. Kirby, and A. H. Shingler, Antimicrob. Ag. Chemother. 1, 283 (1972).
90
METHODS FOR T H E STUDY OF ANTIBIOTICS
[6]
the whole blood by conventional techniques. Titers of about 5000 units of neutralizing ability are not uncommon by this technique, but it must be stressed that not all anti-fl-lactamase sera are of the neutralizing type (see below). Once an active serum has been obtained by this method it is then treated with crude protein prepared from the fl-lactamase-less mutant strain. Ideally the serum should be treated with material made in an identical manner to the crude enzyme, protein being collected from the Sephadex G-75 column at the point at which the relevant fl-lactamase is known to elute. However, it is equally effective to use some of the total cell protein obtained after ultrasonic disruption of the fl-lactamaseless mutant. However, if material that is the product of ultrasonic disintegration without centrifugation is used, losses in the titer of the serum are likely to be high, but if the material at the stage at which it would normally be loaded onto the Sephadex column is employed, absorption is more effective and losses of specific anti-fl-lactamase activity is minimized. The adsorption technique involves adding the absorbing protein to the serum in a series of 0.2-ml quantities until precipitation ceases. Ideally this should lead to no loss in titer of the antiserum, but in practice some loss seems difficult to avoid, and it is usually necessary to compromise between having an absolutely specific serum and one of such low titer that it is virtually useless. In a typical absorption experiment, 0.2-ml quantities of absorbing protein were added at intervals to 5 ml of undiluted serum. The serum was then incubated at 30 ° for 2 hr before the precipitate was centrifuged off at 5000 g for 15 min. In one experiment about 0.75 ml of absorbing protein had to be added to the 5-ml batch of serum before precipitation ceased. In order to sterilize the serum during this procedure, 0.1% (w/v) sodium thiomersalate is added to the serum. Once absorption is complete, the serum is transferred to ampules and freeze-dried in the normal way. III. Examination of fl-Lactamases with Specific Antisera Once antisera to a B-lactamase have been obtained, they can be used to study the enzymes in a number of ways. These include precipitation analyses both in solution and by gel diffusion. But perhaps the most effective is by neutralization of enzyme activity since this can be studied with as little as 0.1 ~g of enzyme in impure preparations.
A. Neutralization Analysis When antibodies interact with enzymes, the activity of the preparation may be altered. However, interaction with an antibody molecule
[~]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
91
need not affect activity, and it is impossible to predict the manner in which an antiserum preparation raised against either a crude or a purified preparation of fl-lactamase will act. Indeed, antisera obtained with samples of the same enzyme preparation in different rabbits may v a r y greatly in their effect, even when the immunization program is identical and carried out in parallel. It follows, therefore, that when antisera are being raised it is important to obtain as large a quantity as possible so that a large series of tests may be made with a serum of uniform properties. Provided an antiserum affects enzyme activity, this interaction m a y be used to provide much information about the nature of a fl-lactamase. Most sera reduce the activity of B-lactamases, 3,~ and this neutralizing effect will be discussed first. However, stimulatory sera, ~ or even sera that stimulate and then inhibit at higher concentration are known, 9 but their use will be discussed later. The most common way to use a neutralizing serum is to study the enzyme/antiserum interaction as a titration of a constant amount of antigen. Increasing amounts of antiserum are added to a series of vessels each containing a fixed amount of antigen. After a period for the antigen/antibody reaction to occur (usually about 10 rain at room temperature) the residual enzyme activity is assayed by a convenient method (usually the iodometric method of Ferret TM as modified by N o v i c k ' ) . With E. coli fl-lactamase, where a serum with titer about 104 units/ml is available, the usual experimental approach is to put about 50-75 units of enzyme in each of a series of conical flasks and immediately add increasing amounts of serum, leaving the first without addition to act as a measure of the untreated enzyme activity. The reaction is allowed to continue for 10 rain. Then 10 ml of a prewarmed solution of benzyl penicillin (1%, w/v, in 0.1 M Na~ H P O J K H 2 P O 4 buffer, pH 5.9) is added. After 6 rain further incubation, 10 ml of standard iodine solution in 2 M sodium acetate buffer, pH 4.2, is added and the excess iodine back-titrated against sodium thiosulfate." Figure 1 shows a typical neutralization curve obtained in this way2 There is a linear decrease in residual enzyme activity with increasing antiserum concentration up to a point (the equivalence point: E--see Fig. 1) at which no further inactivation occurs however much serum is added. The activity remaining after the equivalent amount of serum has been added is known as the "residual activity" and probably represents the activity of the enzyme/antibody complex. The slope of the neutralization curve G. W. Jack and M. H. Richmond, J. Gen. Microbiol. 61, 43 (1970). 9 M. R. Pollock, Immunology 7, 707 (1964). ~oC. J. Perret, Nature (London) 174, 1012 (1954). See also this volume [5]. ~ R. P. Novick, Biochem. J. 83, 229 (1962).
92
METHODS FOR THE STUDY OF ANTIBIOTICS
[6]
F
100~-,,~
'~: .I- \ . 6o-
\o
\o
--
'~EO--~O
20 I 0.02
I 0,04
I 0,06
I 0.08
Antiserum
I 0.1
I 0.12.
: ml
FIG. 1. Neutralization of type I I I a ~-lactamase by anti-type I I I a fl-lactamase serum [G. W. Jack and M. I-I. Richmond, J. Gen. Microbiol. 61, 43 (1970)]. E, equivalence point.
gives the titer of the serum; and if the turnover number of the fl-lactamase is known, this titer m a y be expressed as the number of enzyme molecules neutralized per milliliter of antiserum. In practice 10 min is normally allowed for the enzyme/antibody complex to forIn at room temperature, but a study of the time course of enzyme/antiserum interaction shows that the reaction occurs very rapidly (Fig. 2). Neutralization with more than the equivalent amount of serum is more than 90% complete in 2 min at room temperature. Figure 1 illustrates a typical neutralization curve obtained with an anti-fl-lactamase serum. However, all sera do not neutralize. Figure 3
~
c t00 ]uu .2
6{ E
E E
•
O
/
°~° 2C I 2
I 4
I 6
I 8
I 10
minutes
FIG. 2. Time course of reaction between type I l i a /~-lactamase and a specific antiserum.
[6]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING /~-LACTAMASES
93
241 20(
//
ID
/* /* 40,;I 0.05
i 0.1 Antiserum
| 0.15
I 0-2
: ml
FIG. 3. Stimulation of staphylococcal ~-lactamase activity by a specific antiserum [M. H. Richmond, Biochem. J. 88, 452 (1963)]. E, equivalence point.
shows the effect of addition of increasing amounts of anti-staphylococcal serum to staphylococcal fl-lactamase type A. In this case this particular batch of serum stimulates the activity of the enzyme to a maximum about 4.5 times higher than that of the enzyme without serum. ~ Once again the response is linear with increasing amounts of antiserum, and once more there is an equivalence point (E) above which further addition of serum produces no change in activity. As with neutralization effects, stimulation by antiserum can be used to characterize an enzyme. The slope of the curve (Fig. 3) gives a measure of the titer of the serum, and the extent of stimulation is constant for a given type of fl-lactamase. At present it is uncertain how combination with an antiserum increases the activity of an enzyme. Intuitively one feels that the process must hold the enzyme in a more favorable conformation, but convincing experimental evidence for this sort of mechanism is lacking. Nevertheless one thing seems certain: The binding of stimulatory sera cannot block the active center of the enzyme. Certain sera show even more complex effects with fl-lactamases than the examples shown in Figs. 1 and 3. Pollock has studied the interaction of specific serum with purified fl-lactamase from Bacillus licheni]ormis2 In this example (Fig. 4) addition of antiserum causes stimulation and then inhibition. Such a curve has two equivalence points (El and E.~) but is difficult to use for analytical purposes, although the shape of the curve has great value as a qualitative means of identifying an enzyme type. This complex curve is certainly due to the interaction of the enzyme with an antiserum containing both inhibitory and stimutatory compo-
94
METHODS FOR THE STUDY OF ANTIBIOTICS
[5]
3001 /\,E1 .. 200 >
0
i ,°0,
\,
o
E2
I
I
O.2
o.1 ml
of
I
O.3
antiserum
FIG. 4. Interaction of fl-lactamase from Bacillus licheni]ormis 6346 with specific antiserum [M. R. Pollock, Immunology 7, 707 (1964)]. E,, stimulatory equivalence point; E~, neutralizing equivalence point.
nents. Partial absorption of the serum with enzyme removes the stimulatory component and gives a residual serum which is purely neutralizing2
I. Characterization o] Variants in Naturally Occurring Strains Since the effect of antisera on fl-lactamase activity can be studied with such small amounts of enzyme and since contaminating protein does not interfere significantly with the interaction, tests in solution are an ideal way of looking for molecular variants of fl-lactamase whether of natural or laboratory (i.e., mutational) origin. An example of the detection of natural variants comes from studies that have been made on staphylococcal fl-lactamase. 12,13 The reaction of anti-staphylococcal fllactamase serum with the fl-lactamase found in most clinical isolates of Staphylococcus aureus gives a stimulatory curve already discussed (Fig. 1). However, analysis of a large number of clinical isolates shows that four types of response are to be found (Fig. 5). The majority show the standard stimulation by a factor of about 4.5-fold---known as A-type response--while a substantial minority show no stimulation at all. Indeed in this group (C type) there is no evidence that the serum (which was raised against purified A-type enzyme) interacts with the fl-lactamase at all until a precipitation analysis is carried out. Under these conditions an enzyme/antiserum precipitate is obtained, but this has the expected activity of the amount of enzyme combined in the precipitate. Two other ~2M. It. Richmond, Biochem. J. 94, 584 (1968). ~3V. T. Rosdahl, J. Gen. Microbiol. 77, 229 (1973).
[{)]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
95
20o
"~160 •;
120
o / / -
• ~
• ~
• ~ OA
o
o
o
I
I
/+
i40, ~-~ I
0.1 0.2 0.3
I
Od
Co
I
0.5
ml of antiserum
Fro. 5. Response of the four variants of staphylococcal f~-lactamase after interaction with a serum raised against type A [M. H. Richmond, Biochem. J. 94, 584 (1968)]. A, B, C, D: fl-lactamase types based on their response to serum.
types of response are also found. In the first (type B), the antiserum stimulates, but the titer is about one-fifth of that expressed with A-type fl-lactamase. In fact, further analysis shows that this response is due to expected antiserum binding to a fl-laetamase molecule with about onefifth the turnover number of A-type fl-lactamase. The fourth type of response (type D) gives a lower maximum degree of stimulation (about 1.5-fold at most) with a lower apparent titer. 13 In this case, the turnover number of the fl-lactamase variant is the same as that of A-type enzyme, and therefore the serum interacts with the enzyme with lower affinity. Subsequent experiments have shown that types A, B, and C fl-lactamase from S. a u r e u s (and probably type D as well) are minor variants of a common structure probably with one or a few amino differences in their primary sequence. Similar experiments have been done with fl-lactamases from other bacterial species, and in general these experiments have led to the view that these enzymes tend to be species specific, but that within a given species a relatively large number of minor variants are to be found. 6''4 The cross reaction found between the minor variants within a given species, but the absence of cross reaction between the fl-lactamases from different species suggests that perhaps interaction between enzyme and antiserum is particularly sensitive to changes in the primary sequence of the enzyme protein, and that a change in only one or two residues can be tolerated before the binding of the serum becomes suSstantially impaired. Nor is such a view incompatible with the normal high specificity of antibody molecules. ~ N. Cirri and M. R. Pollock, Advan. Enzymol. 28, 237 (1966).
96
METHODS FOR THE STUDY OF ANTIBIOTICS
[5]
2. The Use o] Antisera to Study Laboratory-Induced Mutations The main objective here, since one starts with the information that any changes in reaction are due to changes in amino acid sequence, is to try to discover whether the mutation has affected only the turnover number of the enzyme, or whether there has also been some alteration in affinity for antibodies. A typical example is the examination of the pen P2 mutant of staphylococcal fl-lactamase. 1~ Bacterial cultures synthesizing this mutant enzyme express about 5% of the normal wild-type level of activity. Comparison of the serum interaction of this mutant enzyme with the wild type shows that the total stimulation obtained is the same (i.e., about 4.5-fold), but that the antiserum titer with the mutant enzyme is about ½oth of that found with the wild type. These results suggest strongly, therefore, that the mutational change in P2 has resulted in a protein with about 5% the turnover number of the wild-type enzyme but with no alteration in the affinity of interaction between serum and enzyme. This result is only an indication and must be confirmed by purification of the enzyme; and indeed in the case of mutant P2 the turnover of the purified mutant enzyme is found to be about 5% of the unmutated parental fl-lactamase. As yet this approach to the study of fl-lactamases is largely unexploited since no systematic analysis of the effect of primary sequence changes on enzyme activity and reaction with antiserum has yet been made. Once this approach is developed, however, the use of sera in an appropriate way will give much useful preliminary information about the properties of mutant enzymes, even if the final situation can be confirmed only by the painstaking isolation and purification of each mutant.
3. Use o] Antisera to Analyze One Component in a fl-Lactamase Mixture The one group of strains where the general rule that fl-lactamases tend to be species specific breaks down in that group of gram-negative bacteria susceptible to R factor infection. In this group it is possible to find bacterial isolates that express two distinct types of fl-lactamase,8 and it can therefore be valuable to have methods for determining the relative amounts of the two enzymes expressed by such strains. One typical example is the use of anti-type I I I a fl-lactamase serum to analyze the enzyme produced by a strain of E. coli which can be shown by electrophoresis of a crude enzyme preparation to make two enzymes, one a cephalosporinase of type Ib and the other an R factor-mediated enzyme (type IIIa). The type Ib enzyme does not react with the anti-type IIIa serum ~ See footnote 4, and also unpublished experiments b y M. It. Richmond.
[6]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
97
whereas the t y p e I I I a e n z y m e - - b e i n g the enzyme against which the serum was raised in the first p l a c e - - g i v e s a neutralization reaction with a residual activity of about 35% (see Fig. 1). About 100 units of the enzyme mixture was titrated with anti-type I I I a serum as a constant antigen titration. The serum partially neutralized the activity of the mixture, and the residual activity was about 67% of the initial activity (Fig. 6). Since (a) only the type I I I a enzyme reacts with the antiserum, and (b) the presence of type I b enzyme does not interfere with the reaction, it is possible to calculate the relative amounts of the two enzymes in the original mixture using the fact that the residual activity obtained with type I I I a enzyme and this antiserum is 35% of the initial activity. I n this particular example, in fact, the calculation shows that the original mixture was made up of about 50 units of type Ib enzyme and 50 units of t y p e I I I a . This approach to analyzing the composition of mixtures of fl-lactamases is very useful, but it is subject to two limitations. First, the antiserum must be specific to one of the two component enzymes; and second, the amount of the two components must be approximately equal. Clearly the total activity of small amounts of type I I I a enzyme in the presence of large amounts of a nonreactive fl-lactamase will show relatively little decrease of activity in the presence of antiserum, and any subsequent calculations must be subject to large errors. 120
\ 8( .>
E
'esidual activity
40
0.1 ml
0.2
I
of a n t i s e r u m
FIG. 6. Neutralization of a mixture of type Ib and type IIIa fl-lactamase with anti-type IIIa serum. E, equivalence point. Type Ib enzyme does not react with anti-type IIIa serum. Moreover this serum neutralizes type IIIa fl-lactamase to 35% residual activity. The actual neutralization observed in Fig. 6 is 35 units out of a total of 103 present; and this corresponds to the neutralization of 54 units of type IIIa fl-lactamase. The type Ib component therefore amounts to 49 units, by difference.
98
METHODS FOR THE STUDY OF ANTIBIOTICS
[6]
B. Precipitation Analysis ~6
Even if antisera to fl-lactamases do not affect enzyme activity, they will precipitate enzyme protein provided the concentration of the reactants is high enough. However, since most enzymes have such high specific enzyme activities it is unusual to have sufficient enzyme protein in a test system to produce an effective precipitate. It follows, therefore, that quantitative precipitation analysis of fl-lactamases is usually confined to rather special circumstances; and it is normally necessary to label the enzyme protein with radioactivity in order to follow the process accurately. Quantitative precipitation analysis has been used effectively to discover whether fl-lactamase I induction in Bacillus cereus was due to the biosynthesis of enzyme de novo, or whether conversion of an inactive precursor was involved.16 In these experiments a culture of Bacillus cereus growing exponentially was induced by adding 6 gg of benzyl penicillin per milliliter, and at the same time a source of 14C-labeled amino acids was added to the culture. Incubation was continued, and samples of the culture were withdrawn at intervals for precipitation analysis. The amounts of fl-lactamase I present in the growth medium in this experiment (the enzyme is an extracellular protein in B. cereus) were far too low for precipitation to occur without the addition of carrier fllactamase. Accordingly, each sample from the culture supernatant was treated with enough purified nonradioactive B. cereus fl-lactamase I to bring the total activity of the sample to 9000 units, followed by a 50% excess of specific antiserum. The precise experimental sequence was carried out as follows.16 Samples of whole bacterial culture (4.0 or 5.0 ml) were pipetted directly into a centrifuge tube containing a few crystals of oxine (8-hydroxyquinoline) and sufficient solid sodium chloride to bring the final concentration to molar strength. After shaking to dissolve the solids, the tubes were cooled rapidly and the bacteria centrifuged down. The penicillinase activity in the supernatant was assayed and enough carried fl-lactamase added to 3.0 ml of the total sample to bring the total penicillinase activity to 9000 units. This amount of enzyme is equivalent to about 3 mg of pure enzyme. The carrier supplemented samples were then dialyzed overnight at 2 ° against 1 mM KH2P04/K~HPO~ buffer, pH 7.0. After overnight dialysis, the carrier-supplemented enzyme preparations were transferred to centrifuge tubes and 0.15 ml of a suspension of "fine-mesh" glass powder (100 mg of glass per milliliter) added to each. The preparation was left at room temperature for 3 min, then the glass powder was centrifuged off and washed once with 5 ml of dis~6M. R. Pollock and M. Kramer, Biochem. J. 70, 665 (1958).
[6]
IMMUNOLOGICAL TECHNIQUES FOR STUDYING ~-LACTAMASES
99
tilled water. The enzyme was then eluted from the glass with two successive washes (2.5 ml and 1.0 ml, respectively) of alkaline 1 M NaC1 at pH 8.5. These two washings were combined and filtered through an Oxoid membrane filter to sterilize them and made up to 5.0 ml with distilled water; their fl-lactamase content was assayed. Enough carrier fl-lactamase to return the total activity of the preparation to 9000 units was then added to 4.0 ml of the eluate. Anti-exo-penicillinase serum was then added to exceed the equivalence point by 50%, and the mixture was incubated overnight at 35% The next morning the precipitate was centrifuged down after cooling to 2 °, resuspended in 2 ml of distilled water, and centrifuged down once more. This precipitate was then prepared for radioactivity measurements in the conventional way. If this procedure is followed meticulously, at least 90% of the fl-lactamase activity is precipitated as an antigen/antibody complex; and the application of these precipitation techniques allowed Pollock and Kramer TM to show conclusively that B-lactamase induction was not due to the activation of an inactive precursor, but was the result of de novo fl-lactamase synthesis. Gel Precipitation and Other Qualitative Procedures The main objective in this article has been to describe the preparation of specific anti-fl-lactamase sera and to show how they may be used for the quantitative study of penicillinases and cephalosporinases. Once the sera have been obtained, however, they may be used for the full range of gel precipitation and immune-electrophoresis procedures available for other enzymes and proteins. These have frequently been reviewed, and the reader is referred to them. 17 One special point must be borne in mind when applying these techniques to fl-lactamases. Several of the enzymes, but notably all the B-lactamase variations synthesized by plasmid carrying strains of Staphylococcus aureus are relatively basic proteins and adsorb strongly to acidic polymers such as agar. Gel diffusion experiments involving these enzymes should therefore be carried out in the presence of 1 M NaC1. Without this no diffusion of enzyme occurs and precipitation, if it occurs at all, is either on the edge of the well or even in the enzyme well itself. Unfortunately, it is impossible to add such high salt concentrates to electrophoretic systems, and so far immune electrophoresis of staphylococcal fl-lactamase has been unsuccessful. These enzymes do not adsorb to starch gels, but the precipitation bands are difficult to locate in this medium unless they are visualized by staining the gels for enzyme activity. The best method of doing this is to treat starch gels after electro1, C. A. Williams and M. W. Chase, Eds., "Methods in Immunology and Immunochemistry," Vol. 3, pp. 118 and 234. Academic Press, New York, 1971.
100
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
phoresis with the reagent used to detect fl-lactamase by colonies growing on starch agar. 1 This method is less effective than might be expected, however, since the reaction spreads rapidly and the precipitation bands appear clearly only for a short time.
[7] Paper Chromatography of Antibiotics By VLADIMIRBETINA I. II. III. IV.
V.
VI.
VII.
VIII.
IX.
X. XI. XII.
Introduction . . . . . . . . . . . . . . . . . . General T e c h n i q u e s . . . . . . . . . . . . . . . . Bioautography . . . . . . . . . . . . . . . . . Paper C h r o m a t o g r a p h y of fl-Lactam Antibiotics . . . . . . . A. Penicillins a n d 6-Aminopenicillanic Acid . . . . . . . . . B. Cephalosporin C F a m i l y , 7-ACA, a n d C e p h a m y c i n s . . . . . C a r b o h y d r a t e Antibiotics . . . . . . . . . . . . . . A. Aminoglycosidic Antibiotics . . . . . . . . . . . . B. Streptothricin Group . . . . . . . . . . . . . . C. Oligosaccharides w i t h C h r o m o p h o r e . . . . . . . . . . D. E v e r n i n o m i c i n a n d L i n c o m y c i n Group . . . . . . . . . E. Other Sugar D e r i v a t i v e s . . . . . . . . . . . . . Macrocyclic Lactone Antibiotics . . . . . . . . . . . . A. N o n p o l y e n e Glycosidic Macrolides . . . . . . . . . . B. Polyene Macrolides . . . . . . . . . . . . . . . C. Other Macrocyclic Antibiotics . . . . . . . . . . . . Quinone Antibiotics . . . . . . . . . . . . . . . A. Tetracyclines . . . . . . . . . . . . . . . B. A n t h r a c y c l i n e s a n d A n t h r a c y c l i n o n e s . . . . . . . . . Amino Acid a n d Peptide Antibiotics . . . . . . . . . . A. Diketopiperazine D e r i v a t i v e s . . . . . . . . . . . B. H o m o p e p t i d e s . . . . . . . . . . . . . . . C. H e t e r o m e r Peptides . . . . . . . . . . . . . . D. Sideromycins . . . . . . . . . . . . . . . . E. Bleomycin Group . . . . . . . . . . . . . . F. A c t i n o m y c i n s . . . . . . . . . . . . . . . G. E c h i n o m y c i n - T y p e Antibiotics . . . . . . . . . . N i t r o g e n - C o n t a i n i n g Heterocyclic Antibiotics . . . . . . . A. M i t o m y c i n Group . . . . . . . . . . . . . . B. P y r i m i d i n e Nucleosides . . . . . . . . . . . . . C. P u r i n e Nucleosides . . . . . . . . . . . . . . O x y g e n - C o n t a i n i n g Heterocyclic Antibiotics . . . . . . . . Alicyclic Antibiotics . . . . . . . . . . . . . . . A r o m a t i c Antibiotics . . . . . . . . . . . . . . A. Chloramphenicol a n d I t s Derivatives . . . . . . . . . B. Novobiocin . . . . . . . . . . . . . . . .
101 102 105 110 110 116 119 119 123 126 128 128 129 129 134 136 137 137 141 144 144 145 146 146 148 150 150 152 152 152 153 154 156 159 159 160
100
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
phoresis with the reagent used to detect fl-lactamase by colonies growing on starch agar. 1 This method is less effective than might be expected, however, since the reaction spreads rapidly and the precipitation bands appear clearly only for a short time.
[7] Paper Chromatography of Antibiotics By VLADIMIRBETINA I. II. III. IV.
V.
VI.
VII.
VIII.
IX.
X. XI. XII.
Introduction . . . . . . . . . . . . . . . . . . General T e c h n i q u e s . . . . . . . . . . . . . . . . Bioautography . . . . . . . . . . . . . . . . . Paper C h r o m a t o g r a p h y of fl-Lactam Antibiotics . . . . . . . A. Penicillins a n d 6-Aminopenicillanic Acid . . . . . . . . . B. Cephalosporin C F a m i l y , 7-ACA, a n d C e p h a m y c i n s . . . . . C a r b o h y d r a t e Antibiotics . . . . . . . . . . . . . . A. Aminoglycosidic Antibiotics . . . . . . . . . . . . B. Streptothricin Group . . . . . . . . . . . . . . C. Oligosaccharides w i t h C h r o m o p h o r e . . . . . . . . . . D. E v e r n i n o m i c i n a n d L i n c o m y c i n Group . . . . . . . . . E. Other Sugar D e r i v a t i v e s . . . . . . . . . . . . . Macrocyclic Lactone Antibiotics . . . . . . . . . . . . A. N o n p o l y e n e Glycosidic Macrolides . . . . . . . . . . B. Polyene Macrolides . . . . . . . . . . . . . . . C. Other Macrocyclic Antibiotics . . . . . . . . . . . . Quinone Antibiotics . . . . . . . . . . . . . . . A. Tetracyclines . . . . . . . . . . . . . . . B. A n t h r a c y c l i n e s a n d A n t h r a c y c l i n o n e s . . . . . . . . . Amino Acid a n d Peptide Antibiotics . . . . . . . . . . A. Diketopiperazine D e r i v a t i v e s . . . . . . . . . . . B. H o m o p e p t i d e s . . . . . . . . . . . . . . . C. H e t e r o m e r Peptides . . . . . . . . . . . . . . D. Sideromycins . . . . . . . . . . . . . . . . E. Bleomycin Group . . . . . . . . . . . . . . F. A c t i n o m y c i n s . . . . . . . . . . . . . . . G. E c h i n o m y c i n - T y p e Antibiotics . . . . . . . . . . N i t r o g e n - C o n t a i n i n g Heterocyclic Antibiotics . . . . . . . A. M i t o m y c i n Group . . . . . . . . . . . . . . B. P y r i m i d i n e Nucleosides . . . . . . . . . . . . . C. P u r i n e Nucleosides . . . . . . . . . . . . . . O x y g e n - C o n t a i n i n g Heterocyclic Antibiotics . . . . . . . . Alicyclic Antibiotics . . . . . . . . . . . . . . . A r o m a t i c Antibiotics . . . . . . . . . . . . . . A. Chloramphenicol a n d I t s Derivatives . . . . . . . . . B. Novobiocin . . . . . . . . . . . . . . . .
101 102 105 110 110 116 119 119 123 126 128 128 129 129 134 136 137 137 141 144 144 145 146 146 148 150 150 152 152 152 153 154 156 159 159 160
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PAPER
CHROMATOGRAPHY
OF ANTIBIOTICS
C. Griseofulvin . . . . . . . . . . . . . . . . . D. Miscellaneous A r o m a t i c Antibiotics . . . . . . . . . . X I I I . Classification a n d S y s t e m a t i c Analysis of Antibiotics b y M e a n s of Paper C h r o m a t o g r a p h y . . . . . . . . . . . . . . . A. Salting-out C h r o m a t o g r a m s . . . . . . . . . . . . B. p H C h r o m a t o g r a m s . . . . . . . . . . . . . . . C. S u m m a r i z e d C h r o m a t o g r a m s ( C h r o m a t o g r a p h i c Spectra) . . . .
101 161 162 162 162 164 168
I. Introduction
Paper chromatography, introduced in this field in 1946 by Goodall and Levi I still remains one of the principal tools used for separation, characterization, and identification of antibiotics. About two thousand antibiotics hitherto known belong to very different groups of organic compounds. It is therefore ahnost impossible to develop universal solvent systems for their paper chromatographic studies. It is also difficult to use universal chemical detection methods such as can be done for other groups of chemically related natural or synthetic compounds, e.g., sugars, amino acids, steroids, sulfonamides. On the other hand, the biological activity of antibiotics can be exploited for their detection on paper chromatograms by means of bioautography. Paper chromatography has become an indispensable tool in the study of antibiotics especially for the following purposes: (1) characterization and identification in the search for new antibiotics; (2) separation of mixtures of antibiotics; (3) quantitative analysis; (4) studies of biogenesis and biosynthesis; (5) selection of producing strains; (6) development of isolation procedures for unknown substances; (7) control of isolation and purification; (8) preparative chromatography; (9) control of pharmaceutical preparations; (10) control of feed additives; (11) structural studies; (12) studies of enzymic degradation or transformations of antibiotics; (13) studies of movement of antibiotics in animals and plants; (14) production of antibiotics as one criterion in systematics and taxonomy of microorganisms; (15) systematic analysis. In this chapter, principles of paper chromatography of antibiotics are described. They include all steps of analysis beginning with the preparation of samples and papers, development techniques, detection methods, and means of documentation. Paper chromatography of the best known groups of antibiotics is given in separate sections. Finally, a section is devoted to paper chromatographic classification and systematic analysis of antibiotics. General theoretical aspects of paper chromatography are not dealt with except for those that directly emerged from studies of antibiotics. R. R. Goodall and A. A. Levi, Nature (London) 158, 675 (1946).
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METHODS FOR THE STUDY OF ANTIBIOTICS
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For the theory of paper chromatography, specialized monographs should be consulted. ~-~ I t was impossible to pack all available data on paper chromatography of antibiotics into such a short chapter, and a selection of the most important applications was necessary. Of the older reviews concerning various aspects of paper chromatography of antibiotics, that by the author of the present chapter could be helpful. ~ More recently, two books on paper chromatography were published. One of them, by Blinov and Khokhlov, 7 is in Russian; the other, in English, is by Wagman and Weinstein. 8 The latter was characterized by its authors as an "antibioticist's vade mecum" and, besides paper chromatography, includes applications of thin-layer and gas-liquid chromatography together with electrophoresis and countercurrent distribution. A chapter dealing with paper and thin-layer chromatography of antibiotics is included in a recent book on pharmaceutical applications of paper and thin-layer chromatography. 9 II. G e n e r a l T e c h n i q u e s
Preparation of Samples Samples of antibiotics may be of varioqs purity, and sometime special procedures are needed before their application to chromatograms. Pure substances are dissolved in appropriate solvents. From laboratory or industrial fermentations, filtrates of the cultivation media containing antibiotics can be used directly. In some cases, antibiotics are present in the biomass of the producing microorganisms and must be extracted with an organic solvent (methanol, acetone, chloroform, etc.) and concentrated. In screening programs it is often desirable to have crude eoncenJ. W. Copius-Peereboom, "Comprehensive Analytical Chemistry," Vol. IIC, Chapter 1. Elsevier, Amsterdam, t971. s G. J. Giddings, "Dynamics of Chromatography, Part I: Principles and Theory". Dekker, New York, 1965. I. M. I-Iais and K. Macek, Eds., "Paper Chromatography," 3rd ed. Academic Press, New York, 1964. E. Heftmann, Ed., "Chromatography," 2nd ed. Van Nostrand-Reinhold, Princeton, New Jersey, 1967. 6V. Betina, in "Chromatographic Reviews" (M. Lederer, ed.), Vol. 7, p. 121. Elsevier, Amsterdam, 1965. 7 N. O. Blinov and A. S. Khokhlov, "Paper Chromatography of Antibiotics" (in Russion). Izd. Nauka, Moscow, 1970. 8G. H. Wagman and M. J. Weinstein, "Chromatography of Antibiotics." Elsevier, Amsterdam, 1973. V. Betina, in "Pharmaceutical Applications of Thin-Layer and Paper Chromatography" (K. Macek, ed.), p. 503. Elsevier, Amsterdam, .1972.
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trates of antibiotics for chromatographic characterization. Crude concentrates of antibiotics produced by fungi are prepared as follows.TM 1. To 50 ml of a filtrate obtained from a cultivation of a producing strain, 50 ml of acetone are added for precipitation of proteins and other substances that might interfere in chromatographic analysis of the antibiotics in the filtrate. The mixture is warmed to 50 ° for 10 min and then cooled to room temperature. After precipitation and filtration, the filtrate is evaporated in vacuo to dryness. The residue is dissolved in 5 ml of 80% aq. acetone, filtered, and used for chromatography. Such concentrates may be stored in a refrigerator. 2. The corresponding mycelium is extracted twice with ethyl acetate in a mixer, the combined extracts are filtered and evaporated in vacuo to dryness. The residue is dissolved in 80% aq. acetone and used for chromatography of antibiotics from the mycelium. Solutions of antibiotics are applied to chromatograms in the form of spots or a streak (1.5-3 cm long).
Form and Development o] Chromatograms Chromatograms may be prepared in three general forms: as sheets, as strips, or as circular papers. Unless special requirements are stated, standard papers are used, for example, Whatman No. 1 or Schleicher and Schuell 2043b. For bioautographic detection, narrow strips of chromatographic papers are recommended. In our laboratory, standard-strips 1 X 35 cm are used, and ascending development is carried out in narrow glass cylinders. The lower end of the chromatogram is immersed to a depth of 1 cm into the solvent system in the cylinder, and the upper end of the strip is fixed with a glass stopper. Besides the ascending development, descending, horizontal, or radial development of chromatograms is used. In descending, ascending, or horizontal development on paper sheets one-dimensional or two-dimensional development may be applied. Electrophoresis in one direction and chromatographic development in the other (electrochromatography) can also be combined,
Solvent Systems As mentioned above, the main difficulty in studies of antibiotics by paper chromatography lies in their chemical diversity. This means that specific solvent systems and detection methods must be worked out for different chemical groups. In general, systems with an aqueous stationary phase and with a stationary hydrophilic organic solvent, respectively, can be used for antibiotics. In the former type of system, water is held 1oZ. Bar~th, V. Betina, and P. Nemec, J. Antibiot. 17, 144 (1964).
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METHODS FOR THE STUDY OF ANTIBIOTICS
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stationary on the paper, and the solvent, which is usually immiscible with water, passes through. In the latter type, paper is impregnated with a polar nonvolatile organic solvent and another organic solvent, immiscible with the stationary phase, is used as the mobile phase. The Zaffaroni's systems belonging to this category are given in the section on nonpolyene glycosidic macrolide antibiotics. Of the nine basic systems recommended by Macek, 11 the six listed below could be tested in paper chromatography of antibiotics which were not yet analyzed chromatographically. Systems for hydrophilic substances: A: Isopropanol-ammonia-water (9 : 1 : 2) B: n-Butanol-acetic acid-water (4: 1:5), the upper phase being used for development and both phases being used to saturate the atmosphere in the tank Systems for slightly hydrophilic substances: C: Formamide/chloroform. In formamide systems, usually a 40% ethanolic formamide solution is used for impregnation of the paper. The formamide may contain 5% ammonium formate or 0.5% phosphoric acid. Chloroform is used as mobile phase which does not need to be saturated with formamide. D: Formamide/benzene-chloroform. The proportion of the two components in the mobile phase is selected from 1 : 9 to 9:1 in accordance with the character of antibiotics to be analyzed. E: Formamide/benzene F: Formamide/benzene-cyclohexane in proportions from 9:1 to 1:9 Detection Methods
Usually, the chromatogram is dried prior to detection in order to remove the residues of the solvent systems from the paper. T h i s is very important when bioautography is used as the detection method, since some solvents m a y be harmful to test microorganisms. For colorless antibiotics, physical, chemical, radiochemical, or bioautographic detections are used to locate their positions on chromatograms. T h e y are reported directly in the sections dealing with individual antibiotics and their groups. Bioautography is described in a separate section. The position of the chromatographed substance is usually expressed as the relative Rs value, i.e., the ratio of the distance of the center of the spot A from the start, and the distance of the solvent front, F, from the start: R~ = A / F . The so-called hRs values (hRr = R~ X 100) are often used for greater clarity, particularly in tables. In certain cases, mainly when the R~ values cannot be calculated, relative R x values can be estimated. R x value is the distance A of a given spot from the start divided by the distance B of substance X from the start: R x = A / B . 1~K. Macek, in "Pharmaceutical Applications of Thin-Layer and Paper Chromatography" (K. Macek, ed.), p. 16. Elsevier, Amsterdam, 1972.
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PAPER CHROMATOGRAPHY OF ANTIBIOTICS
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Finally, R~ values can be calculated according to Eq. (1). RM
=
log[(1/Rs) -- 1]
(1)
m . Bioautography The antimierobial activity of different antibiotics varies; they may have antiviral, antibacterial, antifungal, antiprotozoal, or antialgal properties. In addition, some antibiotics possess cytotoxic, antineoplastic, and other biological effects. Bioautography is based on the biological activities of antibiotics. The possibilities of the bioautographic detection of antibiotics possessing various biological activities are summarized in Table I. In a typical procedure of bioautographic detection of antibiotics on paper chromatograms, chromatographic sheets or strips are placed on the surface of large nutrient agar plates inoculated with microorganisms that are sensitive to the antibiotics being analyzed. After about 15-30 rain the sheets are removed, while the narrow strips may be left on the seeded surface. In both instances antibiotics diffuse from their positions on the chromatograms into the agar layer and inhibit the growth of tile test organisms. The plates are then incubated at an appropriate temperature until a thin film of the growing microorganisms is visible on the agar surface. Clear zones of inhibition are seen in the areas where antibiotics are present. With bacteria and some fungi the incubation period is about 16-24 hr. TABLE I POSSIBILITIES OF BIOAUTOGRAPHY OF ANTIBIOTICS IN PAPER CHROMATOGRAPHYa
Biological activity Antibacterial Antifungal Antiprotozoal Antialgal Antiphage Phage-inducing Antiviral Cytotoxic
Test systems
Effects
Bacteriophages plus host bacteria Mixtures of lysogenic plus indicator bacteria Virus-infected animal cells Monolayer animal cell cultures
Growth inhibition b Growth inhibition b Growth inhibition b Growth inhibition h Absence of plaques Halo of lysis on indicator bacteria Absence of cytopathic action Dead cells
Bacteria Fungi Protozoa
Algae
" Adapted from V. Betina, J. Chromatogr. 7@, 41 (1973). Manifested by zones of inhibition on agar plates and by inhibition of growth in test tubes with liquid media, respectively.
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METHODS FOR THE STUDY OF ANTIBIOTICS
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Bioautography is carried out by similar techniques in most laboratories. Wagman and Weinstein8 recommend Pyrex baking dishes approximately 8.5 inches wide by 13.5 inches long by 1.75 inches deep (21.6 X 34.3 X 4.4 cm) with stainless steel covers. In our laboratory, flat dishes are prepared of glass plates 40 X 27 cm with removable aluminum frames 37 X 24 cm and 2 cm high. The frames are sterilized separately, placed on the plates previously adjusted to horizontal position, and adhered to them by pipetting a part of melted agarized medium for the bottom layer (about 65 °) around their inner edges. Typically, a 300-ml portion of a base medium and a 100-ml portion of seed medium are used. The seed medium is poured on top of the solidified base (bottom) layer. When air bubbles occasionally form on the surface of the agar, a Bunsen burner flame can be passed rapidly over the agar to break them. The plates are then allowed to harden. Metallic or glass covers with filter paper on their inner sides are used to cover the plates during incubation. The filter paper absorbs vapors and prevents their condensation on the cover. A large variety of microorganisms or animal cell cultures may be used to detect various antibiotics under test. A number of useful media for such tests can be found in special books. 1~,13 Selected procedures given below include bioautographic detections of antibacterial, antifungal, phage-inducing, cytotoxic, and antiviral antibiotics. Bioautography o] Antibacterial Antibiotics
When commercial media for bioautography are not available, the following cultivation medium can be usedg: peptone, 10 g; meat extract, 1000 ml; NaC1, 3 g; Na~HP04, 2 g, pH 7.2; agar, 20 g for base layer, and 10 g for seed layer, respectively. The medium is autoclaved at 120° for 20 min. The base medium is cooled to about 65 ° and poured onto an appropriate framed glass plate. The seed medium is cooled to about 45 °, seeded with a suspension of the test organism, mixed well (avoid formation of air bubbles), and poured onto the solidified base layer. Typically, to 100 ml of a seed medium is added 1.0 ml of the working inoculum. Usdin et al. 14 added into the agar medium 2,3,5-triphenyltetrazolium chloride (TTC) which is reduced to a formazan of a red coloration. Zones of inhibition are then better visible on a red background. Other tetrazolium salts may be used. TTC and also 2,6-dichlorophenolindophenol ~2F. Kavanagh, Ed., "Analytical Microbiology." Academic Press, New York, 1963. 13D. C. Grove and W. A. Randall, "Assay Methods of Antibiotics. A Laboratory Manual." Med. Encycl., New York, 1955. 14E. Usdin, G. D. Shockman, and G. Toennies, Appl. Microbiol. 2, 29 (1954)
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are recommended for shortening the incubation time from 16 to 5 hr and for a better visualization of the zones of inhibition as follows. 1'~ The seeded plates with the chromatographic strips are incubated for 4 hr and then sprayed with an aqueous 0.05% solution of T T C and 0.5% solution of 2,6-dichlorophenolindophenol, respectively. After an additional incubation for half an hour, inhibition zones begin to be visible. With TTC, noncolored zones appear on a red background; and with the other redox indicator, blue zones appear on a bleached background. For documentation purposes, test agar plates with inhibition zones on the chromatographic strips can be photographed in polarized light which gives the zones more contrast. 1~ Agar plates can also be photographed by a direct contact with photographic papers. 1~ Color reprints on filter paper can be prepared from the test plates colored by special techniques. TM The direct bioautography of antibacterial substances on paper chromatograms has been described) 9 The developed chromatographic strips are dried in the air, carefully immersed into a soluble agar medium seeded with sensitive bacteria, and then incubated in a moist atmosphere at a suitable temperature. The growing bacterial culture on the strips are then colored using an available bacteriological staining procedure. This technique was shown to be more sensitive than the bioautographic detection on agar plates.
Bioautography o] Anti]ungal Antibiotics With yeasts as test organisms, Sabouraud's glucose medium (glucose, 40 g; peptone, 10 g; distilled water, 1000 ml; pH 5.7) is recommended2 Add 1.5% of agar to base medium and 1.0% of agar to seed medium. The media are autoclaved and handled as are those for bioautography of antibacterial antibiotics. The incubation temperature is 28% Antibiotics active on filamentous fungi can be detected by similar procedures when the seed agar is inoculated with conidia of a sensitive organism, and the plates are incubated at its optimal temperature. Direct bioautography on paper chromatograms is also possible by applying the following procedure, originally used for detecting synthetic fungicides. 2°,2' Developed chromatograms on Schleicher and Sehuell ~5V. Betina and L. Pil~tov£, Oesk. Mikrobiol. (Prague) 3, 202 (1958). ~6N. A. Drake, J. Amer. Chem. Soc. 72, 3803 (1950). 1~Anonymous, Chem. Eng. News 32, 3940 (1954). 1~j. Stephens and A. Grainger, J. Pharm. Pharmacol. 7, 702 (1955). 1, G. Csob£n and G. Szab6, Kiserl. Orvostud. 4, 387 (1952). 2o H. C. Weltzien, Naturwissenscha]ten 45, 288 (1958). .-1 H. M. Dekhuizen, Meded. Landbouwhogesch. Opzoekingsstat. Slaat Gent ~6, 1542 (1961).
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METHODS FOR THE STUDY OF ANTIBIOTICS
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2043b or Whatman No. 3 M N paper are dried in air and sprayed with a conidial suspension of Stemphylium consortiale or Glomerella cingulata in a medium prepared as follows: sucrose, 50 g; NAN03, 5 g; KH2P04 1.25 g; MgS04.7 H20, 1.25 g; distilled water, 500 ml; pH 4.6; 2-2.5 X 105 conidia are used per milliliter of the medium. The chromatograms are then incubated on glass plates in a moist atmosphere for 2-3 days at 25-27% Inhibitory zones indicate the positions of the fungistatic substances tested.
Bioautography o] Phage-Inducing Antibiotics A procedure for the detection of substances inducing X bacteriophage of Escherichia coli K12 was described by Heinemann et al. 22 The top agar layer is inoculated with a suspension of the latter lysogenic bacterium along with a nonlysogenic culture of E. coli C600. After incubation, a halo of lysis on the colonies of E. coli C600 indicates the position of the inducers studied on paper chromatograms.
Bioautography for Cytotoxic Antibiotics 23 Earle's L cells and Eagle's KB cells are grown in suspension culture using Waymouth's medium MB 752/1 supplemented with 10% (v/v) calf serum; 0.3 g/liter of 4000 cps methyl cellulose (Methocel, Dow Chemical Co.) ; 1 g/liter nonionic polymer (Pluronics, Wyandotte Chemical Co.) ; and 1 g/liter anionic surfactant (Darvan No. 2, R. T. Vanderbilt Co., Inc.) in 250-ml conical flasks on a rotating shaker as described by Perlman et al. 24 The L-1210 cells are grown in this medium without shaking as the cells remain in suspension under these conditions when incubated at 37% The KB cells are grown in the shaken culture for 3 days at 37 ° and then harvested by centrifugation. The cells of each culture are then suspended in fresh medium so that the cell count is 4 X 106 cells/ml. Of this cell suspension, 50 ml are added to a flask containing 37 ml of calf serum, 1 g of glucose, and 3 g of melted agar in 120 ml of water; the resulting suspension is poured into a 3-qt Pyrex baking dish and allowed to solidify. The chromatograms are placed on the agar surface for 40 rain and then removed. The dishes are loosely covered with an alumihum cover and incubated at 37 ° for 18-20 hr. The agar surface is flooded with 0.05% solution of 2,6-dichlorophenolindophenol and allowed to stain for 5 min. After the dye has been poured off, the plates are placed in a 37 ° incubator for 40-60 rain. Under these conditions the viable cells 32B. Heinemann, A. J. Howard, and Z. J. Hollister, Appl. Microbiol. 15, 723 (1967). 23D. Perlman, W. L. Lummis, and H. J. Geiersbach, J. Pharm. Sci. 58, 633 (1969). 54D. Perlman, J. B. Semar, G. W. Krakower, and P. A. Diass, Cancer. Chemother. Rep. 51, 255 (1967).
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reduce the dye whereas the dead cells do not, so that the zones of toxicity can be located. Siminoff and H u r s k y ~ used a monolayer of human H e L a cells covered with an agar medium. After incubation with paper chromatograms on the plates, the cells were fixed and stained. According to another method -~G a suspension of tumor cells is simply brought into contact with a chromatogram, and later the color reaction with dehydrogenases is applied using 2,6-dichlorophenolindophenol in order to locate the zones of inhibition.
Bioautography o] Antiviral Antibiotics An adaptation of the Dulbecco virus-plaque technique 27 was described for this purpose as follows. 2s Paper chromatograms are sterilized by means of ethylene oxide and after drying are placed for 5 min or agar overlays of virus-infected cultures. After removal of papers, baking dishes are sealed with Saran Wrap (Dow Chemical Co.) and incubated for 4 days at 36% The cell layer is then stained with a second agar overlay containing indonitrotetrazolium chloride, and within a few hours plaques can be readily observed. When very sensitive tests are needed, the authors recommend applying the chromatograms very soon after virus infection of the cells. When a more accurate determination of the area of antiviral effects is required, but the sensitivity is not important, the application of the chromatograms can be delayed.
Special Cases o] Bioautography For microorganisms which do not grow well on agar media, chromatographic strips can be cut into short pieces; the pieces are placed into a series of test tubes containing a liquid cultivation medium inoculated with sensitive microorganisms. The absence of growth in some test tubes after an incubation period indicates the positions of antibiotics on the chromatograms. This technique was used for antileptospiraF 9 and antiprotozoaP ° antibiotics. When unknown antibiotics in samples from the cultivation media or from the mycelia of the producing microorganisms are studied, it is necessary to know the antibiotic activity of samples against different microorganisms. In such cases, representatives of all sensitive organisms are used for bioautographic detection. Very often the analyzed sample con25p. Siminoff and V. S. Hursky, Cancer Res. 20, 618 (1960). 56A. Oda and T. Yamamoto, dap. J. Exp. Med. 29, 87 (1959). 57R. Dulbecco, Proc. Nat. Acad. Sci. U.S. 38, 747 (1952). ~s E. C. Herrmann and J. P. Rosselet, Proc. Soc. Exp. Biol. Med. 104, 304 (1960). ~ P. Nemec, V. Betina, and J. Durkovsk:~, Naturwissenscha]ten 47, 235 (1960). 30j. Balan, L. Ebringer, P. Nemec, 8. Kov£~, and J. Dobias, J. Antibiot. 15, 157 (1963).
110
METHODS FOR THE STUDY OF ANTIBIOTICS
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rains several antibiotics with different Ry values and with different antimicrobial activities. 1° In the so-called correlative assays, 31 it was demonstrated, by means of paper chromatography and bioautography, that the same component in a certain filtrate from a microbial fermentation inhibited vaccinia virus in vitro and also a yeast, Saccharomyces pastorianus. Other bioautographic techniques may be found elsewhere. 6-9,32
IV. Paper Chromatography of ~-Lactam Antibiotics This group includes penicillins, cephalosporins, and cephamycins. Penicillins and cephalosporins are fungal metabolites, and cephamycins are a new family of antibiotics produced by actinomycetes. Penicillins are N-acyl derivatives of 6-aminopenicillanic acid (6-APA). Cephalosporin C family and cephamycins are derivatives of 7-aminoeephalosporanic acid (7-ACA). Cephalosporin P series, of steroid nature, is included in the section dealing with alicyclic antibiotics. Cephamycins have been known since 1971 (for references, see Stapley et al2 s) and structures of cephamycins A, B, and C have been elucidated. The methyl group in the acetyl residue of cephalosporin C is substituted by other radicals in the cephamycins.
A. Penicillins and 6-Aminopenicillanic Acid Paper chromatography of natural, biosynthetic, and semisynthetie penicillins and of 6-APA is described here. Sampling. Culture filtrates can be applied directly on chromatograms. Penicillins may also be extracted, after chilling the filtrate and adjusting to pH 2, with butyl acetate and back extracted from the organic layer with 0.5% aq. sodium bicarbonate. 34 References to procedures for penicillins in pharmaceutical preparations may be found elsewhere. 9 Solvent Systems. Of many systems described in the literature, those most used have been selected as follows. A: Ether satd. with water on paper buffered with phosphate buffer, pH 5.8 or 6.2, development at 4-5 °1'~5 B: Ethyl acetate satd. with water on paper buffered with McIlwaine's citrate phosphate buffer pH 6.0 s6 ~IL. J. Ha~ka and C. G. Smith, Antimicrob. Ag. Chemother. 1961, p. 677 (1962). s2V. Betina, J. Chromagogr. 78, 41 (1973). 'SE. O. Stapley, M. Jackson, S. Hernandez, S. B. Zimmerman, S. A. Currie, S. Mochales, J. M. Mata, H. B. Woodruff, and D. Hendlin, Antimicrob. Ag. Chemother. 2, 122 (1972). M. Cole, Appl. Microbiol. 14, 88 (1966). M. L. Karnovsky and M. J. Johnson, Anal. Chem. 21, 1125 (1949). V. Betina, Chem. Zvesti (Bratislava) 18, 209 (1964).
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PAPER CHROMATOGRAPHY OF ANTIBIOTICS
111
C: Amyl acetate satd. with water on paper buffered with procaine-citrate buffer pH 5.337 D: n-Butanol-acetic acid-water (12:3 : 5), upper layer 3s E: n-Butanol-ethanol-water (4:1:5), upper layer 38 F: n-Butanol-pyridine-water (1 : 1 : 1) 38 G: 2-Butanol-formic acid-water (75: 15 : 10) 39 H: n-Propanol-water (6: 4) 39 I: n-Propanol-ethanol-water (5: 2 : 3) 39 J : n-Butanol-n-propanol-water (25: 50: 25) 39 K: 27% KH2PO4 pH 6.0 on paper impregnated with 2% liquid paraffin in ether, air dried, and saturated with vapors of butyl acetate 4° L: Both phases of isopropy! ether-isopropanol-water (7:3:10) on paper buffered with phthalate buffer pH 4 or 541 T h e systems A, B, C, and K are convenient for natural and biosynthetic penicillins. With system B, the dependence of the R~ values of natural penicillins on p H of the stationary phase was studied, ~7 and Sshaped curves were obtained showing optimal separation at p H 6 (Fig. 1). The systems D to J and L were used for separation of 6-APA from penicillins, 39 for seminisynthetic penicillins, ~° and for degradation products of penicillins and 6-APA. 42 Detection. For bioautography of natural and biosynthetic penicillins, Bacillus subtilis, Sarcina lutea, or Staphylococcus aureus are recommended. For penicillin N and semisynthetic penicillins active on gramnegative bacteria, an avirulent strain of Salmonella t y p h i m a y be used. 6-APA can be converted to benzylpenicillin as follows. ~8 The chromatogram is sprayed with 5% aqueous N a H C 0 3 , then with 2 - 5 % phenylacetyl chloride in acetone and again with the N a H C Q solution. After drying for a short time in air the chromatographic strip is placed on a plate seeded with S. aureus or B. subtilis. Of chemical detection methods for fl-lactam compounds the following m a y be used. 1. A mixture composed of l0 m M iodine in 3 m M K I (10 ml), 1 M phosphate buffer p H 7.0 (1 ml), and 2% (w/v) sodium starch glycolate in water (9 ml) is used to spray the chromatograms. Compounds with potential sulfhydryl groups (but not the penicillins with a Closed fll a c t a m ring) appear as pale spots on a deep blue background. The paper is then sprayed with a mixture of equal volumes of the starch-iodine solution and a solution of purified penicillinase (e.g., from Bacillus cereus 1000 units/ml). Additional pale spots appear at the site of penicillins ~7H. G. Macmorine, Appl. Microbiol. 5, 386 (1957). s8M. Cole and G. N. Rolinson, Proc. Roy. Soc. Ser. B 154, 490 (1961). 39M. RShr, Microchim. Acta 4, 705 (1965). 4oT. Watanabe, S. Endo, and Y. Iida, J. Antibiot. 15, 112 (1962). 41H. Hellberg, J. Ass. O~c. Anal. Chem. 51, 552 (1968). 42p. H. A. Sneath and J. F. Collins, Biochem. J. 79, 512 (1961).
112
[71
METHODS FOR THE STUDY OF ANTIBIOTICS Rf 1.0
0.8
0.6
0.4
0.2
I
I
1
I
3
~.
5
1
I
I
I
I
6
7
8
9
10
pH
FIG. 1. Separation of natural penicillins. (A) Dependence of RI values on pH of the stationary phase. Solvent system: ethyl acetate saturated with water. Stationary phases: McIlvaine's citrate-phosphate buffers and phosphate buffers, respectively. (B) Separation of penicillins from the same sample using ethyl acetate as mobile phase and McIlvaine's buffer, pH 6.0. Traveling distance of the solvent front, 30 cm. Descending development indicated by the arrow. Bioautography with Bacillus subtilis. Modified from V. Betina, Chem. Zvesti (Bratislava) 18, 209 (1964). whose fl-lactam ring is opened by the penicillinase. Sensitivity with benzylpenicillin is 1 ttg/cm -~. Methicillin is revealed less readily owing to its relative stability to penicillinase, p-Hydroxybenzylpenicillin absorbs iodine before treatment with penicillinase owing to iodination of the phenolic side chain. The method cannot be applied to cephalosporin C . 42
[7]
P A P E R CHROMATOGRAPHY OF ANTIBIOTICS
113
TABLE II PAPER CHROMATOGRAPHIC DATA OF NATURAL AND BIOSYNTHETIC PENICILLINS
RGa in systems b
Penicillins F FH2 G K V X
A at pH 5.6¢ B at pH 6.0d 1.62 2.18 1.00 3.10 -0.17
2.42 2.92 1.00 3.26 -0.11
Rs X 100 in system K b'~ 22 -48 14 36 59
RpenicilliaG. b See solvent systems for penicillins on p. 110. c From S. Yamatodani, in "Papierchromatographie in der Botanik" (H. Linskens, ed.), p. 181. Springer, Berlin, 1955. d Estimated from drawings in V. Betina, Chem. Zvesti (Bratislava) 18, 209 (1964). e From T. Watanabe, S. Endo, and Y. Iida, J. Antibiot. 15, 112 (1962).
a RG
=
2. In a variation of the previous method, the paper is sprayed with 0.5 N N a O H and,dried for 1-15 min (in order to open the fl-lactam ring) before spraying with a reagent composed of a mixture of 1% aqueous starch, glacial acetic acid, and 0.1 N iodine in 4% K I (50:3:1). Decolorization of the iodine reagent by the hydrolyzed penicillins and cephalosporin C yields maximum contrast after 5-10 rain. 4a 3. The chromatogram is immersed into a solution of A g N Q in acetone (1 ml of satd. aqueous AgNO~ added dropwise into 100 ml of acetone, and the precipitate dissolved by adding water). After drying in air, the paper is immersed into a solution of 2.5 ml of 50% N a O H in methanol until there is maximal development of the color reaction. The chromatogram is washed briefly in water and, to decolorize the background, immersed into 6 N NH~OH and washed carefully in tap water. 39 A p p l i c a t i o n s . Mobilities of natural and biosynthetic penicillins in different solvent systems obtained independently by three authors are compared in Table II. 6-APA was separated from natural penicillins 4~ in system E. Mobilities of penicillins in the same system were~: penicillin K, FH~ ~ F, G ~ penicillin 4 ~ penicillin 3 ~ penicillin 2 ~ penicil4~R. Thomas, Nature (London) 191, 1161 (1961). " F. R. Batchelor, F. P. Doyle, J. H. C. Nayler, and G. N. Rolinson, Nature (London) 183, 257 (1959). 4~A. Ballio, E. B. Chain, and F. D. Di Accadia, Nat~re (London) 183, 180 (1959).
114
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
lin 1. RShr, 39 using 9 solvent systems, including G, H, I, and J, characterized phenoxymethyl penicillin, phenoxymethylpenicilloic acid obtained from the former by degradation with penicillinase, 6-APA, and penicilloic acid obtained from 6-APA by degradation with penicillinase. The following R~ values were observed in system F3S: methylpenicillin, 0.58; penicillin X, 0.3; 6-APA, 0.48; penicillin, 0.75. In system E, a-aminobenzylpenicillin had a lower mobility than had a-azidobenzylpenicillin. 46 Hellberg 41 compared 8 penicillins using system L. At pH 5.0, their RI values were as follows: ampicillin, 0.0; benzylpenicillin, 0.23; cloxacillin, 0.56; methicillin, 0.04 oxacillin, 0.50; phenoxymethylpenicillin, 0.33; phenoxyethylpenicillin, 0.55; and phenoxypropylpenicillin, 0.80. At pH 4.0, the separation was less successful. Cole 47 studied a series of reaction mixtures of 6-aminopenicillanic acid with carboxylic acids, amides, and N-acyl derivatives of glycine using system A at pH 6.2 and system E. The chromatograms were detected with Bacillus subtilis. Cole 4s compared several esters of benzylpenicillin and other compounds using paper chromatography in systems D, E, and F. In studying effects of exogenous penicillin V on penicillin biosynthesis, 49 penicillins and 6-APA were identified by applying samples and standards to Whatman No. 1 paper, which underwent an overnight development in system E. After phenylacetylation and drying, chromatograms were placed on agar plates inoculated with B. subtilis, then removed after 15 min contact; the plates were incubated overnight. Duplicate chromatograms were prepared in those studies in which 35S-labeling was used. The RI values of radioactive peaks were compared with those of bioactive zones. The R~ values of 6-APA, penicilloic acid, and penicillin V were 0.19, 0.27, and 0.63, respectively. Penicilloic acid and other biologically inactive penicillins were detected by spraying chromatograms with iodine 43 as described above. Paper chromatography helped to demonstrate that removal of the side chain of penicillins is a minor transformation route in man) ° The resulting 6-APA was detected in urine samples by developing chromatograms in system E and detecting bioautographically with B. subtilis after phenylacetylation (RI = 0.13). Older data concerning paper chromatography of penicillins (biosyn,6E. Hansson, L. Magni, and S. Wahlqvist, Antimicrob. Ag. Chemother. 1967, p. 568 (1968). 4, M. Cole, Biochem. J. 115, 747 (1969). 48M. Cole, Biochem. J. 115, 733 (1969). ~9E. L. Gordee and L. E. Day, Antimicrob. Ag. Chemother. 1, 315 (1972). 50M. Cole, M. D. Kenig, and V. A. Hewitt, Antimicrob. Ag. Chemother. 3, 463 (1973).
[7]
P A P E R CHROMATOGRAPHY OF ANTIBIOTICS
115
thesis by various fungi and their nmtants, commercial preparations, discovery of 6-APA, semisynthetic penicillins, degradation products, etc.) m a y be found elsewhere2 ,s Methods for quantitative analysis of penicillins and 6-APA are described below. 1. ~ A T U R A L AND BIOSYNTHETIC PENICILLINS. Karnovsky and Johnson :'~ used dry filter paper strips buffered with 20% pH 6.2 phosphate buffer. Samples containing 1-2 units of penicillins were applied. After development in system A, the positions and concentrations of the individual components were determined either by measurement of the zones of inhibition produced when the entire strips were laid on a large agar plate seeded with B. subtilis, or by cutting the strips into small uniform squares and measuring the circular inhibition zones produced by the individual squares. A convenient measure of the amount of each penicillin was given by the area under the relevant portion of a standard curve. The bestknown natural penicillins migrated with increasing mobilities as follows: G, F, FH2, and K. 2. LABELEDPENICILLINS. Using the buffer and solvent as in (1), Smith and Allison 51 described a quantitative method for the detection and evaluation of labeled penicillins. The developed chromatographic strips are left in contact with an X - r a y film for several days, and the film is then developed. Two alternative procedures can be then used. (1) Using the radioautograph as a guide, the corresponding strip is cut into sections each containing the whole of one penicillin species. Each section is then extracted by boiling for a few minutes with a very dilute phosphate buffer. An aliquot of each extract is evaporated down on a planchette, and the radioactivity is measured. From the total counts for each penicillin species, the proportions can be readily calculated. (2) Alternatively, again using the radioautographs as a guide, the corresponding paper strips are cut into squares, or, if necessary, rectangles, in such a way as to have each penicillin on a separate square. The squares are then fitted into planchettes and measured radiometrically. 3. 6-AMINOPENICILLANICACID. 6-APA can be estimated quantitatively according to Erickson and Bennett. '~ Penicillins are separated from 6-APA by development, in system E, of chromatograms, which are then air dried and dipped in a solution of 4% phenylacetyl chloride in methyl isobutyl ketone to convert 6-APA to benzylpenicillin. The chromatograms are dried under a hood and then bioautographed with S. lutea. Levels of 6-APA in the samples are determined by plotting zone areas against a standard curve established by treating known amounts of 6-APA in Smith and D. Allison, Analyst 77, 29 (1952). ~2R. C. Erickson and R. E. Bennett, Appl. Microbiol. 13, 738 (1965). 5~ E. L.
116
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
t h e s a m e m a n n e r as t h e samples. T h e e v i d e n c e for t h e presence of 6 - A P A in a r e a c t i o n m i x t u r e is b a s e d on c o m p a r i s o n s w i t h a u t h e n t i c 6 - A P A in r e s p e c t to b o t h c h r o m a t o g r a p h i c m o b i l i t y a n d a n t i b a c t e r i a l p r o p e r t i e s , b o t h before a n d a f t e r p h e n y l a c e t y l a t i o n a n d i n a c t i v a t i o n b y penicillinase.
B. C e p h a l o s p o r i n C F a m i l y , 7 - A C A , a n d C e p h a m y c i n s S o l v e n t S y s t e m s . Selected s o l v e n t s y s t e m s for these c o m p o u n d s are c o m p i l e d below. M o s t of t h e m are used for t h e c e p h a l o s p o r i n C f a m i l y . S y s t e m s K a n d L s e p a r a t e c e p h a l o s p o r i n C, p e n i c i l l i n N, a n d c e p h a l o s p o r i n P. S y s t e m s A a n d S are h e l p f u l in s e p a r a t i n g 7 - A C A a n d its d e r i v atives. S y s t e m s O, P, a n d R are r e c o m m e n d e d for c e p h a m y c i n s . S o l v e n t s y s t e m s for c e p h a l o s p o r i n s , 7 - A C A a n d c e p h a m y c i n s :
A: B: C: D: E: F: G: H: I: J: K: L: M: N: O: P:
n-Butanol-acetic acid-water (4:1 : 5), upper layer 5~ As A, but (5:1:4) 54 As A, but (4:1:2)55 As A, but (3:1:1) 58 As A, but (4:1:4) 57 n-Butanol-ethanol-water (4:1 : 5) 58 n-Butanol satd. with 5 N ammonia ~9 2-Butanol satd. with water 59 2-Butanone satd. with water 59 Methyl ethyl ketone satd. with water e° Methanol-n-propanol-water (6: 2: 1) on paper buffered with 0.75 M phosphate pH 4.0 °1 n-Propanol-0.013 M Na citrate pH 5.5 (7:3) 6~ n-Propanol-water (7:3) 6~ Isopropanol-pyridine-water (65: 5: 30) 63 n-Propanol-pyridine-acetic acid-water (15: 10: 3: 12) 6a n-Propanol-pyridine-acetic acid-CHsCN-water (45: 30: 9: 40: 3 6) 64
~3B. Loder, G. G. F. Newton, and E. P. Abraham, Biochem. J. 79, 377 (1961). 54K. Kariyone, H. Harada, M. Kurita, and T. Takano, J. Antibiot. 23, 131 (1970). 6~j. Kozatani, M. Okui, T. Matsubara, and N. Nishida, J. Antibiot. 25, 86 (1972). 56M. M. ttoehn and C. T. Pugh, Appl. Microbiol. 16, 1132 (1968). 57j. D' A. Jeffery, E. P. Abraham, and G. G. F. Newton, Biochem. J. 81, 591 (1961). ~B. Loder, G. G. F. Newton, and E. P. Abraham, Biochem. J. 79, 408 (1961). 59H. R. Sullivan and R. E. McMahon, Biochem. J. 102, 976 (1967). R. P. Miller, Antibiol. Chemother. 13, 689 (1962). ~ J. L. Ott, C. W. Godzeski, D. Pavey, J. D. Farran, and D. R. ttorton, Appl. Microbiol. 10, 515 (1962). 52A. L. Demain, R. B. Walton, J. F. Newkirk, and I. M. Miller, Nature (London) 199, 909 (1963). ~D. R. Brannon, D. S. Kukuda, J. A. Mabe, F. M. Huber, and J. G. Whitney, Antimicrob. Ag. Chemother. 1, 237 (1972). J. G. Whitney, D. R. Brannon, J. A. Mabe, and K. J. Wicker, Antimicrob. Ag. Chemother. 1, 247 (1972).
[~]
PAPER CHROMATOGRAPHY OF~ ANTIBIOTICS
117
R: I s o p r o p a n o l - w a t e r (70:30), descending development approximately 3.5 hr on 3 M M paper~6 S: E t h y l acetate satd. with aq. N a acetate buffer (0.1 M with respect to N a *) p H 5.2 on paper buffered with same buffer 53
Detections. Bioautography with Bacillus subtilis ATCC 6633 was used for cefazolin, cephalotin, deacetyl cephalotin, cephalosporin C and its deacetyl, lactone, and side-chain derivatives. Sarcina lutea PCI 1001 was used for cephaloglycin and deacetyl cephaloglycin; Staphylococcus aureus (Oxford strain NCTC 6571) for cephalosporin C,. cephalosporin Co, deacetyl cephalosporin C, cephalosporin CA, 7-ACA, and related compounds converted to N-phenylacetyl derivatives; Salmonella typhi for cephalosporin C and its derivatives; Pseudomonas solanaceum Lilly X185 for A16886A, A16886B, cephalosporin C, and deacetyl cephalosporin C; Vibrio percolans MB-1272 for cephamycins A, B, and C. 7-ACA and related compounds must be converted to N-phenylacetyl derivatives by spraying dried chromatograms with pyridine in 50% acetone (v/v) until barely damp. The ehromatograms are then lightly spayed with 2% (w/v) phenylacetyl chloride in acetone, and again with the pyridine solution until a spot of bromocresol green placed on the paper immediately turns blue (pH 5.0). The paper is dried in air for 3-5 min and bioautographed with S. aureus2 G Detection with ninhydrin results in purple spots of cephalosporin CA derivatives. Cephalosporin C is detectable in UV light at 230-400 nm giving dark light-absorbing spots, r'6 Applications. Paper chromatography was one of the important tools in separation and identification of cephalosporins produced by various fungi (for references, see Betina6). It was also used in studies of enzymic degradations of cephalosporins 59,~2,6~,~ A16886A and A16886B, fllactam antibiotics from Streptomyces clavuliger were separated with system p.~4,~5 Cephalosporin C, deacetyl cephalosporin C, and A16886A were separated from each other using system O. 63 Cephamycins were characterized by their mobilities in system R 33 and A16886B also in system p.63 Paper chromatographic data of cephalosporins, their derivatives, and cephamycins are given in Table III. A quantitative paper chromatographic analysis of cephalosporin C and related compounds was described by Miller2 ° An antibiotic prepara6~D. R. Brannon, J. A. Mabe, R. Ellis, J. G. Whitney, and R. Nagarajan. Antimicrob. Ag. Chemother. 1, 242 (1972). C. W. Hale, G. G. F. Newton, and E. P. Abraham, Biochem. J. 79, 403 (1961).
~' C. O'Callaghan and P. W. Muggleton, Biochem. J. 89, 304 (1963). N. Nishida, Y. Yokota, M. Okui, Y. Mine, and T. Matsubara, J. Antibiot. 21, 165 (1968).
118
METHODS FOR THE STVDV OF ANTIBIOTICS
[7]
TABLE III PAPER CHROMATOGRAPHIC DATA Of CEPHALOSPORINS AND CEPHAMYCINS
Rs X 100 in systems a Compounds 7-Aminocephalosporanic acid Cefazolin Cephalosporin C Cephalosporin CA (pyridine) Cephalosporin CA (pyridine) nucleus Cephalosporin Cc Cephalosporin Cc nucleus Cephamycin A Cephamycin B Cephamycin C Deacetyl cephalosporin C N-Phenylacetyl-7-ACAt N-Phenylacetylcephalosporin C N-Phenylacetylcephalosporin Cc
Ab
Cc
14 . -75 04 -O0 . 07 . 09 -26 . . . . . . . . . . --40 . 13 . 23 .
E~
Md
.
. -77 . . 98 .
. -78 . .
. . 85
.
. . . . 57
. . .
. . .
60 . . .
Re
-56
-64 73 44 --
a See solvent systems for cephalosporins, etc., on p. 116. b From B. Loder, G. G. F. Newton, and E. P. Abraham, Biochem. J. 79, 408 (1961). c From J. Kozatani, M. Okui, T. Matsubara, and N. Nishida, J. Antibiot. 25, 86 (1972). From J. D' A. Jeffery, E. P. Abraham, and G. G. F. Newton, Biochem. J. 81,591 (1961). e From E. O. Stapley, M. Jackson, S. Hernandez, S. B. Zimmerman, S. A. Curie, S. Mochales, J. M. Mata, H. B. Woodruff, and D. Hendlin, Antimicrob. Ag. Chemother. 2, 122 (1972). 1 7-ACA = 7-aminocephalosporanic acid. t i o n is dissolved in w a t e r or a m y l acetate, a n d a v o l u m e e s t i m a t e d to c o n t a i n a p p r o x i m a t e l y 0.2 ~g (0.5 ~g for the d e a c t y l a t e d c o m p o u n d s ) is a p p l i e d to each strip of W h a t m a n No. 1 paper. T h e strips are developed in s y s t e m J for 3 hr a t 23 ° a n d detected with B. subtilus. F o r the p a r e n t compounds, the i n c r e m e n t s are 0.05, 0.10, a n d 0.20 ~g. T h e m a x i m u m w i d t h of the zones of i n h i b i t i o n are measured. T h e average m a x i m u m d i a m e t e r s for each of the three s t a n d a r d samples are p l o t t e d a g a i n s t the a m o u n t of a n t i b i o t i c on s e m i l o g a r i t h m i c paper. K n o w i n g the r e l a t i v e specific activities for the v a r i o u s compounds, it is possible to d e t e r m i n e the a m o u n t of p a r e n t c o m p o u n d (cephalosporin C ) , d e a c e t y l c o m p o u n d , a n d l a c t o n e s i m u l t a n e o u s l y in a n u n k n o w n s a m p l e from the one s t a n d a r d curve for the p a r e n t compound. R e a c t i o n m i x t u r e s c o n t a i n i n g 7 - A C A a n d c e p h a l o s p o r i n C are a n a l y z e d for 7-ACA in the same m a n n e r as described a b o v e for 6 - A P A after p h e n y l a c e t y l a t i o n . 52
[7]
PAPER CHROMATOGRAPHY OF ANTIBIOTICS
ll9
V. Carbohydrate Antibiotics I n a c c o r d a n c e with B ~ r d y ' s classification of a n t i b i o t i c s p 9 this group includes a n t i m i c r o b i a l agents c o n t a i n i n g sugar as the sole basic constit u e n t , as well as those where the s u g a r m o i e t y c o n s t i t u t e s the m a i n skelet o n of the molecule. T h e r e f o r e p a p e r c h r o m a t o g r a p h y of aminoglycosides, other glycosides (e.g., s t r e p t o t h r i c i n s , v a n c o m y c i n ) a n d v a r i o u s sugar d e r i v a t i v e s (e.g., l i n c o m y c i n ) is described here.
A. A m i n o g l y c o s i d i c A n t i b i o t i c s Solvent Systems A: B: C: D: E: F: G:
n-Butanol with 2% (w/v) p-toluenesulfonic acid TM 5 % Ammonium chloride in water TM n-Butanol-acetic acid-water (2: l: 1) 71 2 % p-toluenesulfonic acid monohydrate in n-butanol satd. with water 72 n-Butanol-methanol-water-p-toluenesulfonicacid (10 ml:40 ml : 20 ml:l g)7~ Methanol-3% NaC1 in water (2: 1) TM 80% aq. methanol-piperidine (100: 10.5) adjusted to pH 9.0-9.5 with acetic acid75 H: Methanol-water-glacial acetic acid (80:15:5) TM I: 50% Acetone, samples applied in 5% NaC1TM J: n-Butanol-water-piperidine (84:16:2) TM K: Methylethyl ketone-t-butanol-methanol-6.5 N ammonia (l 6: 3:1 : 6)77 L: Methanol-ethanol-conc. HCl-water (50: 25: 6: 19) TM M: n-Propanol-glacial acetic acid-water (9: l: 10) 79 N : n-Butanol-pyridine-water (6: 4 : 3) 80 O: Methanol-water (4: l) plus 3% NaC1 on paper buffered with 0.95 M Na2S()4 plus 0.5 M NariS0481 P: n-Propanol-pyridine-acetic acid-water ( 15: 10: 3 : 12) 8, Q: n-Propanol-water-acetic acid (50: 40:4)81 R: 80% aq. phenol 8~ 0~j. B~rdy, In]orm. Bull. Int. Cent. In]orm. Antibiot. 10, 1 (1973). 7oA. C. Sinclair and A. F. Winfield, Antimicrob. Ag. Chemother. 1961, p. 503 (1962). ~l D. J. Mason, A. Dietz, and R. M. Smith, Antibiot. Chemother. I1, 118 (1961). ~ J. N. Pereira, J. Biochem. Microbiol. Tech. Eng. 3, 79 (1961). ~'~N. Ishida, J. Miyazaki, S. Okamoto, and K. Omachi, J. Antibiol. 6, 1 (1953). 74A. Saito and C. P. Schaffner, Proc. Int. Congr. Biochem. 3rd, 1955, p. 98 (1955). 75T. Miyaki, H. Tsukiura, M. Wakae, and H. Kawaguchi, J. Antibiot. 15, 15 (1962). ~ M. K. Majumdar and S. K. Majumdar, Anal. Chem. 39, 215 (1967). ~TM. K. Majumdar and S. K. Majumdar, Appl. Microbiol. 17, 763 (1969). 74H. Umezawa, S. Takasawa, M Okanishi, and R. Utahara, J. Antibiot. 21, 81 (1968). n K. L. Rinehart, Jr., A. D. Argoudelis, W. A. Goss, A. Sohler, and C. P. Schaffner, J. Amer. Chem. Soc. 82, 3938 (1960). 8oW. T. Shier, K. L. Rinehardt, Jr., and D. Gottlieb, Biochemistry 63, 198 (1969). ~' M. J. Weinstein, G. M. Luedemann, E. M. Oden, and G. H. Wagman, Antimicrob. Ag. Chemother. 1963, 1 (1964).
120
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
S: Chloroform-methanol-17 % ammonia (2 : 1 : 1, lower layer) in atmosphere satd. with vapors of the top layer 82 T: n-Butanol-pyridine-water-acetic acid (6:4:3:1) 83 U: Methanol-water-acetic acid-25% aq. NaC1 (600:212:75:20.8) on paper buffered at pH 384 V: Methanol-5% aq. NaC1 (2:1) on paper buffered at pH 384 W: Ethanol-water-acetic acid-25 % sq. NaC1 (250: 500 : 38: 7.5) s4 Systems A and B are used for actinospectacin, C - F for streptomycin (plus system L), kanamycins, and neomycins. Bluensomyein (glebomycin) gives good results in G-I. Systems J, K, and M are also used for neomycins, N for hybrimycin. Gentamicins are analyzed in systems 0 to S, the latter together with T are used for lividomycin. Butirosins m a y be characterized in T to W. Detections. Staphylococcus aureus, Bacillus subtilis, and Escherichia coli are mostly used for bioautography. Chemical detections given below are also recommended. 1. Streptomycin and other antibiotics containing guanidine groups (primycin, viomycin) are detected according to Szilhgyi and Szab6 s5 as follows. Pull chromatograms through a cold 0.01% a-naphthol solution in methanol containing 5% NaOH. D r y in air for 2--3 min. Pull through a cold 0.5% N-bromosuccinimide solution in 10% ethanol; pull through 40% urea solution for stabilization of the color and then dry in air. 2. Ninhydrin reagent for neomycins. ~7 Spray air-dried chromatograms with a ninhydrin solution (0.25 g of ninhydrin in 10 ml of methanol plus 47 ml of butanol plus 3 ml of water plus 50 ml of pyridine) heat at 80-90 ° for 30 min. 3. Starch-iodine reagent for N-acetylneomycins. 7G Dissolve 1 g of starch and 0.25 g of potassium iodide by heating in 3.5 ml of water and then add quickly 0.5 ml of this solution to 50 ml of pyridine. Use freshly prepared reagent. After transferring the sprayed chromatogram to a water-saturated atmosphere, bluish pink spots are obtained. 4. Ninhydrin reagent for gentamicins. 8: Spray with 0.25% ninhydrin in pyridine-acetone (1:1) and heat at 105 ° for several minutes. Purple or blue spots appear against a white background. Application~. Paper chromatographic data of streptamine derivatives are given in Table IV, and relative mobilities of streptomycins in Table 82G. M. Wagman, J. A. Marquez, and M. J. Weinstein, J. Chromatogr. 34, 210 (1968). S. Umezawa, I. Watanabe, T. Tsuchiya, H. Umezawa, and H. Hamada, J. Antibiot. 25, 617 (1972). H. W. Dion, P. W. K. Woo, N. E. Willmer, D. L. Kern, J. Onaga, and S. A. Fusari, Antimicrob. Ag. Chemother 2, 84 (1972). 8~I. Szilhgyi and I. SzabS, Arzneim.-Forsch. 8, 333 (1955).
[7]
PAPER
CHROMATOGRAPHY
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OF ANTIBIOTICS
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M E T H O D S F O R T H E S T U D Y OF A N T I B I O T I C S
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TABLE V RSTR a VALUES FOR STREPTOMYCINS
RSTR in systems b Antibiotics Streptomycin Dihydrostreptomycin Hydroxystreptomycin Mannosidostreptomycin Mannosidodihydrostreptomycin
D° 1.00 0.62 0.64 0.37 --
Ed 1.00 0.70 -0.40 0.25
a RSTR = Rstreptomycin-
bSee solvent systems for aminoglycosides on p. 119. c From J. N. Pereira, J. Biochem. Microbiol. Technol. Eng. 3, 79 (1961). d From K. Kavanagh, E. Grinnan, E. Allanson, and D. Tunin, Appl. Microbiol. 8. 160 (1960).
V. RI values of gentamicin, kanamycins, neomycins, and their derivatives and of other 2-deoxystreptamine containing antibiotics are compiled in Table VI. Relative mobilities of neomycins and their N-acetyl derivatives m a y be found in Table VII. Relative mobilities of neamine and paromamine as Rp~,......... ine in system T were neamine, 0.47; paromamine, 1.00. 86 Gentamicins are characterized in Table V I I I . Lividomycin is inactivated by P s e u d o m o n a s aeruginosa by the formation of a phosphorylated product. The active and inactive lividomycin in system S had the R~ values 0.77 and 0.82, respectively, s7 I n system T, R l i v i d o m y c i n B of 5"-deoxylividomycin B sa was reported to be 1.1 and Rbutirosin B of 3',4',-dideoxybutirosin B s8 was 1.73. Butirosin complex was differentiated from most aminoglycosidic antibiotics in systems U, V, and W by Rj values of 0.21, 0.5, and 0.71, respectively, s4 Quantitative analysis of neomycins as free bases in samples from fermentations have been described. 77 Paper chromatographic methods for quantitative analysis of gentamicins are also known, s9-91 See also the Addendum. s~K. Tatsuta, E. Kitazawa, and S. Umezawa, Bull. Chem. Soc. Jap. 40, 2371 (1967). 87F. :Kobayashi, M. Yamaguchi, and S. Mitsuhashi, Antimicrob. Ag. Chemother. 1, 17 (1972). 86D. Ikeda, T. Tsuchiya, and S. Umezawa, J. Antibiot. 26, 307 (1973). soG. H. Wagman, E. M. Oden, and M. J. Weinstein, Appl. Mie~iol. 16, 624 (1968). N. Kantor and G. Selzer, J. Pharm. Sci. 57, 2170 (1968). 9, G. H. Wagman, J. V. Bailey, and M. M. Miller, J. Pharm. Sci. 57, 1319 (1968).
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PAPER CHROMATOGRAPHY OF ANTIBIOTICS
123
B. Streptothricin G r o u p S o l v e n t S y s t e m s . T h e systems for this f a m i l y of N - g l y c o s i d i c antibiotics are collected below. Systems A and B are used for separation of streptothricins, geomycin, pleocidin, mycothricins, polymycins, p h y t o baeteriomycin, r a c e m o m y c i n s , griseomycin, nourseomycins, virothricins, etc. (see references in Blinov and KhokhlovT). R a d i a l development is m o s t l y used. A: B: C: D: E: F: G: H: I:
n-Propanol-pyridine-acetic acid-water (15: 10:3 : 12) 92 n-Butanol-pyridine--acetic acid-water (15 : 10:3 : 12) 93 n-Butanol-pyridine-acetic acid-water-t-butanol ( 15: 10: 3 : 12 : 4) 94 75% aq. ethanol on paper impregnated with 0.95 M Na sulfate 95 n-Butanol satd. with water plus 2% p-toluenesulfonic acid 96 80% aq. methanol-piperidine (10: 1) adjusted to pH 9.3 with acetic acid 97 75% Phenol 9s n-Butanol-methanol-ammonium hydroxide-water (10: 4 : 3 : 3) 99 Ethanol-water (3: 1) plus 2% NaC11°°
Detection. T h e detection of antibiotics of this group is possible with S. aureus, B. s~btilis, or E. coli and, chemically, with ninhydrin. Applications. T h e following R I values of streptothricins a n a l y z e d in form of hydrochlorides (and s u l f a t e s - - v a l u e s in parentheses) in system A were obtained: ~°~ streptothricin A, 0.20 (0.17); streptothricin B, 0.14 (0.20); streptothricin C, 0.29 (0.27) ; streptothricin D, 0.35 (0.32); streptothricin E, 0.40 (0.36); streptothricin F, 0.48 (0.43). I n system C, r a c e m o m y c i n s were characterized by Rj values: 1°2 A, 0.33; B, 0.18; C, 0.24; and D, 0.11. R~ . . . . . idin ~ values obtained in system I ~°° are: lemacidin B1, 0.34; lemacidin B2, 0.61; lemacidin B:~, 1.00. P r o d u c t s of h y d r o l y sis of streptothricins, L-fl-lysine, streptolidine, N-guan-streptolidylgulosamine and its h y d r o l y z a t e , were separated in s y s t e m C. '~4 9~M. I. Horowitz and C. P. Schaffner, Anal. Chem. 30, 1616 (1958). ~ A. S. Khokhlov and P. D. Reshetov, J. Chromatogr. 14, 495 (1964). g4A. S. Khokhlov and K. I. Shutova, J. Antibiot. 25, 501 (1972). ,s L. M. Larson, H. Sternberg, and W. H. Peterson, J. Amer. Chem. Soc. 75, 2036 (1953). Y. Saburi, J. Antibiot. Ser. B 6, 402 (1953). ~7K. Akasaki. H. Abe, A. Seino, and S. Shirato, J. Antibiot. 21, 98 (1968). ~Y. Kasukabe, Y. Yamauchi, C. Nagatsu, H. Abe, K. Akasaki, and S. Shirato, .1. Antibiot. 22, 112 (1969). Y. Kono, S. Makino, S. Takeuchi, and H. Yonehara. J. Antibiot. 22, 583 (1969). ~ooE. Gaeumann and F. Benz, U.S. Patent 3,689,816 ; May 14, 1963. ~01A. W. Johnson and J. W. Westley, J. Chem. Soc. London p. 1642 (1962). ~0~H. Taniyama, Y. Sawada, and T. Kitagawa, J. Chromatogr. 56, 360 (1971).
124
METHODS FOR T H E STUDY OF ANTIBIOTICS
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126
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
T A B L E VII RELATIVE MOBILITIES OF NEOMYCINS AND /-AcETYLNEOMYCINS
Compounds
RNeamine in system K a'~
Neamine base Neomycin B Neomycin C N-Acetylneamine N-Acetylneomycin B N-Acetylneomycin C
1.00 0.54 0.30 ----
RN-acetylneamine in system Jb,c ---1.00 0.69 0.35
From M. K. M a j u m d a r and S. K. Majumdar, Appl. Microbiol. 17, 763 (1969). a F r o m M. K. M a j u m d a r and S. K. Majumdar, Anal. Chem. 39, 215 (1967). See solvent systems for aminoglycosides on p. 119.
TABLE VIII RELATIVE MOBILITIES OF GENTAMICINS
Compounds Gentamicin Gentamicin Gentamicin Gentamicin Gentamicin
Rneamine Rgentamicin Bl i n s y s t e m K ~'¢ in system S b'¢
A1 A B X B1
Gentamicin Ca sulfate Gentamicin C1~ sulfate Gentamicin C2 sulfate
-----2.25 3.75 3.07
0.36 0.41 0.52 0.55 1.00 ----
a F r o m M. K. M a j u m d a r and S. K. Majumdar, Appl. Microbiol. 17, 763 (1969). b From G. H. Wagman, J. A. Marquez, J. V. Bailey, D. Cooper, M. J. Weinstein, R. Tkach, and P. Daniels, J. Chromatogr. 70, 171 (1972). See solvent systems for aminoglycosides on p. 119.
C. O l i g o s a c c h a r i d e s w i t h C h r o m o p h o r e
Solvent systems. T h e s y s t e m s u s e d f o r t h i s g r o u p a r e a s f o l l o w s : A: n - P r o p a n o l - w a t e r (40:60) 1°8 B: Methyl ethyl k e t o n e - n - b u t a n o l - w a t e r (30: 5: 65) 1°3 C: M e t h a n o l - w a t e r (80: 20) plus 1.5% of NaC1, on paper buffered with a solution ~o3V. Betina, J. Chromatogr. 15, 379 (1964),
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PAPER CHROMATOGRAPHY OF ANTIBIOTICS
D: E: F: G: H: I: J: K: L: M: N:
127
containing 0.95 M Na~S04 plus 50 mM NariS04, equilibration 3 hr, development 16 h TM n-Butanol-pyridine-n-propanol-acetic ucid-water (20: 10 : 5: 3: 32) ~os 90% phenol-m-cresol-pyridine-acetic acid-water (25: 25:1 : 1 : 25) TM Diethyl ether-ethyl acetate (170: 30) ~0e Isopentenyl acetate-acetone (19 : 1) 10e Butyl acetate-pyridine-water (1 : 5 : 10) 107 Benzene-acetic acid-water (20: 25: 5) ~o8 Benzene-butanol-water (18: 2: 20) los Chloroform-carbon tetrachloride (both satd. with water)-methanol (5 : 4 : 1) ~os Diisoamyl ether satd. with water-butanol (20: 10) 1°8 n-Butanol-dichloroethane-formamide-water (5:45:16:34), lower layer on paper impregnated with upper layer (see Blinov and Khokhlov, 7 p. 126) Methanol-benzene (4:6) 1°9
Detection. U V l i g h t a n d b i o a u t o g r a p h y w i t h S. aureus, B. subtilis, or Corynebacterium xerosis a r e used for a n t i b i o t i c s of this group. Applications. B o t h r i s t o c e t i n a n d v a n c o m y c i n were s e p a r a t e d into two c o m p o n e n t s in s y s t e m s A - C . 1°3,1°4 I n s y s t e m D , r i s t o c e t i n B m o v e s f a s t e r t h a n r i s t o c e t i n A. T h e s a m e s y s t e m can be used for t h e c o m p a r i s o n of ristocetins, r i s t o m y c i n s , a n d v a n c o m y c i n . R i s t o m y c i n is a m i x t u r e of 4 components, ristomycin III and IV being identical with ristocetin A and B, r e s p e c t i v e l y . 1°5 A c t i n o i d i n can be s e p a r a t e d into 6 b i o l o g i c a l l y a c t i v e c o m p o n e n t s w h e n s y s t e m D is used. ~1° C h r o m o m y c i n s a r e c h a r a c t e r i z e d in F, G, a n d f u r t h e r s y s t e m s b y M i z u n o , 1°6 who also s t u d i e d c h r o m o m y c i n A3 d e r i v a t i v e s using s y s t e m H. 1°7 " S u m m a r i z e d c h r o m a t o g r a m " of a b u r a m y c i n in 8 s o l v e n t s y s t e m s has been p u b l i s h e d . ~°3 C h r o m o m y c i n A3, a u r e o l i c acid, a n d m i t h r a m y c i n can be c h a r a c t e r i z e d c h r o m a t o g r a p h i c a l l y in four s y s t e m s , iH O l i v o m y c i n is s e p a r a t e d into four m a i n c o m p o n e n t s b y c i r c u l a r c h r o m a t o g r a p h y in s y s t e m M (see B l i n o v a n d K h o k h l o v , : p. 126). lo~M. P. Kunstman, L. A. Mitscher, J. N. Porter, A. J. Shay, and M. A. Darken, Antimicrob. Ag. Chemother. 1968, 242 (1969). 105M. G. l~razhnikova, N. N. Lomakina, M. F. Lavrova, N. V. Tolstykh, M. S. Yurina, and L. M. Klyuyeva, Antibiotiki 8, 392 (1963). lo~K. Mizuno, J. Antibiot. Ser. B 13, 329 (1960). lot K. Mizuno, J. Antibiot. 16, 22 (1963). 10, E. V. Kruglyak, V. N. Borisova, and M. G. Brazhnikova, Antibiotiki 8, 1064 (1963). ,o~A. Aszalos, R. S. Robison, F. E. Pansy, and B. Berk, U.S. Patent 3,551,561; December 29, 1970. 1,0 E. Borowski, H. Chmara, and E. Jereczek-Morawska, Chemotherapia 12, 12 (1967). ~"D. M. Schuurmans, D. T. Duncan, and B. H. Olson, Cancer Res. 24(1), 83 (1964).
128
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
D. Everninomicin and L i n c o m y c i n G r o u p Solvent Systems. The systems used for these antibiotics are as follows. A: Benzene--petroleum ether (bp 30-60°)-acetone (20: 5: 10), descending development, 2 hr m B: Toluene--n-butanol-water-petroleum ether (bp 30-60 °) (20:1.5:7:1.5), descending, 4.5113 C: Petroleum ether (60-90°)-methanol-ethyl acetate-water (5: 8: 5: 2), 2 hr 113 D: Benzene-chloroform (93:7) satd. with formamide on paper dipped, prior to use, in 25% methanolic formamide, blotted, and air dried for 5 rain to remove methanol114 E: Chloroform-benzene (7: 3) on paper satd. with formamide as stationary phase l~s F: n-Butanol-water (84: 16) 116 G: n-Butanol-acetic acid-water (2:1 : 1) 11~ H: 5% Citric acid in water made to pH 7.0-7.5 with ammonia 11s I: 2-Propanol-2 N HC1 (65:35) 11e Systems A - C are for everninomicins and curamicin, D for halomicins, F for avilamicin, exfoliatin, and curamicin. Systems E - I are for the lincomycin group. However, thin-layer chromatography is mostly used to characterize members of ~he latter group. Paper chromatographic data of the everninomicin group are presented in Table IX. In bioautography, S. aureus and B. subtilis (avilamicin, exfoliatin) are used. With lincomycin, NaIO4-KMnO~ reagent m a y be applied: ~6 Spray the developed chromatograms with a mixture of 2% aqueous N a I 0 4 - 1 % K M n 0 4 in 2% aqueous Na.,C03 (2:1). Yellow spots appear on a purple background. E. Other Sugar Derivatives Diumyeins can be separated with n-propanol-n-butanol-0.5 N ammonium hydroxide ( 2 : 3 : 4 ) ; resulting Rf values for diumycin A and B are 0.32 and 0.45, respectively. 117 More recently, the same system differentiated 4 components with the following Rf values: 11s diumycin A, 0.32; diumycin A', 0.35 ; diumycin B, 0.45; diumycin B', 0.52. m M. J. Weinstein, G. M. Luedemann, E. M. Oden and G. H. Wagman, Antimicrob. Ag. Chemother. 1964, 24 (1965). m G. M. Luedemann and M. J. Weinstein, U.S. Patent 3,499,078; March 3, 1970. m M. J. Weinstein, G. M. Luedemann, E. M. Oden, and G. H. Wagman, Antimicrob. Ag. Chemother. 1967, 435 (1968). ~5 F. Buzzetti, F. Eisenberg, H. N. Grant, W. Keller-Schierlein, W. Voser, and H. Z~hner, Experientia 24, 320 (1968). ~ R. Thomas, G. J. Ikeda, and H. Harpootlian, J. Pharm. Sci. 56, 862 (1967). m E. Meyers, D. S. Slusarchyk, J. L. Bouchard, and F. L. Weisenborn, J. Antibiot. 22, 490 (1969). 1~8W. A. Slusarchyk, J. L. Bouchard-Ewing, and F. L. Weisenborn, J. Antibiot. 26, 39 (1973).
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PAPER CHROMATOGRAPHY OF ANTIBIOTICS
129
TABLE IX PAPER CHROMATOGRAPHIC DATA OF EVERNINOMICIN-TYPE ANTIBIOTICS RI X 100 in systems a Antibiotics Everninomlcln Everninomlcm Everninomicin Everninomicin Everninomicin Everninomlcm HaloInicin A c Halomicin B c Halomicin C c Halomicin D c Avilamicin d Exfoliatin ~ Curamicin ~
i b B
b
Cb Db Eb Fb
A
B
C
0 0
00
0 0
- -
- -
14 28 64 74 74
19 42 61 71 71
08 26 60 82 82
------
------
- -
- -
- -
00
- -
- -
- -
- -
35
- -
- -
- -
- -
60
- -
75
-70 58 60
-. . .
-. . .
. . .
-. . .
D
E
a See solvent systems for everninomicin and lincomycin group on p. 128. b F r o m G. M. L u e d e m a n n and M. J. Weinstein, U.S. P a t e n t 3,499,078; M a r c h 3, 1970. Cited by W a g m a n a n d Weinstein. e c M. J. Weinstein, G. M. Luedemann, E. M. Oden, and G. H. Wagman, Antimicrob. Ag. Chemother. 1967, 435 (1968). F. Buzzetti, F. Eisenberg, H. N. Grant, W. Keller-Schierlein, W. Voser, and H. Z~hner, Experientia 24, 320 (1968). e G. H. W a g m a n and M. J. Weinstein, " C h r o m a t o g r a p h y of Antibiotics," p. 63. Elsevier, Amsterdam, 1973.
VI. Macrocyclic
A. Nonpolyene
Lactone
Antibiotics
Glycosidic Macrolides
T h i s f a m i l y c o m p r i s e s t e n s of a n t i b a c t e r i a l a n t i b i o t i c s f r o m a c t i n o m y c e t e s w h i c h h a v e b e e n i n t e n s i v e l y s t u d i e d b y m e a n s of p a p e r chromatography. Filtrates from fermentations are applied directly onto chromatograms while pure samples are dissolved in methanol or ethanol. Bioautography w i t h B. subtilis o r o t h e r g r a m - p o s i t i v e b a c t e r i a is t h e common detection method. Solvent Systems. T h e s y s t e m s f o r s e p a r a t i o n , c h a r a c t e r i z a t i o n , a n d systematic analysis of this family are listed below. Of these, A-H are m o d i f i c a t i o n s of Z a f f a r o n i ' s s y s t e m s . S i n c e a n t i b i o t i c s o f t h i s f a m i l y a r e m o s t l y of b a s i c c h a r a c t e r , s y s t e m s T - W r e p r e s e n t m o d i f i c a t i o n s o f "pH-
130
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
c h r o m a t o g r a p h y ' ' H g - ~ which helps to s e p a r a t e m i x t u r e s of acidic, s6 basic, or a m p h o t e r i c a n t i b i o t i c s b y selecting o p t i m a l p H v a l u e s of the s t a t i o n a r y p h a s e (see Section X I I I , B on p H c h r o m a t o g r a m s ) . Systems m a r k e d * were on p a p e r p r e t r e a t e d with f o r m a m i d e ; **, on p a p e r p r e t r e a t e d with 50% f o r m a m i d e i n m e t h a n o l , s o l v e n t s y s t e m s satd. with f o r m a m i d e . A: B: C: D: E: F: G: H: I: J: K: L: M: N: O: P: Q: R: S: T: U: V: W: X: Y: Z:
Benzene-cyclohexane (1 : 1)*'122 Benzene*'1~2 Benzene-chloroform (3 : 1)*'122 Benzene-chloroform (1: 1) *'122 Benzene-cyclohexane (1 : 1)**.123 Benzene-cyclohexane (2:1)**.123 Benzene--chloroform (3: 1) **'1~3 Benzene-chloroform (1: 1) **'128 5% Aq. ammonium chloride TM Benzene--methanol (4: 1) 124 1 M Phosphate buffer, pH 7.0 TM 0.05 N (0.175%) ammonia satd. with methyl isobutyl ketone TM 1% Aq. ammonia TM 1% Ammonia satd. with methyl isobutyl ketone TM Methanol-acetone-water (19: 6: 75) 125 80% Methanol-3% NaC1 (1:1) on paper buffered with 0.95 M Na sulfate plus 0.5 M Na bisulfate, descending12~ Propanol-pyridine-acetic acid-water (6: 4:1 : 3) ascending128 Butanol-acetic acid-water (4: 1 : 5), ascending~28 80% Phenol, ascending12~ Benzene on paper impregnated with citrate buffer of pH 4.61~7 Butyl acetate on paper impregnated with citrate buffer of pH 4.0 ~7 Isopropyl ether-methylisobutyl ketone-2 % aq. ammonium carbonate (2: 1 : 2) ~2s Methanol-benzene (20:70) buffered with diisopropylamine-acetic acid ~22 Methanol-benzene (20: 70) buffered with pyridine-acetic acid l~g Methanol-dichloroethane (10: 80) buffered with pyridine--acetic acid 122 Methanol-benzene (20: 70) buffered with pyridine-oxalic acid 129
11,V. Betina, Nature (London) 182, 796 (1958). 1~ V. Betina and P. Nemec, Nature (London) 187, 1111 (1960). 12~M. IglSy, Magyar KJm. Lap]a 18, 622 (1963). 1~ A. Zaffaroni, R. B. Burton, and E. H. Keutmann, Science 111, 6 (1950). l~aT. M. Lees, J. De Muria, and W. H. Boegemann, J. Chromatogr. 5, 126 (1961). 1~4H. Koshiyama, M. Okanishi, T. Ohmori, T. Miyaki, H. Tsukiura, M. Matsuzaki, and tt. Kawaguchi, J. Antibiot. 16, 59 (1963). 1~5C. W. Pettinga, W. M. Starke, and F. R. Van Abeele, J. Amer. Chem. Soc. 76, 569 (1954). 1~ M. J. Weinstein, G. M. Luedemann, G. It. Wagman, and J. A. Marquez, U.S Patent 3,632,750; January 4, 1972. 1~ T. Osato, K. Yagashita, and H. Umezawa, J. Antibiot. 8, 161 (1955). 1~ H. A. Whaley, E. L. Patterson, A. C. Dornbush, E. J. Backus, and N. Bohonos, Antimicrob. Ag. Chemother. 1963, p. 45 (1964). ~2~M. IglSy, A. Mizsei, and I. I-Iorv~th, J. Chromatogr. 20, 295 (1965).
[7]
PAPER CHROMATOGRAPHY OF ANTIBIOTICS
131
Five buffer mixtures for systems W to Z are prepared as shown in accompanying tabulation:
Moles per 100 ml solvent Mixtures
Acid
Base
1 2 3 4 5
0.018 0. 018 0.018 0.009 Nil
Nil 0. 009 0.018 0~018 0.018
A p p l i c a t i o n s . P a p e r chromatographic data of 26 macrotides are compiled in T a b l e X. In system O, samples from erythromycin fermentation show two bioactive spots, one of them belonging to erythromycin B which can be isolated by the use of cellulose column chromatography. 12~ A further spot, detected in the course of the isolation of erythromycin B, helped to discover erythromycin C. 13° System 0 helped in finding two components in a commercial preparation of erythromycin (Ilotycin). T M Ochab et al. 132 described a technique for quantitative analysis of erythromycin in the presence of tetracycline in drugs. IglSy et al. ~29 used their modification of p H chromatography in order to compare erythromycin, magnamycin, oleandomycin, picromycin, and methymycin. On paper chromatography, spiramycins proved to be identical with foromacidins A, B, and C, but foromacidin D differed from the three spiramycins. 133 System E was applied in quantitative analysis of triacetyl oleandomycin. T M P a p e r c h r o m a t o g r a p h y helped to separate m e t h y m y c i n from neom e t h y m y c i n ~'~ and to prove the identity of a m a r o m y c i n and albomycetin with pikromycin. ~36 N i d d a m y c i n was compared with tylosin, carbomycin
,~0p. F. Wiley, R. Gale, C. W. Pettinga, and K. Gerzon, J. Amer. Chem. Soc.79, 6074 (1957) "~ V. Betina, pH chromatography of Antibiotics (in Slovak), p. 106. Thesis, Slovak Acad. Sci., Bratislava, 1960. 13~S. Ochab, D. Malysz, and B. Borowiecka, Chem. Anal. (Warsaw) 8, 597 (1963). I~'~R. Corbaz, L. Ettlinger, E. G~iumann, W. Keller-Schierlein, F. Kradolfer, E. Kyburz, L. Neipp, V. Prelog, A. Wettstein, and H. Z~ihner, Helv. Chim. Acta 39, 3O4 (1956). 1~ It. J. Pazdera, personal communication, 1962. 1~ D. Perlman and .E. O'Brien, Antibiot. Chemother. 4, 894 (1954). "eR. Hfitter, W. Keller-Schierlein, and H. Z~ihner, Arch. Mikrobiol. 39, 158 (1961).
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METHODS FOR THE STUDY OF ANTIBIOTICS
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A, and carbomycin B. 13~ Blinov et al. 1"8 found that some macrolides and other antibiotics gave double spots on chromatograms even when they were not mixtures of two compounds. Acumycin can be differentiated from 9 macrolides in systems A to C and also by thin-layer chromatography. 139 In structural studies of angolamycin, 149 amino sugars were determined chromatographically in hydrolyzates. Mycaminose was identified by paper chromatography in hydrolyzates of acumycin while a neutral deoxysugar was characterized by thin-layer chromatography. 130 Similar spots are detected in hydrolyzates of miyamycin and erythromycin, m Leucomycins A and B, related to spiramycins, can also be studied by means of paper chromatography. ~4= Aminosugars with similar RI values can be detected in hydrolyzates of leucomycins A~, A2, B1, Ba, B4, and .143
B. Polyene Macrolides Polyene antibiotics are applied in methanolic solutions onto chromatograms which can be then detected bioautographically with Saccharom y c e s cerevisiae, Candida a~bicans, or other fungi and by their fluorescence in UV light. S o l v e n t S y s t e m s . The systems used in studies of polyenes are as follows. A: B: C: D: E: F:
Butanol-acetic acid-water (4:1 : 5) 144 5% Dimethylformamide in methanoP** Butanol-ethanol-water (5: 1: 4) 14s Propanol-water (7: 3) 145 As D, but 8:2145 n-Butanol satd. with water 146
137G. Huber, K. H. Wallhgusser, L. Fries, A. Steigler, and H. L. Weidenmiiller, Arzneim.-Forsch. 12, 1,191 (1962). 138N. 0. Blinov, E. F. Oparysheva, I. N. Trudnikova, T. M. Rozanova, and A. S. Khokhlov, Antibioti£i 6, 660 (1961). is, H. Bickel, E. Giiumann, R. Hiitter, W. Sackmann, E. Vischer, W. Voser, A. Weftstein, and H. Z~hner, Helv. Chim. Acta 45, 1396 (1962). 140R. Corbaz, L. Ettlinger, E. G~umann, W. Keller-Schierlein, L. Neipp, V. Prelog, P. Reusser, and H. Z~hner, Helv. Chim. Acta 38, 202 (1955). 141H. Schmitz, M. Misiek, B. Heinemann, J. Lein, and I. R. Hooper, Antibiot. Chemother. 7, 37 (1957). 1,, T. Hata, F. Koga, and It. Kanamori, J. Antibiot. 6, 110 (1953). 1,, T. Watanabe, Bull. Soc. Chem. Soc. Jap. 34, 15 (1961). I~A. Aszalos, R. S. Robison, P. Lemanski, and B. Berk, J. Antibioti. O.l, 611 (1968). i~ A. P. Struyk, I. ttoette, G. Drost, J. M. Waisviz, T. van Eck, and J. C. Hoogerheide, Antibiot. Annu. 1957/1958, p. 878 (1958). I~K. Dornberg, R. Fugner, G. Bradler, and H. Thrum, J. Antibiot. 24. 172 (1971).
[7]
PAPER CHROMATOGRAPHY
OF ANTIBIOTICS
135
G: 20% Ammonium chloride 14~ H: As G, but 3% 14~ I: 75% Aq. phenol 14e J: 50% Aq. acetone ~4~ K : n-Butanol-methanol-water-methyl orange (40 ml : 10 ml : 20 ml : 1.5 g) ~4~ L: n-Butanol-methanol-water (40: 10: 20) 14~ M : Benzene-methanol (80: 20) 14~ N: Distilled water ~4e O: n-Butanol-pyridine-water (60: 40: 30) ~4~ P: Dimethyl formamide-water (10 : 90) ~46 Q: As P, but 50:5014~ R: As C, but 5:1:5147 S: t-Butanol-water (4: 1) 14s T: n-Butanol-ethyl acetate (1: 1) satd. with water ~4s U: Methy! isobutyl ketone-n-butanol-water (50:15 : 3) ~49 V: Methyl isopropyl ketone-water (satd. ca 1.6%) 149 W: Methyl isopropyl ketone-n-butanol-water (50: 10: 3.5) ~49 X : Methyl isopropyl ketone-methanol-water (50:1 : 0.4) ~0 Y: Ethyl acetate-pyridine-water (4: 3 : 2) 1~0 Z: As O, but 6:4:51~° : Methanol-25 % ammonium hydroxide-water (20:1 : 4) ~0 f~: As A, but 20:1:25 TM 7: As Y, but 6:2.5:7 TM : Chloroform-tetrahydrofuran-formamide (50: 50: 5) ~2 ~: n-Butanol satd. with 0.2% aq. acetic acid ~53 T h e a b o v e s y s t e m s are used for p a p e r c h r o m a t o g r a p h y of different groups of p o l y e n e s as follows. T r i e n e s : s y s t e m s A a n d B for trienine. T e t r a e n e s : s y s t e m A - - f o r t e r r a m y c i n , t e t r i n A, a n d t e t r i n B ; C - - f o r pimaricin; D--for pimaricin and tetramyein; F--for endomycins, flavacid, n y s t a t i n , p i m a r i c i n , p o l y f u n g i n s , r i m o c i d i n , t e r r a m y c i n , a n d unamycin; J--for nystatin, tetramycin, and unamycin; R--for amphot e r i c i n A, a n t i m y c o i n A, c h r o m i n , a n d n y s t a t i n ; S - - f o r e n d o m y e i n s and f l a v a c i d ; T - - f o r e n d o m y c i n s ; s y s t e m s G, H, I, K, L, M , N, O, P, a n d Q for t e t r a m y c i n . P e n t a e n e s : s y s t e m L - - f o r e u r o c i d i n ; E a n d F - - f o r m o l d i e i n B ; syst e m s U, V, W , a n d X - - - f o r f u n g i c h r o m i n . 14~C. P. Schaffner, I. D. Steinman, R. S. Safferman, and H. Lechevalier, Antibiot. Antra. 1957/1958, p. 869 (1958). 14SL. C. Vining and W. A. Taber, Can. J. Chem. 35, 1461 (1957). '*~A. C. Cope, R. K. Bly, E. P. Burrows, O. J. Ceder, E. Ciganek, B. T. Gillis, R. F. Porter, and H. E. Johnson, J. Amer. Chem. Soc. 84, 2170 (1962). 1~,R. Bosshardt and H. Bickel, Experientia 24, 422 (1968). 1~'G. R. Deshpande and N. Narasimhachari, Hindustan Antibiot. Bull. 9, 76 (1966). 1~2R. Schlegel and H. Thrum, J. Antibiot. 24, 360 (1971). 15~H. Lechevalier, R. F. Acker, C. T. Corcke, C. M. Haensler, and S. A. Waksman, Mycologia 45, 155 (1953).
136
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
H e p t a e n e s : system E - - f o r amphotericin B and h a m y c i n ; F and J - - f o r h a m y c i n ; L - - f o r aureofungin and h a m y c i n ; Y - - f o r candicidin; Z - - f o r candicidin, h a m y c i n , and p e r i m y c i n ; a - - f o r amphotericin B, ascosin, azacolutin, h a m y c i n , and levorin; f l - - f o r ascosin, candidin, and c h a m p a m y c i n ; e - - f o r candicidins. Octaenes: systems A, O, and Z for ochramycin. Others: systems L, Q, and Z for flavofungin and fungichromin.
C. O t h e r M a c r o c y c l i c A n t i b i o t i c s This section includes aglycosidic n o n p o l y e n e macrolides (e.g., cyanein), r i f a m y c i n s and related compounds, antimycins, azalomycins, etc. S o l v e n t S y s t e m s . Systems used for these antibiotics are as follows. A: Phosphate buffer, pH 7.3, containing 0.1% Na ascorbate satd. with n-amy! alcohol-n-butanol (9: 1) TM B: n-Butanol satd. with phosphate buffer, pH 7.3, containing 0.1% Na ascorbate TM C : Phosphate buffer, pH 8.6, containing 0.1% Na ascorbate satd. with n-butanol TM D: Water containing 3 % ammonium chloride plus 1% ascorbic acid 15~ E: M/15 Phosphate buffer pH 8.6 with or without addition of Na ascorbate on paper impregnated with 2-octyl alcohol 15e F: Cyclohexane-chloroform-water (1 : 8: 2) on Schleicher and Schuell No. 589 (Blue Ribbon Special) impregnated with 0.2 M phosphate buffer at pH 4.1 and air dried. Equilibrate for 2 hr in atmosphere of both phases and develop in organic phase 15~ G: n-Hexane-benzene-acetone-water (30: 10: 18: 32) ~ss H: As G, but (1:3:1:3) 1~9 I: 0.075 N ammonium hydroxide satd. with methyl isobutyl ketone 16° J: Water-ethanol-acetone (7: 2: 1) 16' K: Ethanol-n-hexane (1 : 2) le2 L: Water-acetone (75:25) 183 M: 16% Aq. n-propano1182 u4 p. Sensi, C. Coronelli, and B. J. R. Nicolaus, J. Chromatogr. 5, 519 (1961). ~5 p. Sensi, A. M. Greco, and R. Ballotta, Antibiot. Annu. 1959/1960, p. 262 (1960). 1~S. Sferruzza and R. Rangone, Farmaco, Ed. Prat. 19, 486 (1964). u7 p. Siminoff, R. M. Smith, W. T. Sokolski, and G. M. Savage, Amer. Rev. Tuberc. 75, 576 (1957). 158T. Kishi, H. Yamada, M. Muroi, and K. Mizuno, 163rd Meet. Jap. Antibiot. Res. Ass., September, 1968. 15oT. Kishi, H. Yamada, M. Muroi, S. Harada, M. Asai, T. Hasegawa, and K. Mizuno, J. Antibiot. 25, 11 (1972). ~ C. DeBoer, P. A. Meulman, R. J. Wnuk, and D. H. Peterson, J. Antibiot. 23, 442 (1970). 161D. Kluepfel, S. N. Seghal, and C. Vezina, J. Antibiot. 23, 75 (1970). ~ Y. Sakagami, A. Ueda, S. Yamabayashi, Y. Tsurumaki, and S. Kumon, J. Antibiot. 22, 521 (1969).
[7]
PAPER CHROMATOGRAPHYOF ANTIBIOTICS
137
N: Water-ethanol-acetic acid (70:24:6), 18-20 hr at 30° on Eaton-Dikeman No. 613183 O: Benzene-ethyl acetate (1: 2) TM P: 40% Aq. methanol 1~5 Q: As M, but 20% 1~5 R: n-Butanol-benzene-5 % ammonium chloride (l : 9: 10) 166 S: n-Butanol satd. with water 167 T: n-Butanol-methanol-water (4: 1:5), bottom layer 167
Detection. Methods v a r y greatly for these antibiotics. Rifamycins, besides their original color, are detected with Sarcina lutea, streptovaricins with Mycobacterium ranae, tolypomycin Y with Staphylococcus aureus, geldanamycin with Tetrahymena pyriformis, 1~° antimycins with Saccharomyces cerevisiae, oligomycins with Glomerella cingulata, ~63 aabomycin A and ikutamycin with Piricularia oryzae, 164,165 azalomyein B with S. lutea or Mycobacterium phlei, ~66 cyanein with Candida pseudotropicalis. Applications. Systems A through E are recommended for rifamycins, F for streptovaricins, G and H for tolypomycins, I for geldanamycin, J for antimycins, K and L for hondamycins, N for oligomycins, O for aabomycin, P and Q for folimycin and ikutamycin, R for azalomycin B, S, and T for cyanein (brefeldin A). Chromatographic data of rifamycins and other large macrocyclic antibiotics are summarized in Table XI, and those of other macrolactones in Table X I I . Quantitative paper chromatographic analysis of streptovaricins has been described. 1~8
V I I . Quinone Antibiotics
A. Tetracyclines Pure substances are applied on chromatograms in methanol. Samples from fermentations must be acidified in order to liberate tetracyclines from the mycelium. One such procedure has been described. 169 P h a r m a ceutical aqueous suspensions are acidified with HC1 and diluted with le,~M. H. Larson and W. H. Peterson, Appl. Microbiol. 8, 182 (1960). 1~4German "Offenlegungsschrift" 1,961,746 (1970). Cited by Wagman and Weinstein.~ 1,5y. Sakagami, A. Ueda, and S. Yamabayashi, J. Antibiot. 20, 299 (1967). '~ M. Arai. J. Antibiot. 13, 51 (1960). 1~V. Betina, P. Nemec, J. Dobias, and Z. Bar/~th, Folia Microbiol. (Prague) 8, 353 (1962). 1~W. T. Sokolski, N. J. Eilers, and P. Siminoff, Antibiot. Annu. 1957/1958, 119 (1958). 169j. Vondr£Skov/~and O. ~trauchovh, J. Chromatogr. 32, 780 (1968).
138
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TABLE XII PAPER CHROMATOGRAPHIC DATA OF MACROCYCLIC ANTIBIOTICS
Antibiotics Aabomycin Ab Antimycins c A0 AI A~ Aa A4 A~ A~ Azalomycin B d Cyanein ~
Solvent systems ~ R/ X 100 O J
R S T
Antibiotics
23
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Ikutamycin: Oligomycins h A B C
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See solvent systems for other macrocyclic antibiotics p. 136. b German "Offenlegungsschrift" 1,961,746 (1970). Cited by G. H. Wagman and M. J. Weinstein, "Chromatography of Antibiotics," Elsevier, Amsterdam, 1973. c D. Kluepfel, S. N. Seghal and C. Vezina, J. Antibiot. 23, 75 (1970). d M. Arai, J. Antibiot. 13, 51 (1960). V. Betina, P. Nemec, J. Dobias, and Z. Bar~th, Folia Microbiol. 8, 353 (1962). / Y. Sakagami, A. Ueda, and S. Yamabayashi, J. Antibiot. 20, 299 (1967). a y . Sakagami, A. Ueda, S. Yamabayashi, Y. Tsurumaki, and S. Kumon, J. Antibiot. 22, 521 (1969). h M. H. Larson and W. H. Peterson, Appl. Microbiol. 8, 182 (1960).
m e t h a n o l . T e t r a c y c l i n e s f r o m d r i e d m i l k c a n be e x t r a c t e d w i t h a m i x t u r e of m e t h a n o l - a c e t o n e - H C 1 (49: 4 9 : 2 ) .17o
S o l v e n t S y s t e m s . T h e s y s t e m s u s e d for t h i s f a m i l y a r e as follows. A: B: C: D: E: F:
3.5% NaAsO2 in water 171 n-Butanol-acetic acid-water (4:1 : 5) 172 n-Butanol-conc. NH~OH-water (4: 1 : 5) m As B, but on paper impregnated with E D T A and dried ~73 As C, but on paper impregnated with E D T A and dried 173 n-Butanol-chloroform (9:1) satd. with 0.15 M phosphate buffer on Whatman No. 4 paper impregnated with 3.7% soln. of Chelaton III, dried, and wetted with the aq. phase ~69
17oF. Bozzi and P. Valdebouze, J. Chromatogr. 72, 426 (1972). 1~1S. Caffau and S. Jacobacci, Minerva Med. 53, 3747 (1962). 17~p. p. Regna and I. A. Solomons, Ann. N.Y. Acad. Sci. 53, 229 (1950). 173R. G. Kelly and D. A. Buyske, Antibiot. Chemother. 10, 604 (1960).
140
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
G: Chloroform-nitromethane-pyridine (10:20:3) on paper dampened with McIlvaine's citrate-phosphate buffer pH 3.5 x74 H: Chloroform-n-butanol (4: 1) satd. with McIlvaine's buffer pH 4.5 on paper buffered with same buffer satd. with the organic phase 175 I: n-Butanol satd. with water on Eaton-Dikeman No. 613 paper soaked in 0.3 M pH 3 phosphate buffer and air dried 17e J: Nitromethane-chloroform-pyridine (20: 10: 3) satd. with a soln. composed as follows: 500 ml 0.1 N citric acid, 208 ml 0.2 N disodium phosphate containing 10 mg of Na benzoate as preservative. Paper is moistened with the aq. phase satd. with the organic phase ~77 K: Toluene-pyridine (20:3) satd. with water on paper satd. with aq. citratephosphate buffer, pH 4.2177 L: 0.1% Ammonium chloride on Whatman modified cellulose phosphate cationexchanger paper 17s M: n-Butanol satd. with water (equilibrate 4 hr, develop overnight)179 N: Ethyl acetate satd. with water (equilibrate overnight) 17° O: Chloroform-2-chlorethanol-water (2:1:1) (organic phase, equilibrate overnight) 170 P: Hexane-ethyl acetate (3: 1) on Whatman No. 1 paper buffered at pH 4.5 with 50 mM potassium citrate and hydrated by dipping into an aq. soln. of 80% acetone and air-drying to evaporate the acetone is° Q: n-Butanol satd. with stationary phase on paper treated with 0.3 M phosphate buffer as stationary phase ~sx R: Nitromethane-chloroform-~-picoline (20:10:3) on Schleicher and Schuell 2040b moistened with acetone-McIlvaine's pH 4.6 buffer (30:70)17° Detection. B i o a u t o g r a p h y with B. subtilis or other susceptible organisms is useful in d e t e c t i n g t r a c e q u a n t i t i e s of these antibiotics. M a n y of the t e t r a c y c l i n e s can be detected b y e x h i b i t i o n of a b r i g h t yellow or orange fluorescence u n d e r UV light which is e n h a n c e d b y gentle t r e a t m e n t with a m m o n i a vapors. Iso c o m p o u n d s give blue fluorescence, chlortetracycline has blue fluorescence in s y s t e m D, 1~ 7 - c h l o r o - 6 - d e m e t h y l - 4 d i d e m e t h y l a m i n o t e t r a c y c l i n e gives a dull orange fluorescence w i t h o u t a m m o n i a t r e a t m e n t a n d a green fluorescence after a m m o n i a , a n d 7c h l o r o - 6 - d e m e t h y l - 5 a , 6 - a n h y d r o t e t r a c y c l i n e has a r e d - o r a n g e fluores1T4G. B. Selzer and W. W. Wright, Antibiot. Chemother. 7, 292 (1957). 1~,M. Urx, J. Vondr£Skov£, L. Kova~ik, O. Horsk~', and M. Herold, J. Chromatogr. 11, 62 (1963). 17,N. Bohonos, A. C. Dornbush, L. I. Feldman, J. H. Martin, E. Pelcak, and J. H. Williams, Antibiot. Annu. 1953/1954, 49 (1954). ~T~M. M. Noseworthy, U.S. Patent, 3,009,956; November 21, 1961. m E. Addison and R. G. Clark, J. Pharm. Pharmacol. 15, 268 (1963). 1~j. H. Martin, L. A. Mitscher, P. A. Miller, P. Shu, and N. Bohonos, Antimicrob. Ag. Chemother. 1966, 563 (1967). is0S. L. Neidlemann, R. W. Kinney and F. L. Weisenborn, U.S. Patent, 3,375,276; March 26, 1968. ~slM. Tobkes and R. G. Wilkinson, U. S. Patent 3,549,681 ; December 22, 1970.
[7]
P A P E R CHROMATOGRAPHY OF ANTIBIOTICS
141
cence before and an orange fluorescence after treatment with ammonia. 1S° Chemical detections are as follows. 1. Spray with 5% methanolic solution of ferric chloride, which results in brown spots2 s2 2. Ehrlich's reagent for tetracyclines. 183 Spray with 2% p-dimethylaminobenzaldehyde in 1.2 N HC1 and keep for 5 hr at room temperature. Chlortetracycline gives a dirty yellow and hydroxytetracycline a bluegreen color. Sensitivity 5 ~g. 3. Starch-iodine following N-chlorination283 Spray with diluted Na hypochlorite and air dry. Dip into 95% ethanol and air dry. Spray with starch-iodide (1% soluble starch and 1% KI, 1:1, by volume). Applications. Chromatographic data of tetracyclines, their epimers and derivatives are compiled in Table XIII. Quantitative determination of tetracycline and quatrimycin in pharmaceutical preparations has been described by Selzer and Wright2 TM
B. Anthracyclines and Anthracyclinones Anthracyclines and their tetracyclic agtycons named anthracyclinones have been extensively studied by means of paper chromatography mainly by German and Soviet workers. They can be detected by their color and anthracyclinones also by fluorescence in UV light. Because of their basic character, mobility of anthracyclines in organic solvent systems increases with increasing pH values of the stationary phases on papers impregnated with buffers. T M A principal solvent system and its variations have been described which allow the separation of various anthracyclines, ls~ When antibiotics remain at the origin after development in the principal system dichlorethane-formamide-water (50: 15: 35), n-butanol is added to dichlorethane. For antibiotics traveling with the front of the principal system, tetrachloromethane is added to dichlorethane according to Table XIV. These variations can be used in separating hydrophilic antibiotics of the rhodomycin-mycetin E_, type as well as lipophilic substances of the cinerubin type. Chromatographic data of rubidomycins, cinerubin A, and mycetins are presented in Table XV. 18~H. Fischbach and J. Levine, Antibiot. Chemother. 5, 610 (1955). J83R. M. C. Dawson, D. C. Elliott, W. H. Elliott, and K. M. Jones, "Data for Biochemical Research," 2nd ed., p. 538. Oxford Univ. Press, London and New York 1969. '84G. Z. Yakubov, Y. M. Khokhlova, and N. O. Blinov, Mikrobiologiya 29, 911 (1960). ~G. Z. Yakubov, N. O. Blinov, L. N. Sergeyeva, O. I. Artamonova, and A. S. Khokhlov, Antibiotiki 10, 771 (1965).
142
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TABLE XIV SOLVENT SYSTEMS FOR SEPARATION OF ANTIBIOTICS OF THE MYCETIN--RHODOMYCIN--CINERUBIN GROUP a
Mobile phase Stationary phase
Systems
Tetrachlorohydrocarbon (ml)
Dichloroethane (ml)
n-Butanol (ml)
Formamide (ml)
Water (ml)
A B C D E
48 40 ----
2 10 50 47.5 45
---2.5 5
15 15 15 15 15
35 35 35 35 35
, Combinations recommended by G. Z. Yakubov, N. O. Blinov, L. N. Sergeyeva, O. I. Artamonova, and A. S. Khokhlov, Antibiotiki 10, 771 (1965). R u b o m y c i n is s e p a r a t e d into c o m p o n e n t s A1, A2, A~, Bo, B1, a n d C in s y s t e m s ~s~ A - C : A: Acetone-benzene-water (12: 3: 2) B: Benzene-acetic acid-pyridine--water (10: 11 : 1 : 3) upper layer C: Chloroform on paper impregnated with formamide T h e i d e n t i t y of r u b o m y c i n C w i t h d a u n o m y c i n h a s been confirmed. A n t h r a c y c l i n o n e s c a n be i d e n t i f i e d b y using t h e s y s t e m : benzene s a t u r a t e d w i t h f o r m a m i d e on p a p e r i m p r e g n a t e d w i t h f o r m a m i d e , ls4 C h r o m a t o g r a p h i c d a t a of r h o d o m y c i n o n e s a r e given in T a b l e X V I .
VIII. Amino Acid and Peptide Antibiotics E x c e p t for f l - l a c t a m a n t i b i o t i c s , b i o g e n e t i c a l l y d e r i v e d f r o m a m i n o acids, t h e b e s t k n o w n a n t i b i o t i c s of this r e l a t i v e l y big f a m i l y a r e i n c l u d e d here.
A. Diketopiperazine Derivatives I n b u t a n o l - a c e t i c a c i d - w a t e r ( 4 : 2 : 1 ) t h e R~ of gliotoxin ls~ is 0.75. A r g o u d e l i s 18s s e p a r a t e d m e l i n a c i d i n s I, I I , a n d I I I in b e n z e n e - m e t h a n o l water (1:1:2). 1= M. G. Brazhnikova, N. V. Konstantinova, and V. A. Pomazkova, Antibiotiki 11, 763 (1966). ~sTG. Nanda, A. Pal, and P. Nandi, Curr. Sci. 38, 518 (1969). ~ A . D. Argoudelis, J. Antibiot. 25, 171 (1972).
[7]
PAPER CHROMATOGRAPHY OF ANTIBIOTICS
145
TABLE XV PAPER CHROMATOGRAPHIC DATA OF RUBIDOMYCINS~ CINERUBIN~ AND MYCETINS RI X 100 in systems Antibiotics
A ~,b
B .,c
Rubidomycin A Rubidomycin B Rubidomycin C Cinerubin Ag Mycetin A~ Mycetin A2 Mycetin B~ Mycetin B2 Mycetin C Mycetin D Mycetin E1 Mycetin E2
30 70 90 ----------
10 30 90 ----------
C ~ . ~ 1)a.i ---55 90 75 45 45 15 05 00 00
----95 95 84 83 92 95 90 00
F r o m R. Despois, M. Dubost, D. Mancy, R. Maral, L. Ninet, S. Pinnert, J. Preud'homme, G. Charpentier, A. Belloc, N. de Chezelles, J. Lunel and J. Reaut, Arzneim.-Forsch. 17, 934 (1967). b System A: n-Butanol satd. with water on paper impregnated with 0.15 M p H 4.5 phosphate buffer. c System B: n - B u t a n o l - e t h y l a c e t a t e - w a t e r (2:2: 1) on paper impregnated with formamide. d From N. O. Blinov, G. Z. Yakubov, O. I. Artamonova, and Y. M. Khokhlova, Antibiotiki 7, 1063 (1962). System C: Benzene satd. with formamide on paper treated with formamide. I System D: Acetone. g From N. O. Blinov and A. S. Khokhlov, "Paper Chromatography of Antibiotics," p. 156 (in Russian). Izd. Nauka, Moscow, 1970.
B. Homopeptides A n t h e l v e n c i n s a r e d i s t i n g u i s h e d f r o m n e t r o p s i n a n d d i s t a m y c i n A in the system methanol-5
mM
Na
citrate buffer, pH
i m p r e g n a t e d w i t h t h e s a m e b u f f e r , ls9 B u t a n o l - a c e t i c is a s y s t e m f o r c h a r a c t e r i z a t i o n o f a m i d i n o m y c i n s e p a r a t i o n of k i k u m y c i n
A
(Rs, 0.04)
5.7
(7:3)
acid-water
on paper (2: 1 : 1 )
( R r : 0 . 2 5 - 0 . 2 7 ) 19° a n d
from kikumycin
B
(Rf, 0.72).
TM
18~G. W. Probst, M. M. Hoehn, and B. L. Woods, Antimicrob. Ag. Chemother. 1965, 789 (1966). 1~0S. Nakamura, H. Umezawa, and N. Ishida, J. Antibiot. 15, 163 (1961). 191M. Kikuchi, K. Kumagai, N. Ishida, Y. Ito, T. Yamaguchi, T. Furumai, and T. Okuda, J. Antibiot. 18, 243 (1965).
146
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
TABLE XVI PAPER CHROMATOGRAPHIC DATA OF RHODOMYCINONES
RI × 100 in systems Anthracyclinones
A a'b
B a'c
C" 'a
D,.S
a-Rhodomycinone a-Isorhodomycinone ~-Rhodomycinone ~-Isorhodomycinone ~,-Rhodomycinone 10-Epi--r-rhodomycinone ~-Rhodomycinone e-Rhodomycinone ~'-Rhodomycinone Daunomycinone
10 -27 -64 -77 85 94 30
22 -45 -81 -87 96 100 65
O0 -12 -35 -47 78 88 --
16 16 26 26 53 70 -----
a From N. O. Blinov and A. S. Khokhlov, "Paper Chromatography of Antibiotics" (in Russian), p. 148. Izd. Nauka, Moscow, 1970. b System A: Benzene satd. with formamide on paper treated with formamide. c System B: Benzene-formamide-water (50: 15: 35). d System C: Tetrachloromethane-formamide-water (50: 15:35). e System D: Decaline-tetraline-acetic acid-water (5: 5: 10: 1). s From H. Brockmann and J. Niemeyer, Chem. Bet. 100, 3578 (1967). C h r o m a t o g r a p h y of b a c i t r a c i n , ~92 e d e i n e s , 1~3 c a p r e o m y c i n s , 194 a n d s u b t i l i n s ~95 h a s b e e n r e p o r t e d .
C. Heteromer Peptides C h r o m a t o g r a p h i c d a t a of p o l y m y x i n s a r e c o m p i l e d in T a b l e X V I I . T h i o p e p t i n s c a n be s e p a r a t e d in t w o s y s t e m s ( T a b l e X V I I I ) , a n d t h i o s t r e p t o n h a s b e e n c h a r a c t e r i z e d in t h r e e s y s t e m s b y P a g a n o et al. 196
D. Sideromycins T h e i r o n - c o n t a i n i n g p e p t i d i c a n t i b i o t i c s c a n be c h r o m a t o g r a p h e d in t h e s y s t e m s g i v e n b e l o w . T h e y a r e d e t e c t e d w i t h B. subtilis, S. aureus, ~o~j. Prath, Acla Chem. Scand. 6, 1237 (1952). ~ T. P. Hettinger, Z. K. Borowski, and L. C. Craig, Biochemistry 7, 4153 (1968). 1~ W. M. Stark, C. E. Higgins, R. N. Wolfe, M. M. Hoehn, and J. M. McGuire, Antimicrob. Ag. Chemother. 1962, 596 (1963). ~ G. Alderton and N. Snell, J. Amer. Chem. Soc. 81, 701 (1959). ~**J. F. Pagano, M. J. Weinstein, H. A. Stout, and R. Donovick, Antibiot. Annu. 1955/1956, 554 (1956).
[7]
147
PAPER CHROMATOGRAPHY OF ANTIBIOTICS TABLE XVII PAPER CHROMATOGRAPHIC DATA OF POLYMYXINS R t X 100 in systems ~
Polymyxin
Ab
Be
Cd
Dd
A B D E
18 56 38 54
-43 23 --
39 54 . 51
. 50 .
E~
F~
.
. 73 . 77
. 86 .
46
. 90
Gd .
H~ .
. 37
42 . 42
I~
.
. .
37
jr
K~
54
60
--
--
. 24 . 24
a Solvent systems: A: n - B u t a n o l on paper pretreated with 0.2 M glycine-HCl at p H 2.5, 6-18 hr. B: B u t a n o l - w a t e r - i s o p r o p y l a m i n e (125: 60: 4) C: n - B u t a n o l - a c e t i c acid-water (120: 30: 50) D: As C, b u t 4 : 1 : 5 , upper phase. E: As D, b u t lower phase. F: n - B u t a n o l - p y r i d i n e - a c e t i c acid-water (30 : 20: 6: 24) G: n - B u t a n o l - a c e t i c a c i d - l % aq. N aC1 (120: 30:50) H: As G, b u t 5 % aq. NaC1 I: n - B u t a n o l - p y r i d i n e - a c e t i c a c i d - 1 % aq. NaCl (30: 20: 6: 24) J : t-Butanol-acetic acid-water (74: 3 : 25) K : t - B u t a n o l - m e t h y l ethyl ketone-formic acid-water (8: 6: 3: 3) b H. A. N a s h and A. R. Smashey, Arch. Biochem. Biophys. 30, 237 (1951). ¢ A. G. Mistretta, Antibiot. Chemother. 6, 196 (1956). S. Wilkinson and L. A. Lowe, J. Chem. Soc. (London) (1964) 4107 (1964). M. J. Daniels, Biochim. Biophys. Acta 1li6, 119 (1968).
TABLE XVIII PAPER CHROMATOGRAPHIC DATA OF THIOPEPTIN8 a R j X 100 in systems b Thiopeptin
A
B
Al A2 A3 A4 B
95 85 57 57 00
18 25 52 38 23
a F r o m N. Miyairi, T. Miyoshi, H. Aoki, M. Kohsaka, H. Ikushima, K. K u n u g i t a , H. Sakai, and H. I m a n a k a , 176th Meeting J a p a n Antibiotics Res. Ass., November 20, 1970. Cited b y G. H. W a g m a n and M. J. Weinstein, " C h r o m a t o g r a p h y of Antibiotics." Elsevier, Amsterdam, 1973. b Systems: A: E t h y l a c e t a t e - n - h e x a n e - 2 N a m m o n i u m hydroxide (4: 1:1) B: M e t h a n o l - a c e t i c acid-water (25 : 3 : 72)
148
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
a n d E. coli, b y m e a n s of t h e i r color, or b y r e a c t i o n with ferric chloride a n d p o t a s s i u m f e r r i c y a n a t e . C h r o m a t o g r a p h i c d a t a of 11 s i d e r o m y c i n s are given i n T a b l e X I X .
Solvent Systems A: B: C: D: E: F: G: H: I: J: K: L: M: N: O: P: Q: R: S: T: U:
n-Butanol-acetic acid-water (4:1 : 2) 197 As A, but 1:1:2197 Methanol-0.1 N HC1 (3: 1) 197 n-Propanol-2.5 % NaCl-acetic acid (10: 8: 1) 197 n-Butanol-ethanol-acetic acid-water (25: 25: 3: 47) ~97 Acetone-acetic acid-water (60: 37 : 3) ~97 Phenol-butanol-water (5 : 4: 9) 198 As A, but 4:1:519s Butanol-aminopropanol-water (25:1 : 25) 198 As G, but 3:3:4198 As A, but 1:2:1199 As A, but 5:1:4199 n-Propanol-acetic acid-water (15: 1 : 10) 199 n-Propanol-pyridine-water (15:1 : 10) 199 As A, but 2:1:19°° n-Butanol satd. with water 99° t-Butanol-0.(~04 N HCl-satd. aq. NaC1 (2:1:1)291 Butanol-butyl acetate-glacial acetic acid-water (109: 30: 13: 143), 24 hr 201 As H, but glacial acetic acid ~°1 Isopropanol-0.2 M acetate buffer, pH 6.0 (7:3) ~°9 Ethanol-0.05 M acetate buffer, pH 6.0 (80" 20) 2°2
E. B l e o m y c i n G r o u p C h e l a t e - f o r m i n g peptides of this group are detected b i o a u t o g r a p h i c a l l y with M y c o b a c t e r i u m phlei 2°3 or with cancer cell culture. 2°4 I n T a b l e X X , b l e o m y e i n s a n d p h l e o m y c i n s arc characterized b y t h e i r R~ values. 1~7E. 0. Stapley and R. E. Ormond, Science 125, 587 (1957). 1~8G. Schmidt-K~istner and J. Schmidt, Med. Chem. Abhandl. Med. Chem. Forschungsstraeten Farbenfabrik Bayer A. G. 7, 528 (1963). Chem. Abstr. 60, 1542e (1964). 1~ H. Thrum, Naturwissenscha]ten 44, 561 (1957). P. Sensi and M. T. Timbal, Antibiot. Chemother. 9, 160 (1959). 2olE. Gaeumann, E. Vischer, and It. Bickel, U.S. Patent 3,093,550; June 11, 1963. 202T. H. Haskell, R. It. Bunge, J. C. French, and Q. R. Bartz, J. Antibiot. 16, 67 (1963). 2o8H. Umezawa, K. Maeda, T. Takeuchi, and Y. Okami, J. Antibiot. 19, 200 (1966). 2~ H. Umezawa, Y. Suhara, and T. Ikekawa, Int. Congr. Chemother. Proc. 1963 Stuttgart 3rd, Voh 2, p. 974 (1964).
[7l
PAPER
CHROMATOGRAPHY
I
I
[
I
I
I
I
I
I
I
I
]
I
1
I
I
I
[
OF
149
ANTIBIOTICS
I
i i
I
,-4 c,D
t~
I
o
I ¢D
,2 ©
I
t
I
I
I
I C~
v
.¢ ©
©
5) [~-
~-~
il
t,-
I
~,
]~
;2
Ill t~-
o¢
.¢
~=~
-¢©
[
:~ v
¢~
o9
.~
~.~
~2 ~
~':
150
METHODS FOR T H E STUDY OF ANTIBIOTICS
[7]
TABLE XX PAPER CHROMATOGRAPHIC DATA OF BLEOMYCINS AND PttLEOMYCINS
Bleomycins Bleomycin Ab Bleomycin Bb Bleomycin-Cu chelatesc Cu-At 1 Cu-Bt 1 Cu-At 2 Cu-Bt 2 Cu-At 3 Cu-At 4 Cu-Bt 3 Cu-At 5 Cu-Bt 4 Cu-Bt 5 Cu-At 6
R/ X 100 in system A~ 88-99 66-70 92 71 83 72 85 85 71 86 72 70 88
Rs X 10O in systems Phleomycinsg Phleomycin C Phleomycin D Phleomycin D1 Phleomycin D2 Phleomycin E Phleomycin F Phleomycin G Phleomycin H Phleomycin I Phleomycin J Phleomycin K
B e
CI
73
- -
79 88 88 88 88 76 86 87 88 81
- -
8 8
- -
81 76 84 78 81 73 83
System A: 10% ammonium chloride. H. Umezawa, K. Maeda, T. Takeuchi, and Y. Okami, J. Antibiot. 19, 200 (1966). H. Umezawa, Y. Suhara, T. Takita, and K. Maeda, J. Antibiot. 19, 210 (1966). d T. Ikekawa, F. Iwami, H. Hiranaka, and H. Umezawa, J. Antibiot. 17, 194 (1964). e System B: As A, but 0.5%. 1 System C: As A, but 1%.
F. A c t i n o m y c i n s P a p e r c h r o m a t o g r a p h y , most often with r a d i a l development, is widely used in s t u d y i n g the biosynthesis, separation, identification, and microbial d e g r a d a t i o n of actinomycins. The most used solvent systems and the a c t i n o m y c i n s a n a l y z e d therein are given in T a b l e X X I . Detection is made either by their characteristic color or with B. subtilis.
G. Echinomycin-Type Antibiotics Chromatographic data of 5 antibiotics are collected in Table XXII. These chromopeptolides can be detected with B. subtilia.
[7l
PAPER CHROMATOGRAPHY OF ANTIBIOTICS
151
TABLE XXI SOLVENT SYSTEMS FOR PAPER CHROMATOGRAPHY OF ACTINOMYCINS Systems A B C D
E
Components Ethyl acetate-n-butyl ether-2% aq. naphthalene2-sulfonic acid (1 : 1 : 2) Ethyl acetate-n-butyl ether-10% aq. Na o-cresotinate (1 : 3 : 4) n-Dibutyl ether-n-butanol (5: 1) satd. with 2% aq. ~-naphthalene sulfonic acid, Na salt n-Dibutyl ether-ethyl acetate-2% aq. naphthalene-B-sulfonic acid (3 : 1 : 4) on paper dipped in aq. phase and blotted between sheets of filter paper n-Butanol-pyridine-water (4: 1: 5, upper phase) on Whatman No. 3 MM paper
F
Di-n-butyl ether-s-tetrachloroethane-10% aq. Na o-cresotinate (2: 1:3) G As F, but 5:1:6
Actinomycins analyzed A, B, C, D, ~,b S~, $2, S~~'d A, B, C, D a Co, C1, Cla, C~, C~a, Ca, Caa, C.+'+ A, B, C, Dl.g
Actinomycin, actinomycin monolactone, actinomycinic acidh I, II, III, IV, V, VI, A, B, C, D, X h,~ As with F plus Via, VIb, VL, Via, VIo, C1, C2, C3h, i
H I J K
Di-n-butyl ether-ethyl acetate-10% aq. Na o-cresotinate (2: 1: 3) Isoamyl acetate-5% Na naphthalene sulfonate (l:l) Amyl acetate-10% aq. Na m-cresotinate (1: 1) n-Butyl acetate-di-n-butyl ether (3: 1) satd. with 10% aq. Na m-cresotinate on paper dipped in the aq. layer and blotted Butanol-di-n-butyl ether (2:3) satd. with 10% aq. Na m-cresotinate on paper dipped in the aq. layer and blotted
DIV, BvIc, 63 d,i D, $2, S~a,i U1, U2, Ua, U4J,k Co, Coa, Cl, C~, Ca, Io, Ion, Ib I~, Is, X0, Xoa, X1, Xla, X~, X3, X4, Zo, Z1, Z~, Za, Z4, Z~a'l''~ Con, Co, C1, C2, C3, I0a, Io, It, I2, In, X0, X0a, X1, Xla, X2, X~, X4m
a R. A. Mancher, F. J. Gregory, L. C. Vining, and S. A. Waksman, Antibiotics A n n . 1954]1955, 853 (1955). b F. J. Gregory, L. C. Vining, and S. A. Waksman, Antibiot. Chemother. 5, 409 (1955). J. Kawamata and H. Fujita, J. Antibiot. 13, 295 (1960). d Circular development. +K. H. Zepf, Experientia 14, 207 (1958). f L. C. Vining and S. A. Waksman, Science 120, 389 (1954). a Ascending or circular development. h D. Perlman and A. Capek, J. Antibiot. 21, 421 (1968). i G. G. Rousos and L. C. Vining, J. Chem. Soc. (London, p. 2469 (1956). J M. Furukama, A. Inoue, and K. Asano, J. Antibiot. 21, 568 (1968). k G. Schmidt-K~stner, C. Hackman, and J. Schmidt, German Patent No. 1,126,563; March 29, 1962. z R. Bossi, R. Htitter, W. Keller-Schierlein, L. Neipp, and H. Z~hner, Helv. Chim. Acta 41, 1645 (1958). m H. Brockmann and H. GrSne, Chem. Ber. 87, 1036 (1954).
METHODS FOR THE STUDY OF ANTIBIOTICS
152
[7]
TABLE X X I I PAPER CHROMATOGRAPHICDATA OF ECHINOMYCIN GROUP RI × 100 in systems ~ Antibiotics
A
Echinomycin~,c Levomycina Quinomycin A. Quinomycin C . Triostin b
26 . . . 40
B 54 .
. . .
C
D
E
F
61 .
13
-94 . .
-95 . .
. . 26
. . 76
. . .
.
.
G
55 . . .
H
I
J
63
84
-13 59
. . .
.
.
a Solvent systems: A: Petroleum ether-benzene-methanol-water (66.7: 33.3 : 80: 20) B: 25% Ethanol C: Amyl acetate sat& with water D: di-n-Butyl ether-s-tetrachloroethane--10 % Na o-cresotinate (2: 1 : 3) E! n-Butanol satd. with water F: n-Butanol satd. with 10% acetic acid G: Methyl isopropyl ketone H: Methyl isopropyl ketone-2% aq. toluenesulfonic acid I: Methyl isopropyl ketone-2% piperidine J: n-Butyl ether-s-tetrachloroethane-10 % aq. o-cresotinate (2:1 : 3) b Data in systems A, B, and C from T. S. Maksimova, I. N. Kowsharowa, and U. V. Proshlyakova, Antibiotiki 10, 298 (1965). ° Data in system C from K. Katagiri, J. Shoii, and T. Yoshida, J. Antibiot. 15, 273 (1962). From H. E. Carter, C. P. Schaffner, and D. Gottlieb, Arch. Biochem. Biophys. 53, 282 (1954). ' From T. Yoshida and K. Katagiri, J. Antibiot. 14~ 330 (1961).
IX. Nitrogen-Containing Heterocyclic Antibiotics A. Mitomycin Group Although Bdrdy ~9 included these antibiotics among quinones, they are dealt with separately here. Mitomycin C, porfiromycin, and G-253 series are detected with S. lutea, B. subtilis,lysogenic E. coli,22 or sarcoma 180 cells.111 Chromatographic data of the above antibiotics are given in Table XXIII. B. Pyrimidine Nucleosides S o l v e n t s y s t e m s a n d c h r o m a t o g r a p h i c d a t a of a n t i b i o t i c s b e l o n g i n g to this g ro u p can be f o u n d in T a b l e X X I V . C o m p o u n d s of a m i c e t i n t y p e
[7]
PAPER CHROMATOGRAPHY OF ANTIBIOTICS
153
TABLE XXIII PAPER CHROMATOGRAPHIC DATA OF MITOMYCIN GROUP RI X 100 in systems ~ Antibiotics
A
B
C
D
Mitomycin Cb Porfiromycin b G-253 series ~ G-253A G-253B1 G-253B~ G-253B G-253C G-253C1
57 71
02 13
56 98
49 c --
---
40-50 27-38 13-17 06-10 01-06 O0
m
-
-
---
-
a Solvent systems: A: n - B u t a n o l - w a t e r (84: 16) B: M e t h a n o l - b e n z e n e - w a t e r (1 : 1 : 2) C: E t h y l acetate satd. w i t h water D: Chloroform sat& with water F r o m S. Wakaki, Y. Harada, K. Uzu, A. N. Wilson, E. O. Stapley, F. J. Wolf, and D. E. Williams, Antibiot. Chemother. 12~ 469 (1962). c F r o m N. O. Blinov, G. Z. Yakubov, L. A. Vetlugina, and Y. M. Khokhlova, Mikrobiologiya 30~ 642 (1961). d S. Nomura, H. Y a m a m o t o , H. Umezawa, A. M a t s m n a e , and T. Hata, J. Antibiot. 20, 55 (1967).
Mycobacterium phlei o r E. coli, h i k i z i m y c i n w i t h Pseudomonas fluorescens, b l a s t i c i d i n S i n U V l i g h t , p o l y o x i n s a n d p i o m y c i n are detected with
i n U V l i g h t o r b y m e a n s of n i n h y d r i n r e a c t i o n .
C. Purine
Nucleosides
Solvent Systems. T h e s y s t e m s m o s t l y a p p l i e d f o r t h e s e a n t i b i o t i c s are: A: B: C: D: E: F: G:
n - B u t a n o l satd. w i t h water ~°5 n - B u t a n o l - a c e t i c acid-water (4:1: 1)s°s n - B u t a n o l - f o r m i c acid-water (77 : 10: 13) 305 n - B u t a n o l satd. w i t h a m m o n i a 2°5 n - B u t a n o l satd. with water and containing 2 % of piperidine 2°6 As B, b u t 25:6:25111 Methanol-glacial acetic a c i d - a n h y d r o u s N a acetate (400 ml: 0.14 m l : l . 6 4 g), p H 5.5111
2°5K. Isono and S. Suzuki, J. Antibiot. 13, 270 (1960). ~o~British P a t e n t 935,075. Cited by Blinov and Khokhlov. 7
154
METHODS F O R T H E
H: I: J: K: L:
M: N: O: P: Q: R:
S:
STUDY OF ANTIBIOTICS
[7]
1% Ammonia-n-propanol (1 : 2) s0T 4% Ammonium chloride z°z n-Butanol-methanol-water (4: 1 : 2) s07 Isobutyric acid-0.5 N ammonia (5:3)2°8 60% Aq. n-propanol 2°8 Ethyl acetate-n-propanol-water (7: 1 : 4) ~os Isopropanol-conc. aq. ammonia-0.1 M boric acid (7: 1 : 2) sos As B, but 5:1:42~° As B, but 2:1:12H n-Butanol-ethanol-water (50: 15: 35) s~2 Ammonia-water, pH 10.3213 As B, but 5:2:32~3
D e t e c t i o n . Angustmycins can be detected bioautographically 2~4 with a m y e o b a c t e r i u m 607, or by UV light. Cordycepin was detected by means of sarcoma 180 cell culture, m Tubercidin is detected by UV light or C a n d i d a albicans. ~15 F o r m y c i n is visualized by its characteristic absorption in UV light and purple fluorescence. ~e A p p l i c a t i o n s . R I values of nebularin in systems A, B, C, and D were 0.41, 0.52, 0.43, and 0.41, respectively. 2°5 Angustmycins A and C were separated in system E with R~ values 0.37 and 0.13, respectively. 2°6 In systems F and G, cordycepin has RI values of 0.47-0.52 and 0.21, respectively. 2°~ Characteristic R~ values of t o y o k a m y c i n ~°s in systems H, I, and J were as follows: 0.70, 0.51, and 0.65, respectively. Suhadolnik et al. 2~ characterized 3'-acetamido-3P-deoxyadenosine from a H e l m i n t h o s p o r i u m sp. by its R / v a l u e s in solvents C, D, R, and S as follows: 0.35, 0.28, 0.41, and 0.54, respectively. Chromatographic data of tubercidins and formycins are presented in T a b l e XXV.
X. Oxygen-Containing Heterocyclic Antibiotics "Summarized chromatograms" of kojic acid, mycophenolic acid, patufin, penicillic acid, and usnic acid have been published. I°3 A substance preventing somatic segregation in diploid strains of Penicillium chrysogeso7E. C. Taylor and R. W. Hendess, I. Amer. Chem. Soe. 87, 1995 (1965). ~08G. Acs, E. Reich, and M. Hori, Proc. Nat. Acad. Sci. U.S. 52, 493 (1964). 2°9A. Bloch, R. Leonard, and C. Nichol, Bioehim. Biophys. Aeta 138, 10 (1967). ,ioS. Aizawa, T. Hidaka, N. Otake, H. Yonehara, K. Isono, N. Agarashi, and S. Suzuki, Agr. Biol. Chem. 29, 375 (1965). ~11M. J£rai, G. J6zsa, and J. Koll£r, Aeta Mierobiol. Aesd. Sci. Hung. 11, 203 (1965). ~ T. Sawa, Y. Fukugawa, Y. Schimauchi, K. Ito, M. Hamada, T. Takeuchi, and H. Umezawa, J. Antibiot. 18, 259 (1965). 2~aR. J. Suhadolnik, B. M. Chassey, and G. R. Waller, Biochim. Biophys. Acta 179, 258 (1969). 2~4H. Yiintsen, K. Ohkuma, Y. Ashii, and I-I. Yonehara, J. Antibiot. 9, 195 (1956). ~5K. Anzai, G. Nakamura, and S. Suzuki, J. Antibiot. 10, 201 (1957). ~* M. Hori, E. Ito, and T. Takita, J. Antibiot. 17, 96 (1964).
[7]
155
PAPER CHROMATOGRAPHY OF ANTIBIOTICS
TABLE XXIV PAPER CHROMATOGRAPHIC DATA OF PYRIMIDINE NUCLEOSIDES R / X 100 in s y s t e m s a Antibiotics Amicetin Amicetin B b B a m i c e t i nb Blasticidin S a Hikizimycin' PiomycinY Polyoxinsg A B C D E F G H I
A
B
63 b 86 22 --.
22 c . . --.
. . . . . . . . .
. . . . . . . . . .
C
D
46 . . .
.
.
. . -00 .
. . . . . . . . .
. . . . . . . . .
E
. . 40 --
F
. . . . 40 17
G
.
. . .
. . .
.
.
. . . . . . . . .
H
-36
. ---
---
19 07 09 07 08 18 09 25 23
53 18 27 06 09 38 30 66 61
07 03 03 01 01 03 03 t2 08
a Solvent s y s t e m s : A: n - B u t a n o l satd. w i t h 50 m M p H 7 p h o s p h a t e buffer on paper i m p r e g n a t e d w i t h the buffer soln B: 90 % Aq. n - b u t a n o l C: n - B u t a n o l satd. w i t h water D : n - B u t a n o l - a c e t i c a c i d - w a t e r (2:1 : 1) E: A s D , b u t 1 : 1 : 1 F: As D, b u t 4 : 1 : 2 G: 75% Phenol H: B u t a n o l - p y r i d i n e - w a t e r (4:1 : 2) b T. H. Haskell, A. Ryder, R. P. F o r h a r d t , S. A. Fusari, Z. L. J a k u b o w s k i , a n d Q. R. Bartz, J. Amer. Chem. Soc. 80, 743 (1958). c j . W. H i n m a n , E. L. Caron, a n d C. DeBoer, J. Amer. Chem. Soc. 75, 5864 (1953). d H. Y o n e h a r a and N. Otake, Antimicrob. Ag. Chemother. 1965, 855 (1966). e K. Uchida, T. Ichikawa, Y. Shimauchi, T. Ishikura, a n d K. Ozaki, J. Antibiot. 24, 259 (1971). f N e t h e r l a n d s P a t e n t 67,13997; April 16, 1968. Cited by G. H. W a g m a n a n d M. J. Weinstein, " C h r o m a t o g r a p h y of Antibiotics," Elsevier, A m s t e r d a m , 1973. K. Isono, J. N a g a t s u , K. K o b i n a t a , K . Sasaki, and S. Suzuki, Agr. Biol. Chert1. 31, 190 (1967).
n u m w a s s h o w n to be m y c o p h e n o l i c and other methods antibiotics
acid when
paper
w e r e a p p l i e d . 217 C h r o m a t o g r a p h i c
are given in Table
XXVI.
Patulin
chromatography
data
of the above
and penicillic acid may
~17A. Baillio a n d G. Sermonti, Sci. Re?. Ist. Super. Sanita 1, 8 (1961).
156
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
TABLE XXV PAPER CHROMATOGRAPHIC DATA OF TUBERCIDINS AND FORMYCINS
R / × 100 in systems ~ Antibiotics
K
Tubercidin b Deaminotubercidin b Deoxytubercidin b Tubercidin 5t-monophosphate b Tubercidin 5~-diphosphate b Tubercidin triphosphate b Formycin Formycin B Isopropylidene formycin Isopropylidene formycin B Formycin 5'-phosphatel Formycin B 5'-phosphates
L
79 73 55 . . 81 73 54 50 36 . . 25 . . . . . . . . . . . . . . 54 . . 26 . .
M
N°
57
54 . 70 . . .
. 68 04 . .
0d -. . . .
. . . . . .
. . . .
. .
--
--
-.
--
47 40
47 43 83 81
. . 34 23
. .
QI
. --
.
P~
. .
- See solvent systems for purine nucleosides on p. 153. b G. Acs, E. Reich, and M. Hori, Proc. Nat. Acad. Sci. U.S. 52, 493 (1964). A. Bloch, R. Leonard, and C. Nichol, Biochim. Biophys. Acta 138, 10 (1967). d S. Aizawa, T. Hidaka, N. Otake, H. Yonehara, K. Isono, N. Igarashi, and S. Suzuki, Agr. Biol. Chem. 29, 375 (1965). e M. J~rai, G. J6zsa, and J. Koll~r, Acta Microbiol. Acad. Sci. Hung. 11, 203 (1965). s T. Sawa, Y. Fukugawa, Y. Shimauchi, K. Ito, M. Hamada, T. Takeuchi, and H. Umezawa, J. Antibiot. 18, 259 (1965). be d e t e c t e d w i t h B. subtilis. U s n i c a c i d g i v e s a b r o w n c o l o r w i t h s u l f u r i c acid. K o j i c a c i d a n d m y c o p h e n o l i c a c i d m a y be d e t e c t e d w i t h 3 % m e t h a n o l i c f e r r i c c h l o r i d e r e s u l t i n g in b r o w n i s h r e d a n d d a r k g r e e n color, r e s p e c t i v e l y . 1°3 T h e l a t t e r a n t i b i o t i c c a n also be d e t e c t e d b y m e a n s of b i o a u t o g r a p h y o n v a c c i n i a v i r u s - i n f e c t e d B S C - 1 m o n k e y k i d n e y cells. 21s XI.
Alicyclic Antibiotics
Blinov and Khokhlov 7 compiled solvent systems a n d o t h e r g l u t a r i m i d e s as follows. A: B: C: D: E:
for c y c l o h e x i m i d e
1 M Phosphate buffer, p H 6.8 Benzene-methanol-water (1 : 2: 1) n-Butanol satd. with water Benzene-acetic acid-water (6: 7: 3) Ethyl acetate satd. with water on paper impregnated with 0.1 M phosphate buffer, p H 4.0
218R. It. Williams, L. D. Boeck, J. C. Cline, D. C. De Long, K. Gerzon, R. S. Gordee, M. Gorman, R. E. Holmes, S. H. Larsen, D. H. Lively, T. R. Matthews, J. D. Nelson, G. A. Poore, W. M. Stark, and M. J. Sweeney, Antimicrob. At. Chemother. 1968, 229 (1969).
[7]
157
PAPER CHROMATOGRAPHY OF ANTIBIOTICS F: G: H: I: J: K: L: M: N:
B u t a n o l - a c e t i c acid-water (25: 6: 25) Isoamyl alcohol-5% aq. Na~HP04 (1:2), upper layer M e t h a n o l - a c e t i c a c i d - a n h y d r o u s N a acetate (400 ml:0.14 m1:1.64 g) Butanol said. with 2 N a m m o n i a E t h e r satd. with w a t e r Benzene satd. w i t h water Chloroform said. with water M e t h y l isobutyl ketone said. w i t h water 3 % A m m o n i u m chloride TABLE XXVI PAPER CHROMATOGRAPHIC DATA OF SOME HETEROCYCLIC ANTIBIOTICS
RI X 100 in systems ~ Antibiotics Kojicacid Mycophenolic acid Patulin Penicillic acid Usnic acid
Ab B b C b D b E b F b G b H b i b 81 00 94 89 00
56 90 78 85 60
32 98 91 95 96
00 82 94 00 94
32
93
23
80
64
90
jb
K b Lb M c N ~
68 91
86 84
82 74
79 84
00
00
24
00
28 74
60
93
Solvent systems: A: W a t e r B: n - B u t a n o l satd. w i t h water C: E t h y l acetate said. w i t h water D: Benzene satd. with water E: 3 % Aq. a m m o n i u m chloride F: Isoamyl a c e t a t e - m e t h a n o l - 9 9 % formic acid-water (65:20:5:10), tipper layer G: n - B u t y l a c e t a t e - m e t h y l ethyl ketone-0.15 M p H 7.4 p h o s p h a t e buffer (50: 25 : 5), upper layer H: E t h y l acetate-n-hexane-0.15 M p H 6.0, p h o s p h a t e buffer (65:15:20), upper layer I : Isoamyl a c e t a t e - m e t h a n o l - 9 9 % formic acid-water (40: 20: 10: 30), lower layer J: n - B u t a n o l - m e t h a n o l - w a t e r (40: 10: 50), lower layer K : M e t h a n o l - n - h e x a n e (60:40), lower layer L: Benzene-cyclohexanone-0.15 M p H 7.4 phosphate buffer (5 : 35 : 60), lower layer M: n - P r o p a n o l - a m m o n i a , d = 0.92 (7: 3) N: n - B u t a n o l said. with water plus 2 % p-toluenesulfonic acid and 2 % piperidine. b V. Betina, J. Chromatogr. 15, 379 (1964). c A. Ballio and G. Sermonti, Sci. Rep. Ist. Super. Sanita 1, 8 ( 1 9 6 1 ) . d R. H. Williams, L. D. Boeck, J. C. Cline, D. C. DeLong, K. Gerzon, R. S. Gordee, M. Gorman, R. E. Holmes, S. H. Larsen, D. H. Lively, T. R. Matthews, J. D. Nelson, G. A. Poore, W. M. Stark, and M. J. Sweeney, Antimicrob. Ag. Chemother. 1968, 229 (1969).
158
METHODS FOR T H E STUDY OF ANTIBIOTICS
[7]
TABLE X X V I I PAPER CHROMATOGRAPHIC DATA OF STEROID ANTIBIOTICS a
Rs X 100 in systems b Antibiotics Cephalosporin Cephalosporin Cephalosporin Cephalosporin Cephalosporin Fusidic acid Helvolic acid
P~ P2 P~ P4 Ps
A
B
45 45 00 40 20 00 --
50 . 00 50 . 60 --
C
D
100 .
. 05 100
.
. 100 --
40 . . . . 78 30
F
G
--
72
.
.
. .
. . .
I
65
78
50 --
65 --
. . .
. 11 45
H
. . .
50 --
Compiled from: K. Crawford, N. Heatley, P. S. Boyd, C. W. Hale, B. W. Kelly, G. A. Miller and N. Smith, J. Gen. Microbiol. 6, 47 (1952). H. S. Burton and E. P. Abraham, Bioehem. J. 50, 168 (1952). H. Vanderhaeghe, P. Van Dijck, and P. De Sommer, Nature (London) 205, 4972 (1965). R. P. Elander, R. S. Gordee, R. M. Wilgus, and R. M. Gale, J. Antibiot. 22, 176 (1969). b See solvent systems for steroid antibiotics on p. 159. R I v a l u e s of c y c l o h e x i m i d e ( A c t i d i o n e ) in s y s t e m s A, B, C, a n d D 7 a r e 0.59, 0.4, 0.8, a n d 0.87, r e s p e c t i v e l y . S y s t e m E is used for s e p a r a t i o n of c y c l o h e x i m i d e , d e h y d r o c y c l o h e x i m i d e , a n d a n h y d r o c y c l o h e x i m i d e . ~19 S t r e p t o v i t a c i n s a r e s e p a r a t e d in s y s t e m E into 5 c o m p o n e n t s . 2-~° U s i n g t h e s a m e s y s t e m a n d d e t e c t i o n w i t h S a c c h a r o m y c e s pastorianus, E b l e et al. ~21 o b t a i n e d t h e following R I v a l u e s : s t r e p t o v i t a c i n A, 0.26; s t r e p t o v i t a c i n B, 0.36; s t r e p t o v i t a c i n C1, 0.46; s t r e p t o v i t a c i n C2, 0.53; s t r e p t o v i t a c i n D, 0.62; s t r e p t o v i t a c i n E, 0.72; c y c l o h e x i m i d e , 0.96. I n s y s t e m s F , G, H , a n d I, t h e following R I v a l u e s were f o u n d ~1 for s t r e p t i m i d o n : 0.84, 0.72, 0.76, a n d 0.88, r e s p e c t i v e l y . S y s t e m s J t h r o u g h N can be used for p r o t o m y c i n . S t e r o i d a n t i b i o t i c s can be c h a r a c t e r i z e d in t h e f o l l o w i n g s o l v e n t s y s t e m s : 2~2-~25 *~*L. Dole~ilov~, J. Spi~ek, M. Vondr£~ek, F. Pale6kov£, and Z. Van~k, J. Gen. Microbiol. 39, 305 (1965). ~ W. T. Sokolski, N. J. Eilers, and G. M. Savage, Antibiot. Annu. 1958/1959, 551 (1959). 221T. E. Eble, M. E. Bergy, R. R. Herr, and J. A. Fox, Antibiot. Chemother. 10, 479 (1960). ~ K . Crawford, N. Heatley, P. S. Boyd, C. W. Hale, B. W. Kelly, P. A, Miller, and N. Smith, J. Gen. Microbiol. 6, 47 (1952). ~2~H. S. Burton and E. P. Abraham, Biochem. I. 50, 168 (1952).
[7l
PAPER CHROMATOGRAPHY
A: B: C: D: E: F: G: H: I:
OF ANTIBIOTICS
159
0.02 N Acetic acid in 25% aq. ethanol Diisopropyl ether satd. with 0.1 M Na phosphate, pH 7 Amyl acetate satd. as B Benzene/formamide Ethanol-0.1 N acetic acid-water (2.5: 2: 2.5) Methyl isobutyl ketone satd. with water plus 2% piperidine Methyl ethyl ketone-benzene-water (1 : 5: 1) 7% NaC1 plus 2.5% methyl ethyl ketone in water 17.4 g of K~HPO4 plus 30 ml of ethanol per liter of water.
C e p h a l o s p o r i n s 191 to P2, fusidic acid, a n d helvolic acid m a y be detected with B. subtilis. T h e i r c h r o m a t o g r a p h i c d a t a are presented in Table XXVII.
XII. Aromatic Antibiotics
A. C h l o r a m p h e n i c o l a n d I t s D e r i v a t i v e s S o l v e n t S y s t e m s . M e t h a n o l i e solutions of e h l o r a m p h e n i e o l are applied onto paper. R e c o m m e n d e d s o l v e n t s y s t e m s are listed below. A: B: C: D: E: F: G:
Water satd. n-butanol containing 2.5% acetic acid 226 n-Butanol satd. with water-phenol-pyridine (191 : 5 : 4) 22~ n-Butanol-acetic acid-water (4: 1 : 5) 2,s n-Butanol-pyridine-water (2:1 : 2), 8-10 hr 2.9 n-Butanol satd. with water on paper impregnated with 0.2 M KH2PO4 ~9 Benzene-methanol-water (2:1 : 1) ~3° McIlvaine's buffer, pH 6, on paper impregnated with octyl alcohol, about 1 hr TM
Rr v a l u e s as r e p o r t e d in these systems for c h l o r a m p h e n i e o l are 0.89 in A, 0.92 i n C, 0.96 in D, 0.97 in E. I n s y s t e m G: c h l o r a m p h e n i c o l , 0.30; c h l o r a m p h e n i e o l p a l m i t a t e , 0.0; e h l o r a m p h e n i e o l stearate, 0.0; chlor a m p h e n i c o l suecinate, 0.75. ~4 H. Vanderhaeghe, P. Van Dijck, and P. De Sommer, Nature (London) 205, 4972 (1965). ~-~R. P. Elander, R. S. Gordee, R. M. Wilgus, and R. M. Gale, J. Antibiot. 22, 176 (1969). 2~A. Glazko, W. A. Dill, and M. C. Rebstock, J. Biol. Chem. 183, 679 (1950). ~ I. M. Hais, K. Macek, and V. Francov~, Cesk. Farm. 4, 127 (1955). ~s V. H. Reber and E. Lichtenberg, Helv. Med. Acta 20, 396 (1953). 2~ M. R. Rousselet and R. Paris, Ann. Pharm. Ft. 22, 249 (1964). ,30K. Macek, Chem. Listy 47, 467 (1953). 23~G. Sferruzza and R. Rangone, Farmaco, Ed. Prat. 18, 322 (1963).
160
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
Detection. Chloramphenicol is detected with B. subtilis, E. coli, b y observing a d a r k zone in UV light or chemically as follows. 1. Spray with 15% titanous chloride in 1 N H C I (diluted 1:10) to reduce the nitro group to amino group. While still damp, hang in an atmosphere of Br: for a few minutes to oxidize excess titanous reagent. Then spray with butyl n i t r i t e - n - b u t a n o l - a c e t i c acid (10:5:1), and 2 min later spray with 0.1% N - ( 1 - n a p h t h y l ) e t h y l e n e d i a m i n e dichloride in n-butanol-2 N HC1 (95:5). Pink spots are given by compounds having an aromatic nitro group (see Dawson et al., p. 538).188 2 . S p r a y with 15% SnCl2-conc. H C l - w a t e r (3:15:180), freshly prepared. D r y at room temperature. S p r a y with a solution of 1 g of p-dimethylaminobenzaldehyde in ethanol-conc. HCl-n-butanol (30:30:180). Chloramphenicol gives yellow spots. Decomposition compounds containing an aryl amino group instead of the nitro group of chloramphenicol react with the second reagent without previous reduction with the first reagent (see Dawson et al., p. 538).1s3
B. Novobiocin
Solvent Systems. Systems used for novobiocin and its derivatives include the following: A: 0.1 M KH,PO4 added to 0.1 M NasHPO4 to pH 8.2 or 8.8 on paper impregnated with sec-octanol-methanol (1: 5) and dried 2~* B: 50 mM Boric acid and 50 mM KC1 in water, pH adjusted to 8.2 or 8.8 with 0.5 M NaOH and diluted to 1 liter, on paper prepared as in A 23. C: Benzene-hexane-methyl ethyl ketone-ethanol (45 : 39 : 13 : 3) on paper buffered with 0.2 M phosphate buffer pH 7.7-7.8 and partially dried 233 D: 0.1 M Phosphate buffer pH 8.2 equilibrated with capryl alcohol on paper the lower part of which was dipped in a mixture of capryl alcohol-methanol (1:5) up to the point of application of the sample and blotted 234 E: Isopropyl ether satd. with ethylene glycol on Whatman No. 4 or 20 dipped in ethylene glycol containing 2% of 85% lactic acid, descending 16 hr at 28 °~3~
Detection. Novobiocin is detected with B. subtilis or in UV light. Applications. Circular development in solvent C is used for quantitative analysis of novobiocin, isonovobiocin, and decarbamylnovobiocin. 2~ Chromatographic data of novobiocin and related compounds are compared in T a b l e X X V I I I . Mobilities in system E reported by K o m i n e k 2~5 ~8~G. Sferruzza and R. Rangone, Farmaco, Ed. Prat. 17, 404 (1962). ~3sV. B. Korchagin, V. V. Stepushkina, and Z. E. Voinova, Antibiotiki 11, 107 (1966). ~u E. J. Wolf and R. Nescot, Antibiot. Annu. 1956/1957, 1035 (1957). ~u L. A. Kominek, Antimicrob. Ag. Chemother. 1, 123 (1972).
[71
PAPER CHROMATOGRAPHY OF ANTIBIOTICS
161
TABLE XXVIII PAPER CHROMATOGRAPHICDATA OF NOVOBIOCIN AND RELATED COMPOUNDS Mobil;ties in systems" Ab
Bb
C~
Compounds
pH 8.2
pH 8.8
pH 8.2
pH 8.8
Novobiocin Dihydronovobiocin Isonovobiocin Decarbamylnovobiocin
1.00 0.61 1.12 1.13
1.00 0.66 1.16 1.17
1.00 0.70 1.14 1.23
1.00 0.80 1.14 1.23
Desc. d Asc.~ 22 -33 47
08 -13 20
Df 25 61 ---
a See solvent systems for novobiocin on p. 160. b R .... bloclnvalues from G. Sferruzza and R. Rangone, Farmaco Ed. Prat. 17, 404 (1962). RI X 100 values from V. B. Korchagin, V. V. Stepushkina, and Z. E. Voinova, Ant;blot;k; 11, 107 (1966). a Descending. Ascending. J RI X 100 values from E. J. Wolf and R. Nescot, Antibiotics Ann. 1956/1957, 1035 (1957). are n o v o b i o c i n acid ~ d e c a r b a m y l n o v o b i o c i n ~ i s o n o v o b i o c i n ~ 0 - d e methyldecarbamylnovobiocin ~ 0-demethylnovobiocin.
C.
Griseofulvin
S o l v e n t S y s t e m s . T h e following s y s t e m s are used for griseofulvin a n d r e l a t e d compounds. A: Water satd. butanol-ammonium hydroxide (20: 1) vs chloroform as stationary phase TM B: Butanol satd. water, vs ethyl acetate as stationary phase 2~6 C : Butanol-ethanol-water (5:1 : 4), vs ethyl acetate as stationary phase 2'~6 D: Butanol-satd. water vs methyl ethyl ketone as stationary phase E: Benzene-cyclohexane-methanol-water (5:5:6:4), organic phase; add 0.5% acetic acid after separation 237 I n U V light, griseofulvin gives blue fluorescent spots. I t s RI v a l u e s in solvents A, B, C, a n d D are 236 0.40, 0.22, 0.37, a n d 0.24, respectively. I n s o l v e n t E the following v a l u e s are obtained~37: griseofulvin, 0.90; 4 - d e m e t h y l g r i s e o f u l v i n , 0.70; g r i s e o f u l v i n acid, 0.20; 6 - d e m e t h y l g r i s e o f u l vin, 0.15. ~3~E. G. McNall, Arch. Dermatol. 81, 657 (1960). ~TB. Boothroyd, E. J. Napier, and G. A. Somerfield, Biochem. J. 80, 34 (1961).
162
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
D. Miscellaneous Aromatic Antibiotics "Summarized chromatograms" of alternariol, chloramphenicol, cyclopaldic acid, flavipin, fomecin A, geodin, gladiolic acid, griseofulvin, novobiocin, and quadrileneatin were included in a systematic paper chromatographic analysis of antibiotics. 1°3 Rr values of the above compounds are given in Table XXIX. XIII. Classification and Systematic Analysis of Antibiotics by Means of Paper Chromatography In spite of the discouraging fact that antibiotics are chemically very heterogeneous, attempts have been made to use paper chromatography for classification and systematic analysis of these substances. A. Salting-out Chromatograms Miyazaki et al. 23s classified antibiotics using nine solvent systems: distilled water and increasing concentrations of ammonium chloride (0.5% to saturated solution) in water. The analyzed antibiotics were divided into four groups, and later two additional groups were added by l~ri. 239 According to the influence of the salt concentration on R~ values, the six groups are illustrated by their representatives in Fig. 2 and are defined as follows. In group A, the RI values are not affected by the salt concentration and are always different from zero. Group B includes antibiotics with an R f of zero in distilled water and increasing R~ values with increasing concentration of the salt. In group C the highest RI is in water and increasing concentrations of the salt cause a decreasing tendency of Rr values. To group D belong antibiotics with RI values equal to zero in all nine systems. In group E, RI is equal to zero in distilled water but has then an initial increase with rising concentrations of the salt up to approximately 1.0 in 5% salt concentration; at higher salt concentrations it decreases again but does not reach zero. In group F, the R~ is equal to zero in distilled water, reaches a maximum at the salt concentration of 3-5% and then decreases back to zero. According to results obtained with antibiotics from actinomycetes 28s-24s and with ~8 j. Miyazaki, K. Omachi, and T. Kamata, .I. Antibiot. 6, 6 (1953). 2,9 j . Uri, Nature (London) 183, 1188 (1959). 240K. Isono, S. Yamashita, Y. Tomiyama, S. Suzuki, and H. Sakai, J. Antibiot. 10, 21 (1957). 241K. Ueda, Y. Okimoto, H. Sakai, and K. Arima, J. Antibiot. 8, 91 (1955). ~2 T. Sawazaki, G. Nakamura, M. Kawasaki, S. Yamashita, K. Isono, K. Anzai, Y. Serizawa, and S. Suzuki, J. Antibiot. 8, 39 (1955). ~s F. D6cio de Andrade Lyra and S. de Barros, R e v . Inst. Antlbidt. 4, 67 (1962). Quoted by Blinov and Khokhlov.*
[7]
163
PAPER CHROMATOGRAPHY OF ANTIBIOTICS TABLE XXIX PAPER CHROMATOGRAPHIC DATA OF SOME AROMATIC ANTIBIOTICSa R/ X I00 in systemsb Antibiotics
A
B
C
D
Alternariol
00
85
97
00
Chloramphenicol Cyclopaldic acid Flavipin Fomecin A Geodin Gladiolic acid Griseofulvin Novobiocin Quadrilineatin
78 00 76 63 00 93 00 75 69
88 89 78 57 88 92 92 85 95
95 98 97 60 93 98 96 92 96
00 00 00 00 94 83 93 00 00
E
F
G
H
79
87
92
94
97 52
64 33
85 77
91 53
21 00
79 93
94 94
73 95
I
J
K
L
00
19
38
00
51
00
73
72 --
77 81 00
9"2 90 92
82 76 86
79 91 00 --
a Selected from V. Betina, J. Chromatogr. 15, 379 (1964). b Solvent systems: A: Water B: n - B u t a n o l satd. with w a t e r C: E t h y l acetate satd. with water D : Benzene satd. w i t h water E: 3 % Aq. a m m o n i u m chloride F: Isoamyl a c e t a t e - m e t h a n o l - 9 9 % formic acid-water (65:20:5: 10), upper layer G: n - B u t y l a c e t a t e - m e t h y l ethyl ketone-0.15 M p H 7.4 p h o s p h a t e buffer (50: 25 : 5), upper layer H: E t h y l acetate-n-hexane-0.15 M p H 6.0 p h o s p h a t e buffer (65 : 15: 20), upper layer I: Isoamyl a c e t a t e - m e t h a n o l - 9 9 % formic acid-water (40:20:10:30), lower layer J : n - B u t a n o l - m e t h a n o l - w a t e r (40: 10: 50), lower layer K : M e t h a n o l - n - h e x a n e (60: 40), lower layer L: Benzene-cyclohexanone-0.15 M p H 7.4 phosphate buffer (5 : 35: 60), lower layer. ~ M. M. Albuquerque, F. D~cio Andrade Lyra, O. Gon~alves de Lima, L. Lins de Oliveira, J. S. de Barros Coelho, G. M. Maciel, and M. Da Salete Barros Cavalcanti, Rev. Inst. Antibiot. 6, 35 (1966). Quoted by Blinov and Khokhlov. 7 :45 S. Wakaki, H. Marumo, K. Tomioka, G. Shimizu, E. Kato, H. Kamada, S. Kudo, and Y. Fujimoto, Antibiot. Chemother. 8, 228 (1958). Quoted b y Blinov and Khokhlov/ ~46F. D6cio de Andrade Lyra, O. GonCalves de Lima, C. S. Barros Coelho, M. M. F. de Albuquerque, G. M. Maciel, L. L. de Oliveira and M. C. N. Maciel, Ate. Acad. Brazil. Cienc. 36, 323 (1964). Quoted by Blinov and Khokhlov. ~ ~47y. Waga, J. Antibiot. 6, 66 (1953). 248M. Arai, K. Karasawa, S. Nakamura, H. Yonehara, and H. Umezawa, J. Antibiot. 11, 14 (1958).
164
METHODS FOR THE STUDY OF ANTIBIOTICS
o
~"
i i 0%
i
i
i
i
I
Sat.
Sat.
%
D
to
o
~,
E
,
\
I
I
%
I
I
I
I
I
[7]
I
sat.
$
6 I
I
I
I
I
I
%
I
I
I
S=t.
I
%
I
I
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I
I
I
I
s=t.
O'/o
Sat.
Fio. 2. Classification of antibiotics by means of salting-out paper chromatography into six groups (A-F). Influences of ammonium chloride concentration in water on RI values in the groups demonstrated for representative antibiotics: 1, patulin; ~, gliotoxin; 3, streptomycin; 4, cephalosporin P1; 5, trichothecin; 6, cyanein; 7, oleandomycin; 8, desertomycin. Plotted according to data from P. Nemec, V. Betina, and L. Kova~i6ov£, Folia Microbiol. (Prague) 6, 277 (1961)--curves 1, 2, 4, 5, and 6; J. Miyazaki, K. Omachi, and Z. Kamata, J. Antibiot. 6, 6 (1953)--curve 3; J. Uri, Nature (London) 183, 1188 (1959)--curves 7 and 8. fungal antibiotics by Slovak authors 3°,16~,~49-~ totally 20 antibiotics belong to group A, 21 to group B, 16 to group C, 20 to group D, 4 to group E, and 5 to group F. 7
B. pH Chromatograms
" p H c h r o m a t o g r a p h y ''~19'12° helps to estimate the ionic character of
antibiotics and also the general possibilities of their isolation and separation. I n antibiotics screening, these important data m a y be obtained at the level of samples from cultivation of producing organisms (filtrates or crude concentrates). A series of chromatographic strips is impregnated with buffers in the range of p H 2-10, and an appropriate water-saturated organic solvent =~Dp. Nemec, V. Betina, and L. Kova~i~ov£, Folia Microbiol. (Prague) 6, 277 (1961). 2~ V. Betina, P. Nemec, J. Balan, and 8. KovhS, Chem. Zvesti (Bratislava) 15, 843 (1961). =1V. Betina, P. Nemec, M. Kutkov£, J. Balan, and 8. Kov£5, Chem. Zvesti (Bratislava) 18, 128 (1964). V. Betina, in "Some General Problems of Paper Chromatography" (I. M. Hais and K. Macek, eds.), p. 153. Czechoslov. Acad. Sci., Prague, 1962.
[7]
PAPER CHROMATOGRAPHY
OF ANTIBIOTICS
165
is used as the mobile phase for each antibiotic sample tested. Four types of pH chromatograms are described in our papers. 119,1~°,2~3-2~7 1. RI values from pH chromatograms of acidic antibiotics give S-shaped curves, the maxima of which lie in the acidic range; with increasing pH values of the stationary phase, the R~ values diminish (Fig. 3A). 2. RI values from pH chromatograms of basic antibiotics give S-shaped curves of a reverse character, the maxima of the curves being in the alkaline and the minima in the acidic range (Fig. 3B). 3. Rs values from pH chromatograms of amphoteric antibiotics increase stepwise in the range of lower pH values, and after reaching a certain maximum they begin to diminish. These compounds behave as
FIG. 3A. See p. 167 for legend. ~ V. Betina, Chem. Zvesti (Bratislava) 15, 661 (1961). 2~4V. Betina, Chem. Zvesti (Bratislava) 15, 750 (1961). V. Betina, Chem. Zvesti (Bratislava) 15, 848 (1961). 2~ V. Betina and P. Nemec, Chem. Zvesti (Bratislava) 15, 853 (1961). ~ V . Betina, Chem. Zvesti (Bratislava) 15, 859 (1961).
Fio. 3B.
FIG. 3C.
[7]
PAPER CHROMATOGRAPHY OF ANTIBIOTICS
167
FIa. 3D. FIG. 3. pH chromatograms of acidic (A), basic (B), amphoteric (C), and neutral (D) antibiotics. Chromatographic strips impregnated with buffers of pH values (from left to right) : 2, 3, 4, 5, 6, 7, 8, 9, and 10. Ascending development. (A) Benzylpenicillin in butyl acetate saturated with water; (B) erythromycin in ethyl acetate saturated with water; (C) tetracycline in n-butanol saturated with water; (D) chloramphenicol in ethyl acetate saturated with water. Bioautography with Bacillus subtilis. Zones of inhibition visualized with 2,6-dichlorophenolindophenol according to V. Betina and L. Pil£tov~, Cesk. Mikrobiol. 3, 202 (1958). From V. Betina, pH-chromatography of Antibiotics (in Slovak), Thesis, Slovak Acad. Sci., Bratislava, 1960. bases from p H 2 to the point of the maximal Rr (indicating possibly their isoelectric points or p I values), and from this point on up to p H l0 they behave as acids (Fig. 3C). 4. The p H chromatograms of neutral antibiotics show the same R r values over the whole range of p H values, giving a straight line (Fig. 3D). The S-shaped curves obtained from the p H chromatograms of several acidic antibiotics represent the dependence of R~ values on the following factors: p H of the stationary phase, distribution coefficients between the mobile phase and the stationary phase of these p H values, and pKa values of the antibiotics studied. 25~,254 This means t h a t the suitable p H value for the extraction of an antibiotic from the aqueous phase with an organic
168
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
solvent immiscible with water, and for the eventual reextraction into aqueous phase, could be determined from its pH chromatogram. The optimal pH value of the aqueous phase for the extraction ought to lie in the range of that pH value where the Rr on the pH chromatogram is greatest. Conversely, the optimal pH value for the reextraction into aqueous phase ought to lie in the range of that pH where the Rf curve reaches its minimum. This was verified by the study of the pH chromatograms of several acidic, basic, and amphoteric antibiotics :53-256 and by isolation of an unknown acidic antibiotic based on its pH chromatogram. 251 S-shaped curves of antibiotics with different pKa values are different. This helped to separate a mixture of natural penicillins (see Fig. 1) and to determine dissociation constants from pH chromatograms. 1~1 "pH chromatography" was also successfully applied in separation of various chloronitrophenols.~8 Some red-blue indicator antibiotics behave differently from the above described pH-chromatographic behavior of other antibiotics. Antibiotics P-125 and litmocidin have RI values equal to 1.0 in the pH range 2-8, and then the values suddenly drop to zero at pH 9. With U-267 this sudden change occurs between pH 7 and 8. On the other hand, Rf values of violarin are zero at pH 2-6 and 1.0 at pH 7-10. The peculiar findings are explained by the indicator character of these antibiotics. For example, P-125 depending on pH of the medium can exist in two forms: one soluble in organic solvents giving a red color (corresponding to the high Rr values on the pH chromatogram; and another one soluble in water giving blue color (corresponding to zero Rr values).259
C. Summarized Chromatograms (Chromatographic Spectra) A number of systems have been proposed for paper chromatographic classification and/or identification of specifically related antibiotics. Examples dealing with chemically related compounds may be found in the previous sections. Some others dealing with antibiotics of similar biological properties are discussed here. Ammann and Gottlieb 26° worked out a method for the separation of antifungal antibiotics on the basis of their R~ values in 7 solvent systems. RI values of 15 antibiotics were reported by utilizing a flow chart for the separation of the compounds on the basis of the chromatographic data. z~sZ. Vanek ~nd J. Stan~k, Collect. Czech. Chem. Commun. 28, 264 (1963). u' D. E. Dykhovichnaya, Antibiotiki 8, 939 (1963). A. Ammann and D. Gottlieb, Appl. Microbiol. 3, 181 (1955).
[7]
PAPER CHROMATOGRAPHY OF ANTIBIOTICS
169
Paper chromatography combined with bioautography was shown to be useful in differentiating antibiotics produced by the Bacillus species. 26~ Antibacterial antibiotics including polypeptides produced by other microorganisms were analyzed in 14 solvent systems. A scheme 262 was worked out for classification of over 20 preparations of indicator antibiotics which were divided into five groups. One of the first attempts to use paper chromatography for a general characterization by means of "summarized papergrams" was that of Ishida et al. ~63 Their 8 systems were used mostly in J a p a n and more than 100 antibiotics were characterized until 1970. 7 De Boer et al. 264 and Hoeksema et al. 26~ characterized various antibiotics by means of 6 solvent systems, to which two additional ones were added by DoskoSilov£ and Vondr£Sek, 266 who characterized 44 antibiotics. Omachi 267 and ~ev~ik 26s used l0 solvent systems in order to characterize solubility of unknown antibiotics; 18 antibiotics from streptomycetes were characterized by these systems. :69 Nemec et al. 2~° modified the above series for characterization of 20 fungal antibiotics. Paper chromatographic separation and identification of microbial metabolites including a limited number of antibiotics, using 6 solvent systems, various detection techniques and graphical presentation of Rs values in form of "chromatographic spectra" were described in papers of Reio. 2~-~77 The techniques described above ~63-27° served for paper chromatographic characterization but not for a simultaneous classification of antibiotics. 2~ N. Snell, K. Ijichi, and J. C. Lewis, Appl. Microbiol. 4, 13 (1956). ~ N. 0. Blinov, G. Z. Yakubov, L. A. Vetlugina, and Y. M. Khokhlova, Mikrobiologiya 30, 642 (1961). :6.~N. Ishida, T. Shiratori, and S. Okamoto, J. Antibiot. 3, 880 (1950). ~¢~C. De Boer, A. Dietz, J. R. Wilkins, C. N. Lewis, and G. M. Savage, Antibiot. A~tnu. 1954/1955, 831 (1955). 2~ H. Hoeksema, G. F. Crum, and W. H. De Vries, Antibiot. Annu. 1954/1955, 837 (1955). ~ D . DoskoSilov£ and M. Vondr£Sek, Antibiotiki 6, 649 (1961). :~'~K. Omachi, J. Antibiot. 6, 73 (1953). ~ V. ~evSik, M. Podojil, and A. Vrti~kovh, Cesk. Mikrobiol. (Prague) 2, 175 (1957). :~ Z. Reh~Sek, Antimicrob. Ag. Chemother. 1963, 530 (1964). ~,op. Nemec, V. Betina, and L. KovaSiSov£, Biologia (Bratislava) 16, 375 (1961). 2'~L. Reio, J. Chromatogr. 1, 338 (1958). ~ L. Reio, Chromatogr. Rev. 1, 39 (1959). 2~ L. Reio, J. Chromatogr. 4, 458 (1960). 274L. Reio, Chromatogr. Rev. 3, 92 (1961). ~,5L. Reio, J. Chromatogr. 13, 475 (1964). :76L. Reio, Svensk Kem. Tidskr. 76, 265 (1964). :~ L. Reio, J. Chromatogr. 47, 60 (1970).
170
METHODS FOR THE STUDY OF ANTIBIOTICS
[7]
Drozen 278 discussing the classification and identification procedures of systematic chromatographic analysis in the light of information theory compared two main different methods of analysis, viz. the so-called sequential (stepwise) method and the simultaneous method. Both of them have advantages and disadvantages. Snell et al. 261 classified and identified peptide antibiotics in terms of principles of sequential analysis. Ishida et al. 268 and most of the other authors mentioned above applied the principle of simultaneous analysis in a series of solvent systems. A combination of simultaneous and sequential analysis of antibiotics was elaborated in our laboratory. 1°3 In the first step, antibiotics are analyzed simultaneously in four principal systems and are classified into 5 classes with 14 subclasses. The classification is then completed by the second step using the additional solvent systems. According to this method, it is not necessary to analyze an antibiotic in many solvent systems as is done in simultaneous analysis. On the other hand, it is possible to compare the "summarized chromatogram" of an antibiotic with others belonging to the same class or subclass. The principle whereby the antibiotics were divided into 5 classes with 14 subclasses, in accordance with their R~ values in the principal systems, and the additional systems for individual classes may be found in the original paper. 1°3 Applications of the above systematic analysis in combination with " p H chromatography" and salting-out chromatography have been discussed elsewhere. 2~9 Another combination of simultaneous and sequential analysis is that of Blinov's group. Over 60 antifungal preparations were divided into 4 groups according to their R~ values in two principal systems, and a detailed scheme depending on R~ values in additional systems helped to divide antibiotics in each group. 2s° More than 300 antibacterial antibiotics were classified into 5 groups according to their R~ values in three principal systems and again further division was made in accordance with the R~ values in additional systems. ~sl "Chromatographic maps" were plotted showing positions of individual antibiotics on two-dimensional charts with RI values in butanol and chloroform (both water saturated). Based on the same concept from information theory, the solvents used for antibiotics by Ishida et al. 268 and Betina ~°s were evaluated relative 2T~V. Drozen, in "Some General Problems of Paper Chromatography" (I. M. Hais and K. Macek, eds.), p. 213, Czechoslov.Acad. Sci., Prague, 1962. ~7~V. Betina, Antimicrob. Ag. Chemother. 1966, 637 (1967). ~8oy. M. Khokhlova, A. V. Puchnina, E. F. Oparysheva, L. M. Golovkina, and N. O. Blinov, Izv. Akad. N a u k SSSR Set. Biol. 1966, 433. ~ N. O. Blinov, E. F. Oparysheva, Y. M. Khokhlova, A. V. Lesnikova,K. M. Khryas: cheva, and A. S. Khokhlov, Antibiotiki 13, 283 (1968).
[7]
171
P A P E R CHROMATOGRAPHY OF ANTIBIOTICS
to each other b y m e a n s of s u m m a r i z e d c h r o m a t o g r a p h y . "82 I t was f o u n d t h a t a m o n g the 11 solvents tested, those t h a t gave the most i n f o r m a t i o n were water, w a t e r said. b u t a n o l , 3 % aq. a m m o n i u m chloride, b e n z e n e m e t h a n o l ( 4 : 1 ) , a n d b u t a n o l - m e t h a n o l - w a t e r ( 4 : 1 : 2 ) with a n d w i t h o u t helianthin. A l t h o u g h W a g m a n a n d W e i n s t e i n (ref. 8, p. 2) j u d g e d t h a t " p r o b a b l y the greatest single influence in the s y s t e m a t i c a n a l y s i s was t h a t of B e t i n a , " I feel t h a t a g e n e r a l l y accepted p a p e r c h r o m a t o g r a p h i c systematic a n a l y s i s for h u n d r e d s of a n t i b i o t i c s h i t h e r t o u n k n o w n is still a great t a s k ahead.
Addendum A n t i b i o t i c G-418, a new a m i n o g l y c o s i d e c o n t a i n i n g 2 - d e o x y s t r e p t a m i n e , can be differentiated from other a m i n o g l y c o s i d e s b y c h r o m a t o g -
TABLE XXX R / VALUES OF ANTIBIOTIC G-418 AND OTHER AMINOGLYCOSIDE ANTIBIOTICS a
R s X 100 in systems b Antibiotics G-418 Gentamicin C1 Gentamicin C.~ Gentamicin Cja Neomycin Kanamycin Paromomycin
Ac
Bc
Cc
D~
E d'e
57 57 56 48 0-17 0-28 0-28
35 34 30 22 05 08 07
45 45 45 45 0-12 0-17 0-20
41 74 65 57 03 07 04
05 67 40 21 0 0 0
Adapted from G. H. Wagman, R. T. Testa, J. A. Marqucz, and M. J. Weinstein, Antimicrob. Ag. Chemother. 6, 144 (1974). b Solvent systems: A: 80% Methanol containing 3% NaC1 on paper buffered with 0.95 M Na~SO4 -t- 50 mM NariS04 B: Propanol-pyridine-acetic acid-water (6 : 4:1 : 3) C: 80% Phenol D: Chloroform-methanol-conc. ammonia (1:1:1) on Chromar sheet 500 E: Chloroform-methanol-17% ammonia (2: 1:1) Ascending. d Descending. R¢ = distance of zone from origin/distance from origin to end of paper; where t = 6hr. 282j. Souto and A. G. De Valesi, J. Chromatogr. 46, 274 (1970).
172
METHODS FOR THE STUDY OF ANTIBIOTICS
[8]
raphy in five solvent systems followed by bioautography with aureus A T C C 6538P (Table X X X ) . G-418 and five k n o w n aminoglycosides (verdamicin, gentamicin, k a n a m y c i n , sisomicin, and t o b r a m y c i n ) were h y d r o l y z e d as the free bases in 6 N hydrochloric acid and the p r o d u c t s of hydrolysis were c o m p a r e d b y c h r o m a t o g r a p h y on W h a t m a n No. 1 p a p e r using the following solvent systems: (1) n - b u t a n o l - p y r i d i n e - a c e t i c a c i d - w a t e r ( 6 : 4 : 1 : 3 ) and (2) p r o p a n o l p y r i d i n e - a c e t i c a c i d - w a t e r ( 1 5 : 1 0 : 3 : 1 2 ) . D e t e c t i o n of n i n h y d r i n - p o s i tive spots was carried out b y s p r a y i n g the c h r o m a t o g r a m s with 0.25% n i n h y d r i n in a mixture of p y r i d i n e - a c e t o n e (1:1) and heating at 105 ° for several minutes to develop colored zones. 2s~ Staphylococcus
~3 G. H. Wagman, R. T. Testa, J. A. Marquez, and M. J. Weinstein, Antimicrob. Ag. Chemolher. 6, 144 (1974).
[8] Thin-Layer Chromatography of Antibiotics B y ADORJAN ASZALOS and DAVID FROST
I. Introduction . . . . . . . . . . . . . . . . . . II. Principles and Techniques of Thin-Layer Chromatography (TLC) . A. Adsorption Chromatography . . . . . . . . . . . . . B. Partition Chromatography . . . . . . . . . . . . . C. Apparatus, Adsorbents, and General Technique . . . . . . D. Special Techniques . . . . . . . . . . . . . . . E. Reproducibility of R/Values . . . . . . . . . . . . . F. RM Values . . . . . . . . . . . . . . . . . . G. Preparative TLC . . . . . . . . . . . . . . . . H. Quantitative TLC . . . . . . . . . . . . . . . . III. Thin-Layer Chromatographic Systems for the Separation and Classification of Antibiotics . . . . . . . . . . . . . . IV. Use of Thin-Layer Chromatography in the Biosynthesis and Biotransformation of Antibiotics . . . . . . . . . . . . A. Antibiotic Biosynthesis and Transformation by Fermentation . B. Antibiotic Transformation by Single Enzymes . . . . . . C. Antibiotic Transformation in Vivo . . . . . . . . . . .
.
172 173 173 174 175 179 182 183 184 185 186
.
201 201 206 209
I. I n t r o d u c t i o n
T h i n - l a y e r c h r o m a t o g r a p h y ( T L C ) is a v e r y widely used separation procedure. I t combines the a d v a n t a g e s of paper and column c h r o m a t o g r a p h y , is relatively inexpensive, and is both rapid and simple. T h e usefulness of T L C was first d e m o n s t r a t e d in the separation of lipids, steroids,
172
METHODS FOR THE STUDY OF ANTIBIOTICS
[8]
raphy in five solvent systems followed by bioautography with aureus A T C C 6538P (Table X X X ) . G-418 and five k n o w n aminoglycosides (verdamicin, gentamicin, k a n a m y c i n , sisomicin, and t o b r a m y c i n ) were h y d r o l y z e d as the free bases in 6 N hydrochloric acid and the p r o d u c t s of hydrolysis were c o m p a r e d b y c h r o m a t o g r a p h y on W h a t m a n No. 1 p a p e r using the following solvent systems: (1) n - b u t a n o l - p y r i d i n e - a c e t i c a c i d - w a t e r ( 6 : 4 : 1 : 3 ) and (2) p r o p a n o l p y r i d i n e - a c e t i c a c i d - w a t e r ( 1 5 : 1 0 : 3 : 1 2 ) . D e t e c t i o n of n i n h y d r i n - p o s i tive spots was carried out b y s p r a y i n g the c h r o m a t o g r a m s with 0.25% n i n h y d r i n in a mixture of p y r i d i n e - a c e t o n e (1:1) and heating at 105 ° for several minutes to develop colored zones. 2s~ Staphylococcus
~3 G. H. Wagman, R. T. Testa, J. A. Marquez, and M. J. Weinstein, Antimicrob. Ag. Chemolher. 6, 144 (1974).
[8] Thin-Layer Chromatography of Antibiotics B y ADORJAN ASZALOS and DAVID FROST
I. Introduction . . . . . . . . . . . . . . . . . . II. Principles and Techniques of Thin-Layer Chromatography (TLC) . A. Adsorption Chromatography . . . . . . . . . . . . . B. Partition Chromatography . . . . . . . . . . . . . C. Apparatus, Adsorbents, and General Technique . . . . . . D. Special Techniques . . . . . . . . . . . . . . . E. Reproducibility of R/Values . . . . . . . . . . . . . F. RM Values . . . . . . . . . . . . . . . . . . G. Preparative TLC . . . . . . . . . . . . . . . . H. Quantitative TLC . . . . . . . . . . . . . . . . III. Thin-Layer Chromatographic Systems for the Separation and Classification of Antibiotics . . . . . . . . . . . . . . IV. Use of Thin-Layer Chromatography in the Biosynthesis and Biotransformation of Antibiotics . . . . . . . . . . . . A. Antibiotic Biosynthesis and Transformation by Fermentation . B. Antibiotic Transformation by Single Enzymes . . . . . . C. Antibiotic Transformation in Vivo . . . . . . . . . . .
.
172 173 173 174 175 179 182 183 184 185 186
.
201 201 206 209
I. I n t r o d u c t i o n
T h i n - l a y e r c h r o m a t o g r a p h y ( T L C ) is a v e r y widely used separation procedure. I t combines the a d v a n t a g e s of paper and column c h r o m a t o g r a p h y , is relatively inexpensive, and is both rapid and simple. T h e usefulness of T L C was first d e m o n s t r a t e d in the separation of lipids, steroids,
[81
THIN-LAYER CHROMATOGRAPHY OF ANTIBIOTICS
173
and nucleic acids. The method was soon applied to the separation of other types of compounds, such as alkaloids, barbiturates, antibiotics, and other pharmaceutical compounds. T L C m a y be used in antibiotic research to follow the production of an antibiotic during fermentation or during enzymic transformation, to monitor the purification of an antibiotic, to characterize and classify pure antibiotics and impure antibiotic p r e p a r a tions, and to assay quantitatively antibiotics in bulk or in pharmaceutical preparations. We have divided this article into three sections: in the first, we deal with some theoretical considerations and with the different techniques and materials involved in TLC. In the second section, we describe T L C systems used for the separation, identification, and classification of m a n y antibiotics. The third section deals with the use of T L C in biosynthesis and biotransformation of antibiotics. More details on these subjects and on the use of T L C in antibiotic research can be found in the various references cited. 1-7 Different authors use different nomenclature in texts on TLC. To avoid misunderstandings, we have used the nomenclature of Niederwieser and P a t a k i throughout this chapter, a
I I . Principles a n d Techniques of T h i n - L a y e r C h r o m a t o g r a p h y (TLC) A. A d s o r p t i o n C h r o m a t o g r a p h y
The physicochemical phenomenon of adsorption can be used to separate mixtures of compounds according to the numbers and types of their functional groups. Separation occurs because of differences in the reversi1,,Thin_Layer Chromatography: A Laboratory Handbook" (E. Stahl, ed.), 2nd ed. Springer-Verlag, Berlin and New York, 1969. 2j. G. Kirchner, J. Chromatogr. 82, 31 (1973). 3"Techniques of Thin-Layer Chromatography in Amino Acid and Peptide Chemistry" (G. Pataki, ed.), 2nd Engl. ed. Ann Arbor-Humphrey Sci. Publ., Ann Arbor, Michigan, 1969. 4K. Randerath, "Thin-Layer Chromatography" (transl. by D. D. Libman), 2nd rev. ed. Verlag Chemie, New York, 1966. ~J. Chromatogr., Suppl. 2 (1972) (K. Macek, I. M. Hais, J. Kopecky, J. Gasparic, V. Rabek, and J. Churacek, eds.). Elsevier, Amsterdam, 1973. J. W. Copius-Peereboom, in "Comprehensive Analytical Chemistry" (C. L. Wilson and D. W. Wilson, eds.), Vol. IIc, p. 77. Elsevier, Amsterdam, 1971. ~V. Betina, in "Pharmaceutical Application of Thin Layer and Paper Chromatography" (K. Macek, ed.), p. 503. Elsevier, Amsterdam, 1972. s "Progress in Thin-Layer Chromatography and Related Methods" (A. Niederwieser and G. Pataki, eds.) V.ol. I, p. xii. Ann Arbor-Humphrey Sci. Publ., Ann Arbor, Michigan, 1970.
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ble physical surface forces exerted by the active centers of a solid stationary phase on the functional groups of each component of the mixture. Functional groups that influence adsorption include alcohol, aldehyde, carboxyl, amine, and other polar groups. Unsaturated compounds are adsorbed more strongly than are their saturated homologs, and trans compounds are more strongly adsorbed than are their cis isomers. The formation of hydrogen bonds and the occurrence of steric effects influence adsorption chromatographic behavior markedly. Many materials can be used as adsorbents: starch, cellulose, silica gel, silicic acid, aluminum oxide, magnesium silicate (Florisil®), calcium phosphate, activated carbon, etc. For elution, one may use apolar solvents or mixtures of polar and apolar solvents. Adsorption chromatography is suited best to the separation of lipophilic substances. Very polar compounds, such as sugar-containing antibiotics, must be derivatized (e.g., acetylated) before their chromatographic separation is attempted. The theoretical treatment of the adsorption equilibria existing between different types of compounds that are to be separated chromatographically and the polar groups of different adsorbents is detailed in the literature2 A study of two-component mobile phases has established a relationship between RI values and the mobilities of chromatographed materials. In brief, for a two-component mobile phase, a value (K s) can be calculated from the position of the front of the mobile phase (fl) and that of the slower-moving, more polar component of the mobile phase (a). This K s value also depends on the initial concentrations of the two components. From K s and the initial concentration of the more polar component, the adsorption coefficient of this component can be calculated. As a continuation of the calculation, a connection can be established between K s and the Freundlich equation. Additional calculations and experiments have indicated that there exists a relationship between the RI values of different materials traveling behind the a and fl fronts and the K s of the mobile phase employed. This study is the first step toward expressing a relationship, for different compounds, between the RI value and the mechanism of adsorption. 1° B. Partition Chromatography Partition chromatography, a liquid-liquid chromatographic system, utilizes the differences in solubility in two immiscible solvent systems of the various constituents of a mixture. In this case, members of the P G. Szasz, Gyogyszereszet 15, 10 (1971). lo A. Niederwieser and M. Brenner, Experientia 21, 105 (1965).
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mixture are separated according to the influences on solubility exerted by their functional groups. Although steric effects and formation of hydrogen bonds affect the separation process only weakly, the effects of solvation are pronounced. Substances used for the stationary phase are chiefly water, glycols, formamide, and dimethyl sulfoxide, alone or in combination with each other. As the mobile phase, i.e., the immiscible second phase, any of a variety of solvents or solvent mixtures can be used. In partition TLC, the plates are usually covered with a suspension of the solid support in the stationary phase and are not dried or are only partially dried. But other techniques are also used, such as applying the stationary phase in a 20% solution of acetone, then evaporating the latter. Cellulose, silica gel, or alumina may be used as the solid support. In partition TLC, the chamber is usually saturated with both phases. The theories of partition chromatography, which are based on either equilibrium or kinetic approaches, are well treated in the literature." TM According to the kinetic approach, which is very popular, steady-state conditions must exist during partition chromatography, for the total concentrations of solute in the phases can be described by constants that depend only on the partition coefficients. These coefficients influence peak velocity, spreading, and other chromatographic characteristics. For details and calculations, one should consult the literature.
C. Apparatus, Adsorbents, and General Technique
It is the purpose of this subsection to give a practical outline of the apparatus, adsorbents, and common techniques used in TLC, without specific details. For details, we recommend any of the excellent textbooks on these topics. 1,1~ In TLC, a chromatoplate, which may be either a solid support coated with a stationary phase or a commercially available precoated sheet, is used to provide an adsorbent with the desired characteristics (type, thickness, etc.). The sample to be tested is applied in solution to the chromatoplate, which is then placed into a chamber that contains the mobile phase, 11H. Vink, J. Chromatogr. 18, 25 (1965). 22H. W. Habgood, Annu. Rev. Phys. Chem. 13, 259 (1962). 23C. J. Hardy and F. H. Pollard, ]. Chromalogr. 2, 1 (1959). 24j. W. Copius-Peereboom, i~ "Comprehensive Analytical Chemistry" (C. L. Wilson and D. W. Wilson, eds.), Vol. IIc, p. 5. Elsevier, Amsterdam, 1971. l~"Thin-Film Chromatography" (E. V. Truter, ed.), p. 1. Cleaver-Hume, London, 1963.
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and the latter is allowed to travel the length of the chromatoplate so that the chromatogram is developed. The chromatoplate is removed from the chamber and, after evaporation of the mobile phase, individual spots are detected. Individual spots may be removed from the chromatoplate for further evaluation. In the preparation of a chromatoplate, coating of the hard surface (usually made of glass, aluminum, or plastic) is best achieved by using a special applicator to spread the dry or wet powder, which constitutes the solid support in the case of partition chromatography and the adsorbent in the case of adsorption chromatography. Such special applicators are described in various textbooks. As an alternative, a slurry of the powder may be poured onto the plate, or the plate may be dipped into a suspension of the powder. Usually, a binder, such as gypsum, polyacrylate, polyethylene glycol, or an alkali metal silicate (1-10%), is mixed with the adsorbent in either of the two latter techniques. Many precoated chromatoplates are available commercially, e.g., Analtech and Merck silica gel and cellulose glass plates and Eastman Chromagram plastic sheets, with cellulose, silica gel, or alumina adsorbents. These chromatoplates are available with or without a fluorescent indicator incorporated into them. Many other interesting support and adsorbent systems have been developed for special purposes, but these are beyond the scope of this text. So are the differently shaped support areas used to achieve optimal separation of certain materials. After the layer of powder has been poured or dipped, the suspending solvent (usually water) is removed by heating the plate to 110-150 ° . The desired activity of the adsorbent determines the temperature and time of heating. Plates are stored in a desiccator, and are generally equilibrated with moist air before use. The amount of adsorbed moisture in the chromatoplate has a strong influence on the separation achieved; this factor is discussed in the subsection on the reproducibility of Rs values. The adsorbents or solid supports used in TLC are of many kinds. Nowadays a great variety of well-tested adsorbents is available commercially. Silica gel of various qualities is available, differing in surface area, specific pore volume, activity, and mesh size. Alumina varies in its aluminum hydroxide content and in the process used to prepare it, as well as in activity, pH (in water), and mesh size. Kieselguhr, which is composed of silicic acid and its derivatives, e.g., magnesium and calcium silicates, is often used in TLC. Other inorganic support materials are calcium phosphate, calcium sulfate, glass powder, ferric oxide, zinc oxide, zinc carbonate, bentonite, and activated carbon. Organic supports or adsorbents include cellulose preparations (fibrous and crystalline), cellulose derivatives (acetate and various ethers),
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starch, partially hydrolyzed starch, sucrose, dextran gel, and polyamides (Perlon ® and nylon). Detailed discussions of adsorbents and solid supports may be found in the textbook by Stahl. TM As the second step in TLC, a solution of the material to be examined chromatographically is applied to the chromatoplate at the "start," which is about 0.5-1.0 cm from the lower edge of the plate and about 0.2-0.5 cm from the location of the mobile phase when the chromatoplate is placed into the chamber. The material should be distributed uniformly in a spot of minimal area. The spot should be about 0.5-1.0 cm from either side of the plate, to prevent uneven travel of the spot near the side of the plate. The solvent of the applied material must be evaporated before the plate is developed. Chromatographic separation is very much affected by the way in which the sample is applied to the plate. Many devices have been developed to provide uniform application of the sample to the plate and to permit the use of automated procedures; for details, see the references cited. 17-1.~ After the solvent of the applied material has been evaporated, the chromatogram is developed. As mentioned earlier, the level of the mobile phase should be about 0.2-0.5 cm below the "start." An even flow of the mobile phase and a solvent equilibrium should be achieved before the mobile phase reaches the "start." Most often, TLC employs an ascending technique, i.e., the mobile phase travels upward. The mobile phase is placed in the bottom of the chamber and is left there to achieve an equilibration of the chamber's atmosphere with solvent vapors before the chromatoplate is put into position. The influence of vapor pressure in TLC is discussed in the section on reproducibility of Rr values. A chromatogram can be developed two-dimensionally or even threedimensionally.2° In either case, the chromatoplate is put into the same or a different mobile phase at 90 ° to the prior direction of development. Before the plate is put into a new mobile phase, however, the prior mobile phase is evaporated. Sometimes multiple development is used to achieve better separation, i.e., a second development with the same mobile phase is made in the same direction. 2~-23 l~,,Thin_Layer Chromatography: A Laboratory Handbook" (E. Stahl, ed.), 2nd ed., pp. 6 and 918. Springer-Verlag, Berlin and New York, 1969. 17j. M. Walsh, J. Chem. Educ. 48, 415 (1971). i8 F. K. Klein and H. Rapoport, J. Chromatogr. 47, 505 (1970). 1~H. P. Freeman, J. Ass. O~c. Anal. Chem. 54, 216 (1971). F. Jimeno de Osso', J. Chromatogr. 60, 270 (1971). 2~H. Wiele and E. Horak, J. Chromatogr. 47, 527 (1970). ~2j. D. Craske and R. A. Edwards, J. Chromatogr. 51,237 (1970). _~3G. M. Adams and T. L. Sallee, J. Chromatogr. 49, 552 (1970).
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Chambers used in TLC range in complexity from simple coffee jars to commercially available ground-glass chambers and on to sophisticated so-called BN (Brenner and Niederwieser) chambers for horizontal development and controlled-moisture GS (Geiss and Schlitt) chambers. All these chambers are described in various textbooks and papers. 24-26 In general, these chambers are set up in temperature-controlled rooms, are shielded from sunlight, and are not disturbed during the development of a chromatogram. An interesting technique is drum TLC, in which unlimited migration of the applied materials allows for maximum possible separation of the components. This technique and its comparison with other techniques are well described in the literature. 27 The third step in TLC is evaporation of the mobile phase and detection of the spots. After the chromatogram has been developed, solvents are evaporated either by letting the chromatogram hang, by applying an airstream to it, by heating it, or by some combination of these methods. If the chromatographed materials are colored, the center of each spot is located and the Rr values are calculated (RI = the distance of travel of each spot/the distance of travel of the mobile phase). It is good practice, in the case of identification chromatography, to include reference samples on the chromatogram. If the chromatographed materials are colorless, the spots must be made visible, either by exposure to ultraviolet light, by spraying with a reagent, by spraying with dilute sulfuric acid, followed by heating, or by biological means. Compounds that absorb ultraviolet light and fluoresce can be detected directly; compounds that do not fluoresce under ultraviolet light can be detected against a fluorescent layer of a solid support. Chromatoplates that incorporate a fluorescent indicator are available commercially. Both short- and longwave ultraviolet detection boxes are also available commercially. The type of spray reagent used depends on the nature of the material to be detected. Most organic compounds can be detected when sprayed with 2',7'-dichlorofluorescein, chromic-sulfuric acid solution, or sulfuric acid solution. Exposure to iodine vapors is a technique frequently used for the detection of organic materials. The number of specific spraying agents, is quite large; for details, the reader is referred to textbooks or
~ H. Naghizadeh-Nouniaz and H. Lamotte, Bull. Soc. Chim. Fr. 1971, 1515. ~ R. Takeshita, Chem. Pharm. Bull. 19, 80 (1971). =eA. V. De Thomas, C. R. De Thomas, R. Lazar, and :D. Verrastro, Microchem. J. 16, 52 (1971). ~7D. L. Saunders and L. R. Snyder, in "Advances in Chromatography 1970" (A. Zlatkis, ed.), p. 307. University of Houston, Houston, Texas, 1970.
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to the review by Zweig and Sherma. 2s Spray apparatus, heating equipment, and other helpful accessories are also described in the textbooks. 4,29 If the chromatographed material has been radiolabeled, its location can be detected by the use of commercial equipment designed for this purpose or by autoradiography?°-a2 Biologically active materials can be detected by bioautography, a technique in which developed chromatoplates are placed on agar seeded with a sensitive microorganism, and any clear zones present in the agar after incubation are evaluated in terms of R~ values. 33,34 For a chromatoplate with colored spots, precise RI values can be established by the use of a photodensitometer.
D. Special Techniques Certain cases of TLC require special techniques. Such techniques are numerous, but we will describe here only a few that are used for antibiotics and related materials. Solvents that contain buffers,35 acids, or bases 36 are frequently used in adsorption and partition TLC to effect optimal separation of charged materials. A buffered mobile phase has also been used in reversed phase TLC. 37 Buffers may be incorporated into the support material during the preparation of the plate? 8 Commercially available chromatoplates are sprayed with or immersed in buffers for a short time before they are used. 33,39 The use of these techniques permits the classification of differently charged antibiotics. Gradient TLC is a technique that utilizes a layer having a progressive change or gradient in separating ability derived, for example, from a gradient in pH 4° or in thickness. 41,~2 28 G. Zweig and J. Sherma, Anal. Chem. 44, 52R (1972). 29"Thin-Layer Chromatography: A Laboratory Handbook" (E. Stahl, ed.), 2nd ed., p. 79. Springer-Verlag, Berlin and New York, 1969. 3oj. Gazso and K. G. Bartha, Kiserl. Orvostud. 22, 108 (1970). 31C. O. Tio and S. F. Sisenwine, J. Chromatogr. 48, 555 (1970). E. Cremer and E. Seidl, Monatsh. Chem. 101, 1614 (1970). 33A. Aszalos, S. Davis, and D. Frost, J. Chromatogr. 37, 487 (1968). H. Bickel, E. G~umann, R. Hiitter, W. Sackman, E. Vischer, W. Voser, A. Wettstein, and H. Z~ihner Helv. Chim. Acta 45, 1396 (1962). 35N. D. Gyanchandani, I. J. McGilveray, and D. W. Hughes, J. Pharm. Sci. 59, 224 (1970). 3,,T. Ikekawa, F. Iwami, E. Akita, and H. Umezawa, J. Antibiot. SeT. A 16, 56 (1963). ~ G. L. Biagi, A. M. Barbaro, and M. C. Guerra, J. Chromatogr. 51,548 (1970). '~ W. Sobiczewski and M. Domaradzki, Chem. Anal. (Warsaw) 16, 131 (1971). 3~S. Ochab and B. Borowiecka, Di~s. Pharm. Pharmacol. 21, 359 (1969). 4~E. Stahl and E. Dumont, J. Chromatogr. Sci. 7, 517 (1969). 41B. Warren, J. Chromatogr. 20, 603 (1965). 42 N. G. Bazan, Jr. and C. D. Joel, J. Lipid Res. 11, 42 (1970).
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In gradient-elution TLC, different solvents are mixed together continuously in order to change the eluting power of the mobile phase. This technique has proved very useful for separating nucleotides.4a,44 A new special technique is programmed multiple development (PMD) chromatography. Here, the distance traveled by the mobile phase is controlled electronically by intermittent application of :infrared radiation to evaporate the solvent between consecutive developings. It is claimed that PMD provides a sharper separation of components in a shorter time than dues conventional multiple development.~5 Circular TLC has a resolving power better than that of simple onedimensional TLC in the same solvent system. In circular TLC, the mobile phase is introduced at the center of a circular chromatoplate and the materials are spotted on different radii of the plate, equidistant from the center. During chromatography, the materials travel toward the periphery. The technique has been used to separate closely related materials, such as the quinomycin and actin0mycin antibiotics. 46 Reversed-phase partition chromatography is a special kind of liquidliquid chromatography in which the less polar phase of a pair of immiscible solvents is used as the stationary phase. Separation occurs because of differences in the solubility of the components of a mixture. However, in contrast to their behavior in adsorption or partition chromatography, in reversed-phase partition chromatography compounds that are more polar migrate ahead of those that are less polar. The support material, e.g., cellulose or silica gel, is impregnated with the stationary phase, which can be paraffin or silicone oil. This impregnation is best accomplished by letting a 5-10% ether or acetone solution of the stationary phase migrate the length of the plate. The solvent used for developing the chromatogram is usually a 25-50% mixture of water and a watermiscible solvent, such as methanol, acetone, acetic acid, or acetonitrile. This technique has worked very well in separating different cephalosporins 47 and penicillins,4s with silicone oil used as the stationary phase and a mixture of Veronal buffer (pH 7.6) and acetone used as the mobile phase. The R~ values obtained permitted deductions about the biological activities of the various cephalosporin derivatives examined. Ion-exchange TLC is frequently used for the separation of charged 48T. Wieland,G. Luben, and H. Determann, Experientia 18, 431 (1962). 44E. Randerath and K. Randerath, J. Chromatogr. 16, 126 (1964). Chem. Eng. News 51, 14, August 13, 1973. J. Shoji, J. Chromatogr. 26, 306 (1967). 4~G. L. Biagi, A. M. Barbaro, M. C. Guerra, and M. F. Gamba, J. Chromatogr. 44, 195 (1969). A. E. Bird and A. C. Marshall, J. Chromatogr. 63, 313 (1971).
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materials. Separation is effeeted by the formation of reversible ionic bonds between the stationary phase and the charged materials. The rate of migration of a compound is influenced by the net charge on its ionized groups and by their dissociation constants. It is possible, therefore, to fractionate mixtures of ionic compounds by a proper selection of the pH and ionic strength of the mobile phase. CM cellulose, DEAE cellulose, ECTEOLA cellulose, or polyethylene imine cellulose is frequently used as the stationary phase. The general technique involved in ion-exchange TLC is similar to that for regular TLC. Ion-exchange TLC on ECTEOLA and DEAE cellulose has been used for the fractionation of nucleosides~9; ion-exchange TLC on resin has been used for the separation of amino acids and of water-soluble basic antibiotics. ~°,5~ Sephadex is used as the stationary phase in TLC to separate materials of different molecular weights. Sephadex has been used successfully, in combination with bioautographic evaluation, to separate a mixture of 17 antibiotics2 ~ Reflectance spectroscopy is frequently used in both qualitative and quantitative TLC. It had been found that analytically useful data could be obtained by the application of reflectance spectroscopy to samples in powdered form. The method was easily extended to TLC. Reflectance spectroscopy can be used, in particular cases, to overcome two shortcomings of TLC, namely, that Rs values cannot always be reproduced well and that quantitative removal of the sample from the developed chromatogram is tedious and sometimes results in decomposition of the sample. In brief, the method involves the scanning of the chromatogram and the recording of a reflectance spectrum. ~,5~ Materials that fluoresce or absorb in the visible or the ultraviolet region can be scanned directly. Other materials are made visible by spraying them with chromogenic reagents. Reflectance spectroscopy has been used for qualitative and quantitative estimations of aspirin, ~5 vitamins, ~6 and opium alkaloids, among others. For a discussion of the theory of reflectance spectroscopy 57 and for de49K. Randerath, Angew, Chem. 74, 484 (1962). 5oj. K. Pauncz, J. Antibiot. Set. A 25, 677 (1972). 51T. Devenyi, J. Bati, J. Kovacs, and P. Kiss, Acta Biochim. Biophys. Acad. Sci. Hung. 7, 237 (1972). 52 M. H. J. Zuidweg, J. G. Oostendorp, and G. J. K. Bos, J. Chromatogr. 42, 552 (1969). 5a H. Struck, It. Karg, and H. Jork, J. Chromatogr. 36, 74 (1968). 5, H. Z. Jork, Z. Anal. Chem. 221, 17 (1966). 55V. T. Lieu and M. M. Frodyma, Talanta 13, 1319 (1966). ~5M. M. Frodyma and V. T. Lieu, Anal. Chem. 39, 814 (1967). ~ R. W. Frei, in "Progress in Thin-Layer Chromatography and Related Methods" (A. Niederwieser and G. Pataki, eds.), Vol. II, p. 1. Ann Arbor Sci. Publ. Ann Arbor, Michigan, 1971.
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scriptions of the apparatus available, the reader is referred to the literature. 58 T L C has also been used as a pilot technique for rapid-column chromatography, as detailed by Geiss and Schlitt. ~9
E. R e p r o d u c i b i l i t y o f R j V a l u e s
Because T L C is very often used to characterize and identify unknown compounds, the reproducibility of Rt values is of great importance. A major step forward in this field was taken when Stahl introduced standard materials and techniques to TLC. However, it is still accepted that, even under the most rigorous conditions, R~ values are not to be regarded as definite proofs of identity. The influences of several factors affecting the reproducibility of Rr in T L C are described briefly below. For detailed discussions, one m a y consult the proceedings of a symposium devoted to reproducibility in paper and thin-layer chromatography 6° and the discussion by De Zeeuw21 Solvent equilibrium in the adsorbent and in the vapor phase is a very important factor with respect to the reproducibility of R~ values22-6. The influence of this factor can be attributed to several physicochemical events that take place during TLC. The separation of components of solvents occurs at the moving front on the adsorbent, as in frontal analysis; from a mixed-vapor phase, preferential adsorption of a solvent occurs on the " d r y " part of the layer. The solvent preferentially adsorbed as a vapor may be different from the fastest component of the moving solvent system; adsorption and evaporation of the mobile phase occur in the "wet" part of the layer. The extent to which these physicochemical events affect chromatography depends on several factors, such as vapor pressure, relative affinities of the adsorbent for the components of the solvent system, conditions and geometry of the chamber, and saturation ~8H. Ganskirt, in "Thin-Layer Chromatography: A Laboratory Handbook" (E. Stahl, ed.), 2nd ed., p. 142. Springer-Verlag, Berlin and New York, 1969. F. Geiss and H. Schlitt, ]. Chromatogr. 82, 5 (1973). eo"Proceedings of the Third International Symposium on Reproducibility in Paper and Thin-Layer Chromatography" (M. Lederer, K. Macek, and I. M. Hais, eds.), J. Chromatogr. 33, (1968). e, R. A. DeZeeuw, in "Progress in Thin-Layer Chromatography and Related Methods" (A. Niederwieser and G. Pataki, eds.), Vol. III, p. 39. Ann Arbor Sci. Publ., Ann Arbor, Michigan, 1972. 62T. Takeuchi, Y. Suzuki, and Y. Yamazaki, Bunseki Kagaku 19, 926 (1970). a j. H. Dhont, J. C. de Beauveser, and G. G. Kuijpers, J. Chromatogr. 60, 265 (1971). *~F. Geiss, J. Chromatogr. 53, 620 (1970).
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parameters. These factors influence each other in a complicated way, as has been shown in detailed studies by De Zeeuw2 ~,6(~ In addition to the solvent and vapor phases, the qualities of the adsorbent also influence the reproducibility of RI values; these qualities include particle size, pore volume and diameter, number of active sites at the surface, and adsorption capacity. The adsorption capacity is related to the moisture content of the adsorbent. This physisorbed water prevents the adsorption of other less-polar solvents and, thus, makes the adsorbent less active. The amount of physisorbed water in silica gel, for example, depends on the number of surface hydroxyl groups c'7 and the extent to which the plate is exposed to a moist atmosphere during chromatography2 s,~'' To achieve good separation of components and good reproducibility of R~ values, unsaturated rectangular chambers arc used with multicomponent solvent systems and sandwich chambers are used with single solvents. 7° Temperature seems to affect reproducibility in a rather complicated way, 7~ but running distance affects it only when a multicomponent solvent system is used. 72 Additional factors that influence reproducibility are the load of the sample, the thickness of the adsorbent layer, and the spotting technique used. 6°,6~ Brodasky 73 compared the reproducibility of Rr values of antibiotics (actithiazic acid, chloramphenicol, and chartreusin) in paper chromatography and T L C (silica gel and cellulose). The use of a numerical description of Rr patterns in this correlation study revealed that the R f values in paper chromatography were more reproducible than those in TLC.
F. RM Values The principle underlying the use of RM value in chromatographic structural analyses of compounds, e.g., derivatives of antibiotics, is that the chromatographic behavior of a molecule in a specific chromatographic system results from the thermodynamic properties of the molecule. If either the chromatographic system or the molecule is altered, the chromatographic behavior will change. R. A. De Zeeuw, Anal. Chem. 40, 915 (1968). R. A. De Zeeuw, Y. Chromatogr. 33, 227 (1968).
67j. A. Hockey, Chem. Ind. (London) 1965, 57. 6sM. S. J. Dallas, J. Chromatogr. 17, 267 (1965). F. Geiss and H. Schlitt, Naturwissenscha]ten 50, 350 (1963). ,o R. A. De Zeeuw, Y. Chromatogr. 33, 222 (1968). 71M. Brenner, in "Stationary Phase in Paper and Thin-Layer Chromatography" (I(. Macek and I. M. Hais, eds.), p. 67. Elsevier, Amsterdam, 1965. 72M. Brenner and A. Niederwieser, Experientia 16, 378 (1960). ~3T. F. Brodasky, Anal. Chem. 36, 996 (1964).
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The distribution of a molecule in two phases depends on the change of free energy necessary to transport one molecule from one phase to the other, which is actually the sum of the free energies needed to transport the individual functional groups of the molecule from one phase to the other. I t is possible to derive a mathematical relationship between the partition coefficient of a molecule and its chemical potential, or rather the sum of chemical potentials of the individual functional groups of the molecule. It is equally possible to derive a mathematical relationship between the R f value and the partition coefficient of a molecule. Because the chemical potential of a molecule may be considered as the sum of chemical potentials of the individual functional groups in the molecule, the A R . value of the entire molecule is the sum of partial AR~ values for its constituent functional groups. The foregoing assumption is true, however, only if the proper conditions in chromatography have been selected, as detailed by Bark. 74 Despite the difficulties that are inherent in the elucidation of structure by the use of R~ values, this method has been used to study the structures of steroids, 7~,76 acids, esters, 77 and amines. 7s This method can be used to elucidate the structure of antibiotic derivatives, to determine, for example, how an antibiotic has been modified by certain fermentation conditions. G. P r e p a r a t i v e T L C Among the chromatographic methods, T L C is one of the most suitable for use in the preparation of pure materials in milligram or submilligram quantities. For this purpose, layers 0.5 to 1.0 mm thick are used, in combination with an ascending technique. A 5-10% solution of the material is placed at the "start" as a band, rather than as a spot; the solution may be applied manually or by use of microspray instruments. Plates of 20 X 20 cm are preferred. After the chromatogram has been developed, the separated materials are recovered, usually by scraping each zone from the plate with a spatula and subsequently extracting the removed material. Microvacuum cleaners and similar pieces of apparatus are available for removing the material from the chromatogram. After extraction, it "L. S. Bark, in "Progress in Thin-Layer Chromatography and Related Methods" (A. Niederwieser and G. Pataki, eds.), Vol. I, p. 1. Ann Arbor-Humphrey Sci. Publ., Ann Arbor, Michigan, 1970. 7~I. E. Bush, "The Chromatography of Steroids," p. 31. Pergamon, Oxford, 1961. TeB. P. Lisboa, Steroids 6, 605 (1965). ,TE. R. Reichl, Mikrochim. Acta 1965, 683 (1965). ~8A. Cee and J. Gasparic, Mikrochim. Acta 1966, 295 (1966).
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THIN-LAYER CHROMATOGRAPHY OF ANTIBIOTICS
185
is advisable to purify the material by redissolving and filtering it. The purity of the isolated compound is determined by rechromatographing of an aliquot. Special techniques have been worked out for transferring materials from T L C to other analytical systemsJ 9 Preparative T L C can be used for the isolation of the antibiotic tumimycin2 ° In that case, small quantities of the antibiotic, produced by shake-flask fermentation, are separated from related inactive metabolites. Eastman chromatoplates are used and 2-3 mg of pure antibiotic can be recovered from a 20 X 20 cm plate. Chloramphenicol can be separated in pure form, via preparative TLC, from each of several different chloramphenicol preparations. 81 Preparative T L C on cellulose has also been used to separate and isolate several purines for further analytical evaluation, s~-
H. Quantitative T L C T L C is frequently used as an analytical procedure for the quantitative estimation of materials such as antibiotics. D a t a obtained by evaluation of chromatoplates are usually based on relative measurements, rather than on absolute ones, because of inaccuracies inherent in the system. In general, T L C provides two ways to make quantitative estimates of materials: (1) comparing the sizes or intensities of spots on a chromatoplate; and (2) determining the concentration in solution of materials t h a t have been eluted from the stationary phase. In the former instance, care nmst be taken to obtain spots of regular shape and to compare only spots that have migrated the same distance on the same chromatoplate. The comparisons may be made by direct measurement of spot size, by photodensitometry, or by measurement of clear zones after bioautography. In evaluating spot size directly, it has been found that the square root of the spot area is a linear function of the logarithm of the weight of the chromatographed material at that spot. s3 Photodensitometry can be used in quantitative estimations of the antibiotics siccanin s4 and tetracycline2 5 A spectrofluorimeter has been 7~"Thin-Layer Chromatography: A Laboratory Handbook" (E. Stahl, ed.), 2nd ed.,
p. 102. Springer-Verlag, Berlin and New York, 1969. 8°U.S. Patent 3674867 (1972). 81K. C. James and R. H. Leach, Y. Pharm. Pharmacol. 22, 607 (1970). s2K. Doerner and H. Manzke, Z. Klin. Chem. Kiln. Biochem. 9, 57 (1971). ~' S. J. Purdy and E. V. Truter, Analyst 87, 802 (1962). s4M. Arai, K. Hamano, K. Nose, and K. Nakano, Sankyo Kenkyusho Nempo 20, 93 (1968). C. Radecka and W. L. Wilson, J. Chromatogr. 57, 297 (1971).
186
METHODS FOR THE STUDY OF ANTIBIOTICS
[8]
used for detecting and providing quantitative estimations of different penicillins present in quantities of 0.3-0.6 mg. 86 Amounts of biologically active materials, such as antibiotics, can be estimated by bioautography. This technique is based on measurement of the size of the zone of inhibition of growth of a microorganism. The inhibition is caused by the active compound on the chromatoplate, which is placed on agar that has been seeded with a sensitive microorganism. The technique has been used, for example, for quantitative estimation of oleandomycin,s7 A semiquantitative estimation of nystatin was made by means of the combination of TLC and bioautography. Eastman Silica Gel Chromagram sheets, the solvent system butanol:acetic acid:water (60:15:30), and Saccharomyces cerevisiae as the test organism were used in that study2 s Quantitative measurements made of material that has been eluted from the stationary phase are of several types: weight determinations, spectrophotometry, fluorometry, bioassay, vapor chromatography, and gas-liquid chromatography. In each case, care must be exercised that the eluent does not extract part of the stationary phase, such as the binder. The combination of TLC and solution spectrophotometry is routinely used for the determination of tetracyclines,89 and has been used for simultaneous quantitative determinations of chloramphenicol, sulfadimethoxine, and tetracycline2° For further details on quantitative TLC, the reader should consult the review paper by Seller and Moeller21
III. Thin-Layer Chromatographic S y s t e m s for the Separation and Classification o f Antibiotics
The first section of this chapter outlined briefly the principal theoretical considerations and techniques of TLC. Several of the references cited concerned the application of certain techniques to the study of antibiotics. In this section, we shall describe TLC systems used for the study of specific antibiotics. We hope to facilitate for the reader the selection of a 86j. E. Sinsheimer, D. D. Hong, and J. It. Burckhalter, J. Pharm. Sci. 58, 1041 (1969). 87M. V. Kalinina and E. I. Surikova, Antibiotiki 13, 112 (1968). A. Aszalos,unpublished, 1970. ~' L. Lodi, G. Meinardi, and E. Rossi, Farmaco Ed. Prat. 24, 759 (1969). N. N. Lombardi, J. Dobrecky, and C. D. Rosa De Carnevale Bonino, Rev. Asoc. Bioquim. Argent. 33, 174 (English) and 20 (Spanish) (1968). 91N. Seller and H. Moeller Chromatographia 1969 (6) 273 and 1969 (7) 319.
[8]
THIN-LAYER CHROMATOGRAPHY OF ANTIBIOTICS
187
TLC system appropriate for use with a particular antibiotic or type of antibiotic. For that reason, we shall describe studies in which large numbers of antibiotics or antibiotic analogs are involved. Some of the earliest work on general TLC systems used for antibiotics was done by Umezawa's group? 6 Merck Silica Gel G adsorbent, spread on a 20 X 20-cm plate, is used with an ascending technique. The solvent system is varied with the type of antibiotic being investigated. Spots in the chromatogram are generally made visible by the application of a 10% permanganate solution, followed in 10 min by the application of a 0.2% bromphenol blue solution. For macrolide antibiotics, the chromatogram is sprayed with 10% sulfuric acid solution, then heated to 80 ° for 5-10 min. For separating water-soluble basic antibiotics, the solvent systems propanol : pyridine: acetic acid: water (15 : 10: 3:12) and chloroform : methanol: 17% ammonia (2:1 : 1) are used. Peptide antibiotics are separated by use of the solvent system butanol:acetic acid:water (3:1 : 1). Polyene antibiotics are separated by use of the solvent system ethanol: cone. ammonia:water (8:1:1) and nucleoside antibiotics by use of the solvent system ethyl acetate:methanol (2:1). Antibiotics of the macrolide group are separated by use of any of three solvent systems-ethanol: cone. ammonia :water (8:1 : 1), butanol: acetic acid :water (3:1 : 1), and ethanol:water (4:1). Table I lists the solvent systems and the RI values of the antibiotics investigated by Ikekawa e t al. 36 Alumina (Merck, G) has also been used in the separation of such closely related antibiotics as the actinomycins; a regular ascending technique and a running time of 30-60 min are utilized22 No detecting reagent need be used with these colored antibiotics and, if the chromatography and subsequent elution are done in the dark, quantitative recovery of the antibiotics is possible. Methanol is used as the eluting solvent. Table II lists the solvent systems and the R f values of the individual actinomycins. One study, dealing originally with 84 antibiotics and later extended to 91, classified them according to their mobilities in different solvent systems and used this classification to help identify the antibiotics? ~ This technique has the advantages of rapidity, the use of easily available Eastman Silica Gel Chromagram sheets, and applicability to crude preparations of antibiotics. Since characterization and classification of the antibiotics are based on their mobilities in different solvents, rather than on their R~. values, it becomes unnecessary, once the system has been standardized in a particular laboratory, to include standard antibiotics among the substances to be chromatographed. 9~G. Cassani, A. Albertini, and O. Ciferri, J. Chromatogr. 13, 238 (1964).
188
METHODS FOR THE STUDY OF ANTIBIOTICS
[8]
TABLE I TLC OF ANTIBIOTICS ON SILICA GEL G (MERCK) IN SIX SOLVENT SYSTEMSa Antibiotic
RI
Antibiotic
Butanol: acetic acid :water (3 : 1 : 1) Spiramycin 0.08 Mitomycin C Trichomycin 0.17 Carbomycin Nystatin 0.18 Aureothricin Oleandomycin 0.29 Leucomycin Amphotericin A 0.33 Tylosin Pimaricin 0.34 Tertiomycin B 0.36 Thiolutin Unamycin A Amaromycin 0.38 Etamycin Pyridomycin 0.38 Pentamycin Erythromycin 0.39 Actinomycin C 0.41 Tertiomycin A Pikromycin 0.44 Actinomycin J Telomycin 0.50 Enteromycin Porfiromycin Amphomycin 0.53 Acidomycin Chloroform: methanol : 17 % ammonia (2: 1 : 1) Kanamycin Viomycin 0.11 Paromomycin Streptothricin 0.26 Aminosidin Neomycin B 0.51 Blasticidin S Catenulin 0.60 Zygomycin 0.62 Propanol: pyridine: acetic Paromomycin 0.40 Aminosidin 0.40 Neomycin B 0.46 Streptothricin 0.52
acid:water (15: 10:3: 10) Catenulin Kanamycin A Zygomycin A
Ethanol: conc. ammonia:water (8: 1: 1) Nystatin 0.18 Bacitracin Amphotericin B 0.33 Pentamycin Pimaricin 0.34 Antimycin ttomomycin 0.42 Blastmycin Trichomycin 0.45 Novobiocin Bacitracin Pyridomycin Mitomycin C Porfiromycin Aureothricin Mitomycin C Porfiromycin Aureothricin
Ethanol:water (4: 1) 0.13 Thiolutin 0.18 Althiomycin 0.45 Etamycin 0.50 Antimycin A 0.57 Ethyl acetate: methanol (100: 15) 0.38 Chromomycin A8 0.48 Thiolutin 0.63
Rf
0.54 0.55 0.58 0.58 0.59 0.63 0.65 0.66 0.67 0.68 0.68 0.73 0.73 0.74 O. 65 0.68 0.68 0.70
0.54 0.55 0.56
0.58 0.67 0.72 0.82 0.82 0.64 0.78 0.80 0.81
0.65 0.70
T. Ikekawa, F. Iwami, E. Akita, and H. Umezawa, J . Antibiot. Set. A 16, 56 (1963).
[81
T H I N - L A Y E R CHROMATOGRAPHY OF ANTIBIOTICS
189
TABLE II TLC OF ACTINOMYCINS
ON ALUMINA G (MERCK) IN THREE SOLVENT SYSTEMS a
RI of actinomycin Solvent system
C~
C~.
C~
F~
F3
E t h y l a c e t a t e : sym-tetrachloroethane: w a t e r (3 : 1 : 3) ( b o t t o m p h a s e )
0.44
0..51
0.58
0.21
0.35
Ethyl acetate: di-n-butyl ether :water (3 : 1: 3) (top phase) Ethyl acetate:di-n-butyl ether:water (2 : 1 : 2) (top phase)
0.40
0.46
0.53
0.23
0.29
0.28
0.30
0.33
0.10
0.13
a A. Cassani, A. Albertini, and O. Ciferri, J. Chromatogr. 13, 238 (1964). Eastman Chromagram sheets, silica gel type 6060, are partially deactivated, prior to use, by exposure to air at room temperature (50% R H ) for 24 hr. Lined, simple glass chambers are used for development. Samples are prepared in three different ways to assure that every type of antibiotic in a fermentation broth can be recovered in at least one of the preparations. The samples are prepared as follows: (a) 6 ml of whole fermentation broth are extracted with 3 ml of butanol, alone or in the presence of either 0.6 ml of 6 N HC1 or 0.5 ml of 2 N NH4OH. A 0.02-ml sample of each butanolic extract is applied to the Chromagram sheet by means of a micropipette; (b) 10 ml of isopropanol is shaken with 6 ml of whole broth for 15 rain, then centrifuged. A 0.04-ml sample of the supernatant is applied to the Chromagram sheet by means of a micropipette; (c) a 10-ml sample of the supernatant from the isopropanolic solution is dried in vacuo. The residue is triturated twice with 1 ml of ethanol. To the material insoluble in ethanol are added 2 ml of water and 2 ml of acetone. After centrifugation of the resulting suspension, a 0.02-ml sample of the supernatant and a 0.02-ml sample of the earlier ethanolic solution are applied to Chromagram sheets. The chromatoplates are then developed in three main solvent systems: (a) methanol; (fl) 10% methanol in chloroform; (7) chloroform. Bioactive spots are detected by means of bioautography, as detailed below. Antibiotics are classified into four primary groups, according to their mobilities in the three main solvent systems--Group I: antibiotics showing no mobility in solvents a, fl, or ),; Group II: antibiotics moving only in a; Group I I I : antibiotics moving in a and #, but not in ,/; and Group IV: antibiotics moving in a, fl, and 7The application of 11 additional solvent systems to the members of the four primary groups yields 15 subgroups (see Table I I I ) . The Rr values of the antibiotics tested are shown in Table IV.
190
METHODS FOR THE STUDY OF ANTIBIOTICS
SOLVENT SYSTEMS IN
Group
TABLE I I I ITLC CLASSIFICATION
[8]
OF ANTIBIOTICSa
System Main Solvent Systems Methanol 10% Methanol in chloroform Chloroform
II III IV
Additional solvent systems Pyridine: water (1 : 1) Ia Pyridine:water: absolute ethanol (1 : 1 : 1) Ib Pyridine: water: absolute ethanol (1 : 1 : 3) Ic Butanol : methanol (1 : 1) IIa Chloroform : methanol (1 : 1) IIb Absolute ethanol IIc Methanol : benzene (12: 88) IIIa Methanol : benzene (6: 94) IIIb Methanol : benzene (4: 96) IIIc Methanol : benzene (1 : 99) IVa Methanol: benzene:chloroform (1 : 49: 50) IVb
A. Aszalos, S. Davis, and D. Frost, J. Chromatogr. 37 487 (1968). F o r bioautography, developed and dried C h r o m a g r a m sheets are placed on filter p a p e r resting on seeded agar plates and left there for the entire incubation period (18 hr at 37°). The microorganisms used for bioautography are listed in T a b l e V. The system described above is used in several industrial laboratories for the p r i m a r y classification and probable identification of unknown antibiotics. The system is equally suited to the classification of pure and crude preparations of antibiotics. The m a n y different types of antibiotics included in this study m a k e the results useful for the design of T L C systems for identifying additional antibiotics. Similar T L C systems are used in combination with card-file data systems or, more elegantly, computer programs to achieve probable identifications of unknown antibiotics. Although the final identification of an antibiotic must be based on exact chemical analysis, the application of TLC, bioautography, and computer analysis to antibiotic materials extracted from fermentation broths under basic, neutral, and acidic conditions makes it possible to restrict the probable identity of the antibiotic to only a few choices. A similar study of 42 antibiotics available commercially in France classified them into four groups on the basis of their mobilities in ten different solvent systems2 a The fact t h a t chemically related antibiotics ~ J. P. Schmitt and G. Mathis, Ann. Pharm. Fr. 28, 205 (1970).
[8]
191
THIN-LAYER CI-IROMATOGRAPHY OF ANTIBIOTICS T A B L E IV TLC OF ANTIBIOTICS ON EASTMAN CHROMAORAM SHEETS IN VARIOUS SOLVENT SYSTEMSa
Subgroup
I-1
I-2 (Ia)
I-3 (Ia, Ib) I-4 (Ia, Ib, Ic)
Antibiotic
Gentamicin D Gentamicin C1 Gentamicin C~ Humatin Neomycin Trienine Aminosidin Dihydrostreptomycin Hygromycin B Streptomycin Streptothricin Viomy cin Kanamycin Rubiflavin Actinorubin Polymyxin B Ristocetin Vancomycin
Rs values in specific solvent systems b
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
fl
3'
Ia
Ib
Ic
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0-0.8 0.25 0.1 0.15 0.3 0.3-0.5 0-0.3 0-0.2 0.6 0.7 0.9 0.3-0.8
0 0 0 0 0 0 0 0 0 0 0 0 0-0.2 0-0.15 0.65 0.56 0.7 0.7
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.2 0.4 0.7 0-0.6
IIa
Ib
IIc
0 0 0 0 0.08 0 0.22 0.2 0 0.5 0-0.15 0.3 0.1 0.6 0.55 0-0.1 0.22 0-0.4 0-0.3 0.55 0-0.5
0 0 0 0 0 0.3-0.4 0 0.15 0.15 0.2 0.1 0.2 0.1 0.6 0.53 0-0.1 0.I2 0-0.25 0-0.3 0.5 0-0.2
0 0 0 0 0 0 0 0 0 0 0 0 0.05 0.6 0.62 0.05 0.06 0-0.2 0-0.15 0.45 0-0.2
a II-1
II-2 (a, IIa, or IIb) II-3 (a, IIa, IIb)
II-4 (~, IIa, IIb, IIc)
Candidin Gramicidin S Prasinomycin Duramycin Amphomycin Candicidin Mycostatin Bacitracin Galirubin Subtilin Trichomycin UnamycinA Azacolutin Cephalothecin Fungichromin Hamycin Lucensomycin Oxytetracycline Rimocidin Septacidin Tetracycline
0-0.32 0-0.2 0.28 0.6 0.35 0.4-0.6 0.5 0.23 0.1 0.7 0.5 0.5 0.7 0.8 0.58 0-0.6 0.4 0-0.4 0.5 0.6 0-0.3
¢~ v 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
(Continued)
192
METHODS FOR THE STUDY OF ANTIBIOTICS
[8]
TABLE IV (Continued) Subgroup
Antibiotic
R1 values in specific solvent systems ~ t~
III-1 (a, t~)
III-2 (a, ~, IIIa)
III-3 (a,/~, IIIa, IIIb)
III-4 (a,/~, IIIa, IIIb, IIIc)
Fungimycin Novobiocin Oleandomycin Rhodomycetin Saramycetin Amicetin Chromomycin A3 Citrinin Erythromycin Filipin Gelbecidin Nogalamycin Rhodomycin Streptozotocin Toyocamycin Tuberculin Anisomycin Fusanin B Isarine Lincomycin Puromycin Streptovitacin Sulfocidin Thiostrepton Tylosin Actidione Actinomycin C2 Actinomycin C3 Celesticetin Chloramphenicol Echinomycin Esperin Javanicin Mitomycin Nocardorubin Streptovaricin
0-0.75 0.71 0.48 0.05 0.62 0.6 0.7 0.72 0.42 0.6 0.7 0.46 0.73 0.6 0.65 0.62 0.5 0.65 0.6 0.41 0.7 0.41 0.65 0.62 0.7 0.64 0.82 0.82 0.76 0.72 0.7 0.75 O. 7 O. 72 O. 55 0.79
~,
0-0.2 0 0.7 0 0.3 0 0.05 0 0.62 0 0.28 0 0.3 0 0.8 0 0.26 0 0.15 0 0.3 0 0.4 0 0.5 0 0-0.4 0 0.28 0 0.22 0 0.35 0 0.58 0 0.6 0 0.25 0 0.55 0 0.25 0 0.5 0 0.56 0 0.6 0 0.67 0 0.8 0 0.75 0 0.74 0 0.57 0 0.7 0 0-0.5 0 O. 68 0 O. 48 0 O. 5 0 0.78 0
IIIa
IIIb
0 0 0 0 0 0 0 0 0 0 0.1 0 0.1 0 0.16 0 0.1 0 0.18 0 0.1 0 0.3 0 0.3 0 0-0.2 0 0 0.1 0.05 0 0.2 0.1 0.2 0.1 0.4 0.15 0-0.2 0-0.1 0.25 0-0.15 0.23 0.1 0.85 0-0.5 0.25 0.15 0.35 0.1 0.5 0.22 0.5 0.26 0.5 0.26 0.4 0.25 0.3 0.15 0.5 0.22 0.2 0.09 O. 72 O. 71 O. 25 O. 1 O. 45 O. 3 0-0.5 0-0.4
IIIc 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.2 0.05 0.05 0.05 0.09 0.05 0.05 O. 71 O. 05 O. 25 0-0.4
fell into t h e s a m e group led to t h e s u g g e s t i o n t h a t such T L C g r o u p i n g c a n p r o v i d e i n f o r m a t i o n a b o u t t h e g e n e r a l c h e m i c a l c h a r a c t e r i s t i c s of an a n t i b i o t i c . A suspension of 25 g of K i e s e l g u h r G ( M e r c k ) in 55 m l of w a t e r is p o u r e d onto a 20 X 20-cm glass p l a t e to m a k e a 0 . 2 5 - m m t h i c k layer. A f t e r b ei n g d r ie d at r o o m t e m p e r a t u r e for 1 hr, t h e p l a t e s are
[8]
193
THIN-LAYER CHROMATOGRAPHY OF ANTIBIOTICS
TABLE IV (Continued) Subgroup
IV-1 (c~,f~, 3,)
IV-2 (~, ~, ~, IVa)
IV-3 (a, f~, % IVa, IVb)
Antibiotic
Actinobolin DON Psicofuranine Spiramycin Etamycin Oligomycin Thiolutin Vernamycin A Bostrycoidin Chartreusin Clavacin C-73 Fusarubin Griseofulvin Tyrothricin Usnic acid
R1 values in specific solvent systems b a
~
"r
IVa
IVb
0.42 0.78 0.7 0.6 0.8 0.43 0-0.6 0.6 0.76 0.74 0.7 0.8 0.3 0.75 0.7 0.75
0.2 0.76 0.15 0.4 0.8 0.43 0.65 0.63 0.73 0.65 0.65 0.7 0.73 0.75 0.7 0.75
0.08 0.65 0-0.1 0-0.3 0-0.2 0.32 0.35 0.2 0.26 0.15 0.3 0.55 0.45 0.52 0.25 0.75
0 0 0 0 0.1 0.2 0.3 0.12 0.22 0.16 0.25 0.4 0.23 0.5 0.7 0.5
0 0 0 0 0 0 0 0 0.15 0.1 0.16 0-0.22 (). 15 0.25 0.1 0.45
a A. Aszalos, S. Davis, and D. Frost, J. Chromatogr. 37, 487 (1968). bSee Table III. heated at 120 ° for 1 hr to activate them, and are then stored in a desiccator. Each antibiotic is dissolved in chloroform, methanol, or water and applied to the plates in 1- to 3-~1 quantities. After development of the chromatoplates, the antibiotics are identified by their Rf values and by the colors visible after the plates have been sprayed with Mathis-Schmitt solution2 ~ The latter reagent is made of p-dimethylaminobenzaldehyde (1 g), antimony trichloride (20 g), conc. hydrochloric acid (20 ml), and 95% ethanol (100 ml). The sprayed chromatoplates are heated at 120 ° for about 1 hr. Tables V I and V I I show the Rs values of the antibiotics in the two groups of solvent systems utilized. An investigation of the separation of macrolide antibiotics by the use of a T L C system consisting of acetone:water (98:2) and silica gel G adsorbent showed that ten members of this antibiotic family could be separated and identified24 The R~ values of the antibiotics are: erythromycin, 0.18; pikromycin, 0.20; narbomycin, 0.23; foromacidin A, 0.27; foromacidin B, 0.38; foromacidin C, 0.42; angolamycin, 0.77; tylosin, 0.8; carbomycin, 0.9; and acumycin, 0.9. The antibiotics are identified by bioautography, with Bacillus subtilis as the test organism. A filter paper is placed on the seeded agar plate before the developed chromato94j. p. Schmitt and G. Mathis, Ann. Pharm. Ft. 26, 727 (1968).
194
METHODS FOR THE STUDY OF ANTIBIOTICS
TABLE V ANTIBIOTICS, QUANTITIES APPLIED, AND TEST MICROORGANISMS USED FOR BIOAUTOGRAPHY WITH ITLC ~
Antibiotic Actidione Actinobolin Actinomycin C2 Actinomycin Ca Actinorubin Amicetin Aminosidin Amphomycia Anisomycin Azacolutin Bacitracin Bostrycoidin Candicidin Candidin Celesticetin Cephalothecin Chartreusin Chloramphenicol Chromomycin Aa Citrinin Clavacin C-73 Dihydrostreptomycin DON Duramycin Echinomycin Erythromycin Esperin Etamycin Filipin Fungichromin Fungimycin Fusanin B Fusarubin Galirubin Gelbecidin Gentamicin D Gentamicin C1 Gentamicin C~ Gramicidin S Griseofulvin Hamy cin Humatin Hygromycin B Isarine
Volume of antibiotic solution, 1% conc. Microorganism used for (gl) bioautography 4 5 2
2 10 10 10 5
20 30 10 2 10 2
10 5 3 1
20 10 3
20 20 40 20 5 3
10 10 30 5 2
10 4
10 20 3 3 3 5
60 10 10 10 10
Saccharomyces cerevisiae Staphylococcus aureus S. aureus S . aurcus S. aureus S. aurcus S. aurcus S. aureus S. cerevisiae Saccharomyces p a s t o r i a n u s S. aureus Bacillus subtilis C a n d i d a albicans C. albicans S. aureus S. aureus S. aureus S. aurcus, Escherichia coli S. aureus S. aureus E. coli C. albicans E. coli S. aureus B . subtilis S. aureus S. aureus S. aureus S. aureus S. cerevisiae C. albicans S. cerevisiae B . subtilis S. aureus S. aureus S. aureus S. aurcus S. aureus S. aureus S. aureus C. albicans C. albicans E. coli S. aureus •. aureus
[8]
[8]
THIN-LAYER CHROMATOGRAPHY OF ANTIBIOTICS TABLE V (Continued)
Antibiotic Javanicin Kanamycin Lincomycin Lucensomycin Mitomycin Mycostatin Neomycin C Nocardorubin Nogalamycin Novobiocin Oleandomycin Oligomycin Oxytetracycline Polymyxin B Prasinomycin Psicofuranine Puromycin Rhodomycetin Rbodomycin Rimocidin Ristocetin Rubiflavin Saramycetin Septacidia Spiramycin Streptomycin Streptothricin BI1 Streptovaricin Streptozotocin Streptovitacin Subtilin Sulfocidin Tetracycline Thiolutin Thiostrepton Tyrothricin Toyocamycin Trichomycin Trienine Tubercidin Tylosin Unamycin A Usnic acid Vancomycin Vernamycin A Viomycin
Volume of antibiotic solution, 1% conc. (~1) 10 5 3 5 5
10 10 1 1
10 10 1 1
30 3
20 20 2 3
10 5 1
10 20 40 5
10 10 5
40 10 5 1 3 5
10 10 10 10 5 5
25 10 10 5
10
Microorganism used for bioautography
B. subtilis S. aureus S. aureus S. cerevisiae S. aureus C. albicans B. subtilis S. aureus S. aureus S. aureus S. aureus S. cerevisiae S. aureus E. coli S. aureus S. aureus S. aureus S. aureus S. aureus S. cerevisiae S. aureus S. aureus Paecilomyces varioti Trichophyton mentagrophytes S. aureus S. aureus S. aureus S. aureus S. aureus S. pastorianus S. aureus S. aureus S. aureus S. aureus S. aure~s S. aureus C. albicans S. aureus S. aureus C. albicans C, albicans S. cerevisiae B. subtilis S, aureus S. aureus Proteus vulgaris
A. Aszalos, S. Davis, and D. Frost, J . Chromatogr. 37, 487 (1968).
195
196
METHODS FOR THE STUDY OF ANTIBIOTICS
[8]
TABLE VI TLC OF ANTIBIOTICS ON KIESELGUHR G (MERCK) IN FOUR SOLVENT SYSTEMSa'b
Antibiotic Triacetyloleandomycin Griseofulvin ¥irgimycin Pristinamycin
Solvent I
Solvent II Solvent I I I Solvent IV
Novobiocin (Na) Dihydronovobiocin (Na) Dihydronovobioein Fusidic acid (Na) Rifamycin Penicillin G (Na) Cephalothin (Na) Cephaloridine Chloramphenicol Propiocine Erythromycin Spiramycin
0.14 0.98 0.58 0.33 0.57 0.92 0.38 0.12 0.60 0.79 0.99 0.95 0.22 0.20 0.66 0 0 0
0.98 0.98 0.79 0.35 0.82 0.87 0.61 0.59 0.66 0.67 0.17 0.30 0.38 0.25 0.76 1 0.50 0.55
Oleandomycin (PO4) Lineomycin. (HC1) Kitasamycm (tartrate) Cycloserine Hydroxymethylgramicidin Tyrothricin
0 0 0 0 0 0
0.15 0.80 0.85 0.33 0.67 0.58
0.51 0.78 0.79 -0.80 -0.92 0.90 0.95 0.98 0.88 0.91 0.83 0.26 0.87 0.52 0.52 0.35 0.56 0.18 0.42 0.77 0.86 0.97 0.66
Bacitracin Pimariein Nystatin Trichomycin Tetracycline Methylenecycline (HC1) Oxytetracycline Demethylchlortetracycline (HC1) Chlortetracycline (HC1) Rolitetracycline Colimycin (SO~) Polymixin B (SO4) Streptomycin (SO4) Dihydrostreptomycin (SO4) Neomycin (SO4) Kanamycin .(SO4) Paromomycm (SO4) Framycetin (SO4) Gentamicin (SO4) Viomycin (SO4)
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0.54 0.59 0.55 0.57 0.47 0.53 0.45 0.52 0.49 0.47 0.33 0.18 0.08 0.06 0 0 0 0 0 0
0 0 0.10 -O 0.51 0 0 0 0 0 0.48 0.44 0.12 0.66 0 0 0.10 0.10 0.29 0 0.87 0 0
1
0 O 0 0 0.23 0.33 0.40 0.26 0.25 0.23 0 0.10 0.90 0.91 0.95 0.83 0.91 0.91 0.46 0.83
a j. p. Schmitt and G. Mathis, Ann. Pharm. Ft. 28, 205 (1970). b Solvent I: chloroform: methanol: acetic acid (90: 8: 2) Solvent II: chloroform : methanol: water (80: 20: 25) Solvent III: butanol: acetic acid: water (50: 25: 25) ; before use of this solvent system, chromatoplates were impregnated with pH 3 buffer (potassium phosphate) Solvent IV: water:sodium citrate:citric acid (100"20:5)
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TABLE VII TLC OF ANTIBIOTICS ON KIESELGUHR G (MERCK) IN SIX SOLVENT SYSTEMSa
Antibiotic Triacetyloleandomycin Griseofulvin Virgimycin Pristinamycin Novobiocin (Na) Dihydronovobiocin (Na) Dihydronovobiocin Fusidic acid (Na) Rifamycin Penicillin G (Na) C e p h a t o t h i n (Na) Cephaloridine Chloramphenicol Propiocine Erythromycin Spiramycin Oleandomycin (PO4) Lincomycin (HCI) K i t a s a m y c i n (tartrate) Cycloserine H y d r o x y l m e t h y l g r a m icidin Tyrothricin Bacitracin Pimaricin Nystatin Trichomycin Tetracycline Methylenecycline (HC1) Oxytetracycline Deraethylchlortetracycline
Ether
Ethyl Methao acetate Acetone nol Ethanol
Water
0.01 0.30 0.08 0.15 0.05 0.05 0.20 0.10 0 0 0 0 0.55 0.35 0.10 0 0.05 0 0 0.03 0.45 0 0 0 0 0 0 0 0 0 0
0.15 0.70 0.30 0.30 0.20 0.20 0.40 0.30 0.03 0 0 0 0.60 0.60 0.30 0 0.15 0 0 0.15 0.70 0 0 0 0 0 0 0 0 0 0
0.90 1 0.90 0.80 0.95 0.95 0.95 0.80 1 0 0 0 0.95 1 0.75 0.10 0.55 0.03 0.25 0.95 0.80 0.20 0 0 0 0 0 0 0 0 0
0.75 0.70 0.80 0.80 0.85 0.85 0.90 0.80 0.85 0.85 0.80 0 0.90 1 0.75 0.40 0.70 0.30 0.75 0.90 0.90 1 1 0.30 0 1 0.20 1 1 0.20 1
0.50 0.70 0.90 0.70 0.90 0.90 0.95 0.70 0.80 0.50 0.65 0. l0 -1 0.65 0.15 0.90 0.20 0.80 1 0.95 1 1 0.02 0.50 0 0.65 0.10 0.75 0.50 0.25
0 0 0.10 0.20 0 0 0.45 0 0.65 0.80 0.80 0.20 -0.90 0.20 1 0.10 0.10 0.15 0.60 0.80 0 0 0 0.40 0 0 0 0.10 0.10 0.10
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0
1 0.20 0 0 0 0 0 0 0 0 0 0
0.25 0.30 0 0 0 0 0 0 0 0 0 0
0. I0 0. I0 0 0 0.40 0.40 0.50 0.70 0.65 0.40 0.25 0.60
(HCI) Chlortetracycline (HCI) Rolitetracycline Colimy cin (SO4) Po|ymyxin B (S04) Streptomycin (SO4) D i h y d r o s t r e p t o m y c i n (SO4) Neomycin (SO4) K a n a m y c i n (SO4) P a r o m o m y c i n (S04) F r a m y c e t i n (SO4) Gentamicin (SO4) Viomycin (SO4)
J. P. S c h m i t t a n d G. Mathis, Ann. Pharm. Fr. 28, 205 (1970).
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METHODS FOR THE STUDY OF ANTIBIOTICS
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plate is placed on it. Carbomycin and acumycin, despite their identical R~ value (0.9), can be differentiated by color after the ehromatoplate has been sprayed with Fischbach-Levine reagent2 ~ In another study, designed for the bioautographic evaluation of bioactire materials, Sephadex was used in the separation of 17 antibiotics22 To prepare the chromatoplates, 33 g of Sephadex G-15 is mixed with 100 ml of 25 mM phosphate buffer (KH.~PO4-Na0H, pH 6.0) containing 0.5 M NaC1. After an interval of at least 30 rain, the Sephadex suspension is spread in 0.5 mm thickness on 20 X 20-cm glass plates. The plates are dried at room temperature for 1 hr, then kept in a moist chamber for 24 hr before use. When used, the plates are mounted at a 30 ° inclination and covered with paper flaps presoaked in the buffer solution. The chromatoplates are used in a modified Determan sandwich arrangement, in which strips of filter paper are placed along the upper edges of the Sephadex-coated plate and a second glass plate is placed atop them, leaving a small space between the Sephadex layer and the upper plate. The two plates are clamped together with specially designed clamps. Samples of the material to be chromatographed (2 ~l) are applied, by micropipette, through a sample slit in the upper glass plate; the slit is then covered with tape. Prior to the chromatographic run, the mobile phase is kept immobilized by continuous wetting of the upper paper flap with the buffer solution. For chromatography, the same buffer solution is allowed to flow from a reservoir into the upper part of the sandwich via the paper flaps, then out the lower part of the sandwich into another reservoir. The run is initiated by filling the upper reservoir, and lasts about 60-80 rain. Replicate chromatograms are produced to permit bioautography with any desired number of test microorganisms. Table VIII shows the relative rates of travel of 17 antibiotics. Bioautography is performed by placing the developed chromatoplates on seeded agar for 30 rain, then incubating the agar plates for 18 hr at 30% Several other works are valuable in the selection of TLC systems suitable for use with certain groups of antibiotics. One TLC system, used by Ochab and Borowiecka, 96 separates the macrolide antibiotics carbomycin, erythromycin, narbomycin, oleandomycin, and spiramycin. It is difficult to separate these antibiotics by paper chromatography, but they can be separated successfully on silica gel G TLC plates. The mobile phase may be isobutanol:acetic acid:water (3:1 : 1), butanol:acetic acid: water: dioxane (6: 2: 2 : 1), butanol : acetic acid: water: methanol: nitromethane (6: 2: 2:1 : 2), or dichloromethane: methanol : benzene (4:1:1). Spots are detected by spraying the chromatoplate with a 0.05% 9~It. Fischbach and J. Levine, Antibiot. Chemother. 3, 1159 (1953). S. Ochab and B. Borowiecka, Diss. Pharm. Pharmacol. 20, 449 (1968).
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TABLE VIII TLC OF ANTIBIOTICSON SEPtIADEX a Rate of travel relative to that of penicillin G
Antibiotic Penicillin G Penicillin V Propicillin (L and Naf cillin Ampicillin Dicloxacillin Tetracycline Oxytetracycline Chlortetracycline Neomycin Kanamycin Staphylomycin Streptomycin Oleandomycin Pimaricin Nystatin Amphotericin B
D)
1.0 0.8 0.9 0.5 1.4 0.5 0.7 0.7 0.6 1.8 2.0 0.6 1.7 1.7 0.7 0.2 0.2
" M. H. J. Zuidweg, J. G. Oostendorp, and G. J. K. Bos, J. Chromatoor. 42, 552 (1969). solution of xanthydrol in glacial acetic acid, then with 2 ml of cone. HC1 and, finally, with a 10% solution of phosphomolybdic acid in ethanol. Buri 97 developed a T L C system for separating semisynthetic cephalosporins. H e used silica gel G as the adsorbent, i s o p r o p a n o l : m e t h a n o l : p H 5 buffer (30:105:15) as the mobile phase, and iodine-starch spray as a detecting reagent. Nishimoto et al. 9s recommend the use of silica gel treated with disodium E D T A in a T L C system to separate various tetracyclines, viz. tetracycline, anhydrotetracycline, 4-epitetracycline, 4-epianhydrotetracycline, chlortetracycline, anhydrochlorotetracycline, isochlorotetracycline, oxytetracycline, anhydrooxytetracycline, and ~ and fl apooxytetracyclines. The thin-layer chromatographic behavior of 16 antibiotics was examined on the adsorbents silica gel G, aluminum oxide G, and cellulose M N 300 (Polygram) by D o b r e c k y et al. 99 T h e y obtained good separa~Tp. Buri, Pharm. Acta Helv. 42, 344 (1967). ,s y. Nishimoto, E. Tsuchida, and S. Toyoshima, Yakugaku Zasshi 87, 516 (1967). g9j. Dobrecky, E. A. Vazquez, and R. Amper, SAFYBI (Soc. Arg. Farm. Bioquim. Ind. 8, 204 (1968).
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METHODS FOR THE STUDY OF ANTIBIOTICS
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TABLE IX TLC OF BAsic WATER-SOLUBLEANTIBIOTICSON CELLULOSE300 (MN) a Antibiotic
RI
Glebomycin Streptomycin Dihydrostreptomycin Hydroxystreptomycin Netropsin Amidinomycin Gentam ycin
0.41 0.44 0.44 0.32 0.51 0.53 0.35 0.28 0.20 0.26 0.21 0.17 0.15 0.23 0.15 0.15 0.15 0.10 0.10
Streptothricin Viomycin Kanamycin A Kanamycin B Kanamycin C Paromomycin Zygomycin Catenulin Neomycin Fradomycin
Y. Ito, M. Namba, N. Nagahama, T. Yamaguchi, and T. Okuda, J. Antibiot. Ser. A 17, 218 (1964).
tions of rifamycin, amphotericin B, griseofulvin, polymyxin G, viocin, gentamycin, and gabromycin, which are ordinarily difficult to separate. One of the earliest separations of water-soluble antibiotics by T L C was achieved by Ito et al. 1°° Their recommended adsorbent is cellulose powder; the mobile phase consists of propanol:pyridine: acetic acid:water (15:10:3:12), and the developing reagent is ninhydrin or oxidized nitroprusside. This T L C system is an alternative to that recommended by Ikekawa et al26 for the separation of water-soluble basic antibiotics. Table I X shows the Rs values of the antibiotics studied by Ito et al.
A very good review of solvent systems useful in T L C of the penicillins, aminoglycosides, tetracyclines, macrolides, peptides, and other antibiotics was recently published by V. Betina. T M An excellent book by Wagman 1~,y. Ito, M. Namba, N. Nagahama, T. Yamaguchi, and T. Okuda, J. Antibiot. Ser. A 17, 218 (1964). lolV. Betina, in "Pharmaceutical Application of Thin-Layer and Paper Chromatography" (K. Macek, ed.), p. 502. Elsevier, Amsterdam, 1972.
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and Weinstein 1°~ on the chromatography of antibiotics contains descriptions of paper and thin-layer chromatographic systems suitable for use with more than 1100 antibiotics and their derivatives.
IV. Use of Thin-Layer Chromatography in the Biosynthesis and Biotransformation of Antibiotics Analytical techniques, such as TLC, are used to follow the formation of antibiotics or the changes in an antibiotic molecule caused by enzymic activity. The enzymic activity m a y occur during fermentation, m a y be that of an isolated enzyme on a particular antibiotic, or m a y occur in vivo. The fermentation m a y occur in the production of antibiotics in a laboratory shake flask or in large industrial fermentors, or m a y involve the use of entire microorganisms to biotransform (derivatize) previously isolated antibiotics. Isolated enzymes are used in both laboratory and industrial applications to bring about specific changes in antibiotics. Studies in vivo are generally of the metabolic fate of an administered antibiotic, and include determinations of excretion and tissue distribution.
A. Antibiotic Biosynthesis and Biotransformation by Fermentation There are many instances in which TLC has been used to follow the fermentation of an antibiotic. For example, Johri 1°3 used TLC to follow the shake-flask fermentation of the antibiotic cyathin. In that study, TLC was used to assess the composition of a fermented antibiotic complex, and the data obtained were used in the selection of carbon and nitrogen sources required for optimal production of the most active component of the complex. In the initial fermentation studies with the fungus Cyathus helenae, difficulties had been encountered in producing a high yield of the most active component of the antibiotic complex, cyathin C5. Although a maximal yield of the entire complex could be achieved by varying the physical parameters of the fermentation conditions, it proved necessary to change the carbon and nitrogen sources and the ratio of their relative concentrations before a maximum yield of component C5 could be obtained. Log aliquots of different fermentation batches, from 24 to 40 days old and varying in the above parameters, were extracted with ethyl acetate. The extracts were taken to dryness and the residues were dissolved in methanol. Samples of the methanolic solutions, 50 td, were spotted on silica gel G (Merck) plates, 20 X 20 cm, the layer being ~o~G. H. Wagman and M. J. Weinstein, "Chromatography of Antibiotics," Elsevier, Amsterdam, 1973. '°3B. N. Johri, J. Chromatogr. 56, 324 (1971).
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METHODS FOR THE STUDY OF ANTIBIOTICS
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0.5 mm thick. The plates were prepared according to the method of Stahl, using a Shandon applicator, and were heated to 100 ° for 30 min before use. The mobile phase was benzene:acetone:acetic acid (75:25:1), and spots were detected by spraying the chromatoplate with a 30% sulfuric acid solution. Reference samples of previously isolated components of the antibiotic complex were also spotted on each plate. These components had been tested earlier for their biological activities, and for their RI values in this TLC system. Quantitative estimations of the individual components of the complex produced by each fermentation were obtained from measurements of the areas of the spots. Results indicated that C5, the most active component of the complex, disappeared slowly with time in most of the fermentation media. Johri was able to select the best combination of carbon and nitrogen sources and the best time for isolation of the C5 component. In a similar type of study, the biosynthesis of actinomycins by Strept o m y c e s antibioticus was followed by TLC. Nishimura and Bowers T M sought to determine whether L-thiazoline-4-carboxylate would be incorporated into actinomycin, possibly at the site of the proline residue. They found that L-thiazoline-4-carboxylate was incorporated, and thereby formed two new actinomycins that could be separated by TLC. The sample for chromatography was prepared by extracting the culture broth with ethyl acetate, taking the extract to dryness, and dissolving the residue in acetone. The acetone solution was applied in a narrow line 3 cm from the edge of a 5 X 20-cm silica gel G plate, according to the method of Katz et al. 1°5 The mobile phase was ethyl acetate:acetone (2:1), which was allowed to run to the upper edge of the plate. The colored bands formed (4.2, 10.5, and 12.5 cm from the origin) were eluted from the plate with methanol after the chromatogram had dried. Each spot was analyzed for chemical composition, to help reveal the structure of the actinomycins that had been formed. The enzymic hydroxylation of the antibiotic brevicid was studied by Zarnach et al., ~°8 who used TLC to evaluate the products of different fermentation conditions. This study was undertaken because the toxicities of mono- and dihydroxylated brevicid are greater than that of brevicid, and fermentation conditions had to be established in which the hydroxylated compounds are not produced. It was shown that fermentation conditions in which no hydroxylated forms of brevicid are produced can be changed, by the addition of folic acid and Fe 3÷salt, to conditions in which l ~ j . S. Nishimura and W. F. Bowers, Biochem. Biophys. Res. Commun. 28, 665 (1967). 1~ E. Katz, A. B. Mauger, and H. Weissbach, Mol. Pharmacol. 1, 107 (1965). 1°~J. Zarnach, H. TSnjes, and H. PStter, Pharmazie 20, 503 (1971).
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they are produced. From these experiments, it was possible to establish the mechanism by which brevicid is hydroxylated. In following the formation of fermentation products by TLC, Zarnach et al. used Kieselgel H (Merck) adsorbent. The mobile phase was ethyl acetate:chloroform: methanol (2:6:2). Spots were detected by spraying the chromatogram with a 5% solution of silver nitrate in ethanol:water (9: 1). Under these chromatographic conditions, the R~ values of brevicid, its monohydroxylated derivative, and its dihydroxylated derivative were 0.75, 0.64, and 0.58, respectively. Other details of the TLC technique employed here are described by Zarnach and Pfeifer. 1°7 In screening antibiotics for antitumor activity, Schuurmans et aI. l°s employed a bioautographic system involving mammalian cells. Although the antibiotics were chromatographed on paper, there is no reason why the same results could not have been achieved by the use of TLC. Sarcoma 180 and Detroit 6 cells were incorporated into an agar medium and paper-strip chromatograms of the antitumor antibiotics were placed on the surface of the agar for 1 hr. For TLC, the chromatoplates would be placed on sterile filter paper resting on the agar, and left there for a period that would be determined by the physical characteristics of the antibiotics, e.g., solubility. The steps that follow removal from the agar of both the chromatoplate and the filter paper are those used with the paper-strip chromatograms: the agar is incubated for 2 days, under special conditions, and antitumor activity is detected by measuring the zone of inhibition of cellular dehydrogenase activity, as revealed by resazurin dye incorporated into the agar medium. Yoshida et al. 1°9 used TLC to separate newly formed quinomycins in an experiment that sought to replace the chromophore (qu-inoxaline-2carboxylic acid) of this antibiotic with another chromophore, e.g., quinolinic acid, and to examine the products of such replacements for their biological activities. S t r e p t o m y c e s strain 731-I was used in shake-flask fermentors and the antibiotics formed were extracted into ethyl acetate. After initial purification of the extract by column chromatography, three biologically active materials were separated by TLC. One of the materials was identified as quinomycin A, and the other two were shown to be analogs in which one or two quinoxaline-2-carboxylic acid moieties had been replaced by quinolinic acid. In the TLC system used, silica gel G (Merck) was the adsorbent and methyl ethyl ketone was the mobile phase. In this system, quinomycin A had an R~: of 0.34 and the Rf values of the two new analogs were 0.52 and 0.70. The antibiotics were eluted lo7j . Zarnach and S. Pfeifer, Pharmazie 19, 216 (1964). ~o8D. M. Schuurmans, D. T. Duncan, and B. H. Olson, Ccl~.cer Res. 24, 83 (1964). 1o~T. Yoshida, Y. Kimura, and K. Katagiri, d. Antibiot. Set. A 21,465 (1968).
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METHODS FOR THE STUDY OF ANTIBIOTICS
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from the chromatoplate with chloroform for further attempts at identification. Korchagin et al. 11° used TLC to estimate the amounts of antibiotic produced under various conditions of large-scale biosynthesis of erythromycin, penicillin G, penicillin V, vancomycin, and fusidine. Silica gel G plates were used in each case, but the solvent systems varied with the antibiotic. For erythromycin, they used methylene chloride:benzene:methanol (31 : 10:15) ; for penicillin G, butanol: butyl acetate: acetic acid: methanol: phosphate buffer, pH 5.8 (80:15: 40:5:24) ; for penicillin V, acetone: acetic acid :water (0.5: 5: 3.2) ; for vancomycin, phenol :ethanol:dimethylformamide:acetic acid (15:20:20:45); and for fusidine, ethyl acetate saturated with water: methanol (9: 1). Abou-Zeid m used TLC to analyze the large-scale fermentation of tetracyclines. The-use of silica gel G plates impregnated with pH 3.0 phosphate buffer, butanol saturated with water as the mobile phase, and bioautography allowed quantitative determinations of tetracycline and chlortetracycline in fermentation broths. One particular aspect of TLC applied to fermentation broths, viz. preparation of the antibiotic sample, is worth discussing in detail. Direct spotting of a fermentation broth is not advisable, because the many materials in solution make it difficult to achieve chromatographic separation of the antibiotic in question; R~ values are not reproducible, and a great deal of streaking can occur. In most cases, it is necessary to prepare a sample more suitable for spotting than is the whole broth. The mode of preparation of such a sample depends largely on the physicochemical nature of the antibiotic. If the antibiotic is solvent extractable, a simple or double extraction with ether, benzene, chloroform, ethyl acetate, or butanol, under standard extraction conditions, will usually yield a sample suitable for spotting. Apolar antibiotics can be extracted with ether; as the polarity of the antibiotic increases, the other solvents, listed above in order of increasing polarity, should be used. The ideal solvent will permit complete extraction of the antibiotic without extraction of any other material. If the antibiotic can be extracted into benzene, butanol should not be used, since its use would enrich the content of unwanted inert materials in solution to be spotted. Most of the time, a compromise selection of solvents has to be made, since it is hard to find a single solvent the use of which results in selective and quantitative extraction. A fermentation medium usually contains an antifoam agent, the presence of which is undesirable in TLC. Antifoam agents are soluble in all ~le V. B. Korchagin, L. I. Serova, Z. I. Vtorova, I. I. Vagina, E. Z. Olpinska, and S. P. Dementieva, Epidemiol. Mikrobiol. In]ek. Bolesti 8, 50 (1971). m A. Abou-Zeid Indian J. Pharm. 32, 59 (1970)
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solvents that are used to extract antibiotics. It may not be necessary to separate the antifoam material from the fermentation medium, if the ratio of the concentration of the antifoam agent to that of the extracted antibiotic is not too great and if the mobile phase used is such that the antifoam agent has a much higher R~ value than does the antibiotic. However, if the antifoam material causes streaking in TLC or problems in bioautography, prior separation is advisable. Such separation can, many times, be achieved by triturating with hexane the solvent extract previously taken to dryness. Most antibiotics do not dissolve in hexane and, under suitable trituration conditions (volume, temperature), cosolubilization of the antibiotic with the antifoam agent can be minimized. In certain cases, an apolar antibiotic cannot be extracted with a solvent like hexane because the antibiotic is an intracellular product. In such a case, a solvent like butanol must be used, one that releases the antibiotic from the mycelium by changing the physical status of the mycelium. The solvent chloroform warrants some comment. We have found that this solvent extracts from a fermentation broth many more unwanted materials than does the more polar solvent ethyl acetate. However, antibiotics that are only slightly extractable into ethyl acetate are sometimes extracted quantitatively into chloroform. If an antibiotic is not directly extractable into a solvent, other manipulations are necessary to produce a solution suitable for spotting in TLC. It is possible that the antibiotic is of an acidic nature and that the fermentation broth has a pH of 8.0 or higher. The acidic antibiotic is then in a salt form and cannot be extracted into a solvent. Acidification of the broth to pH 3.0 or lower liberates the antibiotic by converting it to a free acid, thus making it solvent extractable. The same types of considerations are valid for basic antibiotics, but these can usually be extracted without prior adjustment of pH, since fermentation broths are usually alkaline. More manipulations are necessary to prepare a solution suitable for spotting if the antibiotic is very water soluble. If the antibiotic is an extracellular product, the whole broth may be centrifuged and the clear supernatant can be spotted. If the resulting chromatogram is not satisfactory, the clear supernatant can be taken to dryness and then extracted with methanol, acetone, or water. The aqueous extract can be mixed with acetone or another solvent to precipitate unwanted proteins. If the antibiotic is intracellular, it can best be liberated by mixing the whole broth with an equal volume of isopropanol. After centrifugation of this mixture, the supernatant can be spotted directly or can be concentrated by taking it to dryness and triturating it as described above.
206
METHODS FOR THE STI)'D¥ OF ANTIBIOTICS
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Samples may be prepared by means other than extraction. For example, in the case of water-soluble, basic antibiotics (e.g., neomycin, gentamycin, kanamycin), the supernatant of the centrifuged whole broth can be treated with a cabionic-exchange resin, and the antibiotic can then be eluted from the resin. However, these means are beyond the scope of this article and will not be discussed further. Appropriate new methods for using TLC to analyze the large-scale fermentation of other antibiotics can be constructed from the techniques for sample preparation outlined above and from the TLC systems suggested by various authors, as cited in the second section of this article.
B. Antibiotic Transformation by Single Enzymes Although the monitoring of fermentation processes by TLC is complicated by the multiplicity of metabolites produced and by the interference of other components of the fermentation broth, it is comparatively easy to use TLC to monitor a reaction catalyzed by a single enzyme. For one thing, the target of a single enzyme reaction is usually only one compound, and it is easy to prepare a relatively clean solution of this compound for TLC. Knowledge of the physical properties of the substrate compound should make it easy to prepare samples suitable for spotting. Second, the formation of one or two products of the enzymic reaction, most likely ones with known R~ values, should create no difficulties in following the reaction chromatographically over an extended period. One of the enzymes that is used in isolated form for transforming antibiotics is penicillin amidase or penicillin amidohydrolase (EC 3.5.1.11). This enzyme, which is sometimes called penicillin acylase, is produced as a constitutive enzyme by many gram-negative bacteria and by microfungi. The same enzyme also hydrolyzes certain amide side chains of cephalosporins. The significance of this enzyme is primarily in chemotherapy, and derives from the fact that bacteria producing penicillin amidase are resistant to the action of natural, fermented penicillins and cephalosporins. On the other hand, some semisynthetic penicillins and cephalosporins are not attacked by this enzyme and have a greater chance to be chemotherapeutically useful in the treatment of microbial infections. Besides having chemotherapeutic significance, penicillin amidase is used on an industrial scale to split penicillins and cephalosporins obtained by fermentation in order to produce 6-APA, 7-ACA, and 7-ADCA. These products are then used in the synthesis of amidaseresistant penicillins and cephalosporins. The use of extracellular amidase in culture filtrates to obtain 6-APA on a large scale is described in a British patent. 112 1~ British P a t e n t 892,144 (1962).
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Use of whole cultures of different microorganisms that produce an amidase capable of splitting cephalosporin C into the side chain and 7-ACA has been described in a Merck patent. 11~ More elegantly, extracellular amidase, or intracellular amidase obtained after disruption of the source microorganism, is used attached to an insoluble carrier. 114,~15 Attachment of the enzyme to an insoluble carrier has the potential advantage of repeated use of the enzyme after its recovery from the reaction medium by simple filtration. The reaction is carried out until complete hydrolysis of the penicillin or cephalosporin has been achieved, and the completeness of the process can be followed by TLC. In the preparation of a sample for TLC, a small portion drawn from the reactor is acidified to pH 1.5-2.0 and is then extracted with an organic solvent, like methylene chloride. This extract, along with a sample of the compound to be hydrolyzed, can be applied directly to a chromatoplate. TLC systems useful for this purpose are numerous; very good results have been achieved by use of Analtec Silica Gel GE chromatoplates and the solvent system butanol:acetic acid:water (75:7:21). ~6 In that system, the Ri values of penicillin G, cephalosporin C, and N-phenacetyl 7-ADCA are 0.6, 0.82, and 0.75, respectively. The last compound can be split by amidase, used as a culture filtrate or on a solid support, e.g., cellulose. For a 0.1 M concentration of the substrate and an enzyme concentration that saturates the substrate, the reaction is complete in 10-12 hr at 25 °. To follow this reaction, a 0.5-ml sample is drawn from the reactor and mixed with 0.5 ml of methylene chloride; the pH of the mixture is then adjusted to 1.8. From the organic phase, a 20- to 30-~I quantity is spotted on the silica gel plates. After the chromatogram has been developed with the mobile system described above, spots are visualized under ultraviolet light or the dried chromatogram is sprayed with a 1% molybdic acid aqueous solution and heated gently. Completion of the enzymic reaction can be detected by this TLC system with a precision of 1 hr. The TLC system described above can also be used to monitor processes in which milk containing penicillin is treated with penieillinase ;~7 the importance of penicillin-free milk for direct consumption or for cheesemaking is well established. ~1~ Semisynthetic penicillins and cephalosporins resistant to both fl-lactamase and amidase are, as noted earlier, important from the point of view m French Patent 1357977 (1964). 114Hungarian Patent 150,782 (1963). m W. Marconi, F. Cecere, F. Morisi, G. Della Penna, and B. Rappuoli, J. Antibiot. Set. A. 26, 228 (1973). m A. Aszalos, unpublished, 1972. 1~ J. D. Ridgway, J. Soc. Dairy Technol. 13, 197 (1960). m H. R. Chapman, Dairy Ind. 21, 970 (1956).
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METHODS FOR THE STUDY OF ANTIBIOTICS
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of chemotherapy. TLC can be a very helpful tool in the evaluation of these synthetic derivatives, which may be incubated with the soluble enzyme, or with the enzyme on a solid support, or with a bacterial culture; 119 the disappearance of the antibiotic is followed by TLC. TLC systems useful for such studies include Kieselguhr G (Merck) as adsorbent with chloroform: methanol •water (80: 20: 25) or butanol: acetic acid:water (50:25:25) as the mobile phase, and Eastman Silica Gel Chromagram sheets as adsorbent with ethanol or butanol:methanol (1:1) as the mobile phase. Spots can be visualized under ultraviolet light or by spraying the chromatoplate with a 1% molybdic acid aqueous solution and heating it gently. Testing antibiotics for resistance to the actions of fl-lactamase or amidase can also be carried out by fluorescence TLC. 86 A few micrograms of the penicillin derivative are dissolved in 0.5 ml of phosphate buffer (pH 7.0) and the enzyme, also in phosphate buffer, is added. After 30 min of incubation, the sample is treated with 9-isothiocyanoacridine, then freeze-dried; the lyophilate is taken up in ethanol. A 1-ml aliquot of the ethanolic solution is spotted on a silica gel G plate, 0.25-ram thick. Dimethylformamide: chloroform:28% ammonium hydroxide (10:5 : 4) is used as the mobile phase. After development, the chromatoplate is dried at 106°C for 15 min and spots are located by the use of a spectrofluorimeter. An absence of difference in intensity between penicillin spots prepared before incubation and those prepared after 30 min of incubation indicates that the antibiotic is resistant to the action of penicillinase. The possibility of using TLC to study the biotransformation of antibiotics can also be visualized from the work of Okamoto and Suzuki. 1~-° These authors studied the inactivation of chloramphenicol, dihydrostreptomycin, and kanamycin by enzymes obtained from cells harboring R factor. The extent of inactivation of the antibiotics was measured by turbidometric assay of bacterial growth. Satisfactory results would also have been obtained by spotting log samples of the reaction on Eastman Silica Gel Chromagram sheets and measuring the zones of inhibition on bioautograph plates after chromatography. The solvent system recommended for TLC of chloramphenicol is methanol:benzene (12:88), that for dihydrostreptomycin and kanamycin is pyridine: water (1 : 1). TLC was used by Spaeren e t al. ~2~ to check the final purity of biosynthesized gramicidin S. The study involved following the incorporation of [~4C]valine into gramicidin S by cell-free enzyme systems obtained ~lgM. Nishida, Y. Yokota, M. Okui, ¥. Mine, and T. Matsubara, J. Antibiot. Ser. A 21, 165 (1968). ~ S. Okamoto and Y. Suzuki, Nature (London) 208, 1301 (1965). 1~ U. Spaeren, L. O. FrChlm, and S. G. La Land Biochem. J. 102, 586 (1967).
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209
from early- and late-phase cell growth. The extracted gramicidin is chromatographed by two-dimensional TLC on silica gel (medium H, according to Stahl), with ethyl acetate:pyridine:acetic acid:water (60:20:6:11) and n-butanol:acetic acid:water (25:6:25) serving as sequential mobile phases. Gramicidin S is eluted from the chromatoplate (in 90% yield) with ethanol:0.2 N HC1 (9:1). The results indicate that gramicidin S is synthesized in a manner different from that involved in the synthesis of cell proteins. Other studies of the biosynthesis of antibiotics by cell-free enzyme system in which TLC can be used to follow the enzymic processes are described by Katz and Weissbach. 12: C. Antibiotic Transformation in V i v o
Investigations of the use of TLC in connection with the action of enzymes on antibiotics in vivo center on the fate of antibiotics after their administration to living organisms. Antibiotics are secreted by these organisms, are accumulated in certain organs, and may undergo chemical transformations. Investigations of these phenomena are the concern of a rapidly developing branch of science, pharmacokinetics. Sensitive analyrical tools are needed in these investigations and TLC, especially when combined with radioisotope technique, has proved to be very useful. TLC is used to separate the antibiotic under investigation from its metabolites and from other substances present in body fluids or tissues. Care must be exercised to avoid artifacts during the handling of samples and during chromatography, if one is to draw valid pharmacokinetic conclusions. The preparation of samples for TLC usually involves the selection of a chromatographic system, extraction, or other methods of preparation, and the removal of interfering substances, such as proteins, inorganic salts, lipids, and excess carbohydrates. The chromatographic method for detecting antibiotics and their metabolites is usually one that allows good separation of the antibiotic from other substances. If metabolites have been formed that are more polar than the antibiotic, progressive changes are made in the solvent system and adsorbent to help find the most suitable system. When a radioactive antibiotic has been administered, finding a suitable system for detecting the metabolites formed in vivo is relatively easy. All the radioactive spots move from the origin and are separated from each other in a good TLC system, and the chromatography can be followed by autoradiography or radiometry. Following the fate of antibiotics that have not been radiolabeled is more difficult, and the separation of metabolites must be carried out empirically. 1~.~E. Katz and H. Weissbach,Develop. Ind. Microbiol. 8, 67 (1966).
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METHODS FOR THE STUDY OF ANTIBIOTICS
[8]
One of the most difficult tasks in developing a method to study the metabolism of an antibiotic is choosing an extraction process. To find all metabolites by the use of TLC, it would be ideal to apply the biological material directly to the chromatoplate. In most cases, however, this is not possible, because of the low concentrations of the antibiotic and its metabolites and because of interference by other substances in the chromatographic separation. In those cases in which the antibiotic passes into the urine in high concentration, the volume of urine may be small enough to permit direct application of a urine sample to the chromatoplate. For samples of blood, it is usually necessary to remove at least the proteins. More rewarding than such direct application, however, is the extraction of biological materials and the application of the extracts to the chromatoplate. Homogenization of the biological material followed by extraction at different pH's with solvents of different polarity is a helpful procedure. When solvents of high polarity are used, interfering materials that have been extracted simultaneously are separated by gel filtration, or in some other way, before the extract is applied to the chromatoplate. When large amounts of lipid are co-extracted with the antibiotic, special chromatographic systems should be used, e.g., reversedphase or adsorption chromatography with apolar solvents. For charged metabolites, ion-exchange chromatography is used. Some antibiotics become conjugated (derivatized) in v i v o , and may be detected more conveniently £fter hydrolysis. For details of the use of TLC in pharmacokinetic studies, see the summarization by Titus. ~23 Conjugation of antibiotics and other changes, like degradation, are the results of enzymic action on the parent compounds. Enzymes that are involved in the metabolic changes of different drugs are detailed by Brodie and Gillette. 12~ Some examples of enzymes located in the microsomes are the monooxygenases, nitrogen-oxygen and sulfur dealkylating enzymes, reducing enzymes, and nucleic acid diphosphate glucuronyl transferases. Other enzymes, not located in the microsomes, are esterases, alcohol and aldehyde dehydrogenases, monoamine oxidases, methyltransferases, and acetyltransferases. Studies dealing with quantitative and qualitative examinations of antibiotics that have passed through humans and animals are summarized by Clarke, 1~ whose book lists the TLC systems that have been found 121E. O. Titus, in "Handbook of Experimental Pharmacology" (B. B. Brodie and J. R. Gillette, eds.), Part 2, p. 147. Springer-Verlag, New York, 1971. ~ "Handbook of Experimental Pharmacology (B. B. Brodie and J. R. Gillette, eds.), Part 2. Springer-Verlag, New York, 1971. ~ "Isolation and Identification of Drugs in Pharmaceuticals, Body Fluids, and Postmortem Materials" (E. G. C. Clark, ed.). Pharmaceutical Press, London, 1969.
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THIN-LAYER CHROMATOGRAPHY OF ANTIBIOTICS
211
most useful in identifying antibiotics in pharmacokinetic studies. For example, the system recommended for use with the antibiotics framicetin, neomycin, streptomycin, viomycin, kanamycin, tetracycline, vancomycin, spiramycin, oleandomycin, lincomycin, and cephaloridine uses 20 X 20-cm glass plates covered with a 0.25-mm layer of a slurry of 30 g of silica gel G in 60 ml of water, then dried at 110 ° for 1 hr. Samples are applied as 1% solutions in 2 N acetic acid. The mobile phase is conc. ammonia:methanol (1.5:100). The chromatographic chamber is 21 )4 21 X 10 cm and is covered with filter paper to assist evaporation. Antibiotics may be localized on the developed chromatoplates by ultraviolet light or by spraying the plates with acidified iodoplatinate, Dragendorff reagent, potassium permanganate, or some other suitable reagent. Nagasawa e t al. ~26 used TLC to investigate the metabolism in rabbits of 9-(3'-amino-3'-deoxy-fl-D-ribofuranosyl)-6-dimethylamino-9H-purine, the aminonucleoside of the antibiotic puromycin. The investigation was prompted by the finding that this aminonucleoside has a different toxicity in the rabbit than in human, rat, and mouse. The difference in toxicity was attributed to an enzyme system found in the rabbit that is capable of performing a nonspecific N-methyl transfer in a variety of structurally unrelated amines. This N-methyltransferase enzyme can be found in rabbit lung, but not in the lungs of rats or mice. Studies revealed that, in each of several different TLC systems, a metabolite of the parent compound moved in a manner similar to that of synthetic 9-(3'-amino-3'deoxy-fl-D-ribofuranosyl)-6-methylamino-9H purine, but different from that for derivatives of the aminonucleoside that were mono- or dimethylated in the 3' position. The TLC system used by Nagasawa e t al. consisted of silica gel HF~9 or silica 7-GF, with a mobile phase of 88% formic acid: absolute ethanol : water (3: 16:3). For details about the isolation of metabolites from rabbit urine by use of this TLC system, the reader should consult the original article. TLC was also used by Di Carlo e t al. ~7 in pharmacokinetic studies in man of the antimicrobial agent, oxolinic acid, i.e., 5-ethyl-5,8-dihydro8-oxo-l,3-dioxolo[4,5,8]-quinoline-7-carboxylic acid; in these studies, a new metabolite was isolated. Urine samples were fractionated to isolate radiolabeled metabolites of oxolinic acid. The urine was treated with B-glucuronidase, and the unchanged oxolinie acid was removed by filtration. The filtrate was then extracted with chloroform and the extract, after being concentrated, was chromatographed on a thin-layer plate. For TLC, silica gel was used, with a mobile phase of ethanol:conc, am126H. T. Nagasawa, F. N. Shirota, and G. S. Alexander, J. Med. Chem. 15, 177 (1972). 12TF. J. Di Carlo, M. C. Crew, and R. C. (]reenough, Arch. Biochem. Biophys. 1¢~7, 503 (1968).
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METHODS FOR THE STUDY OF ANTIBIOTICS
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monia:water (12:1:7). The fastest moving component, with an RI of 0.67, was found to be a new metabolite, and was shown by ultraviolet, infrared, and nuclear magnetic resonance spectroscopy to be 1-ethyl-l,4dihydro-6-methoxy-7-hydroxy-4-oxo-3-quinoline. It was theorized that this compound was present as the glucuronide in the urine and had been formed by the action of microsomal enzymes that opened the original 1,3-dioxolo ring to form the 6-hydroxy-7-hydroxy compound, and by subsequent methylation by catechol-o-methyltransferase, with S-adenosylmethionine serving as a methyl donor. The identity of the compound was also demonstrated by TLC, using synthesized materials as reference standards, in a system that consisted of 5 X 20-cm glass plates coated with a 0.25-ram thick layer of silica gel G bound with CaS04 and the mobile phase described earlier. Spots were detected under ultraviolet fluorescence or after spraying of the chromatogram with Folin-Ciocalteu 128 reagent or ferric ferricyanide solution. The latter reagent is freshly prepared from 1.0 g of ferric chloride and 50 mg of potassium ferricyanide dissolved in 10 ml of water. These methods of detection permitted the establishment of identify of the new metabolite and the corresponding synthetic material on the basis of Rs values and colors of the spots. Finally, the work of Fischer and Riegelman 129 provides another example of the use of TLC in pharmacokinetic studies. These authors developed a quantitative determination of the antibiotic griseofulvin and its metabolite griseofulvin-4'-alcoho], in plasma extracts by direct fluorescence measurement on thin-layer chromatoplates. For scanning in ultraviolet light, they employed a Photovolt TLC densitometer equipped with a mechanical stage for automatic scanning, a source of ultraviolet light, a primary filter, and a photomultiplier source unit. For details of the scanning technique, the reader should consult the original article. In the preparation of TLC plates, a stock suspension of 6% technical colloidal boehmite alumina (Baymal) was prepared by high-speed agitation in a Waring Blendor. A 50-ml portion of this suspension was diluted with • 50 ml of 85% ethanol, then 30 g of silica gel (Merck) was admixed. A Colab applicator was used to cover 5 X 20-cm glass plates with this suspension. After 15 min at room temperature, the plates were activated by heating them to 110 ° for 1 hr. To prepare samples for chromatography, an aliquot of heparinized rabbit blood was centrifuged and the plasma was extracted with ether. After it had been concentrated, the ether solution was applied to the TLC plates by means of a push-botton disD. Waldi, in "Thin-Layer Chromatography: A Laboratory Handbook" (E. Stahl, ed.), 2nd ed., p. 498. Springer-Verlag, Berlin and New York, 1969. 12~L. J. Fischer and S. Riegelman, Y. Chromatogr. 21, 268 (1966).
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213
p e n s e r ( B r i n k m a n ) f i t t e d w i t h a 0.1-ml H a m i l t o n s y r i n g e t h a t d e l i v e r s 0.002 m l of s o l u t i o n ; t h e T L C p l a t e w a s p l a c e d 1 m m b e l o w t h e syringe. T h i s p r o c e d u r e p r o v i d e d s p o t s of u n i f o r m size. A s t a n d a r d d e v e l o p i n g t a n k , l i n e d w i t h filter p a p e r , was used for c h r o m a t o g r a p h y , a n d t h e m o b i l e p h a s e w a s e t h e r : a c e t o n e ( 3 : 2 ) . I n t h i s s y s t e m , t h e R I of griseof u l v i n was 0.62 a n d t h a t of g r i s e o f u l v i n - 4 ' - a l c o h o l was 0.50. T h e d r i e d p l a t e s were s c a n n e d a f t e r 12 h r a n d d e t e r m i n a t i o n s were m a d e in duplicate. Q u a n t i t a t i v e e s t i m a t i o n s of t h e a n t i b i o t i c a n d its m e t a b o l i t e were m a d e b y c a l c u l a t i n g t h e a r e a s u n d e r t h e curves p r o d u c e d b y r e c o r d e d s c a n n i n g of t h e fluorescent spots. T h e e s t i m a t e s a g r e e d well w i t h a s s a y s of s i m i l a r l y s p o t t e d T L C p l a t e s t h a t h a d n o t been c h r o m a t o g r a p h e d . T h e p h a r m a c o k i n e t i c conclusion of t h i s s t u d y was t h a t g r i s e o f u l v i n - 4 ' - a l c o h o l , i n a c t i v e in vitro, is c o n v e r t e d r a p i d l y in vivo to b i o l o g i c a l l y a c t i v e griseofulvin.
[9] Gas-Liquid Chromatography of Antibiotics By KIYOSHI T s u J i a n d JOHN H . ROBERTSON I. Introduction . . . . . . . . . . . . . . . . . II. Amino-Cyclitol Antibiotic . . . . . . . . . . . . . Spectinomycin . . . . . . . . . . . . . . . . III. Amino-Glycoside Antibiotics . . . . . . . . . . . . A. Gentamicin . . . . . . . . . . . . . . . . B. Kanamycin . . . . . . . . . . . . . . . . C. Lividomycin . . . . . . . . . . . . . . . . D. Neomycin . . . . . . . . . . . . . . . . . E. Paromomycin . . . . . . . . . . . . . . . IV. Aromatic Antibiotics . . . . . . . . . . . . . . A. Chloramphenicol . . . . . . . . . . . . . . . B. Griseofulvin . . . . . . . . . . . . . . . . V. Glutarimide Antibiotic . . . . . . . . . . . . . . Cycloheximide . . . . . . . . . . . . . . . . VI. Lincomycin-Clindamycin Family . . . . . . . . . . . A. Lincomycin . . . . . . . . . . . . . . . . B. Clindamycin . . . . . . . . . . . . . . . . C. Clindamycin Phosphate . . . . . . . . . . . . . D. Clindamycin Palmitate . . . . . . . . . . . . . VII. Macrolide Antibiotic . . . . . . . . . . . . . . Erythromycin . . . . . . . . . . . . . . . . VIII. Penicillin . . . . . . . . . . . . . . . . . . IX. Tetracycline . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . .
214 215 215 217 217 218 220 220 228 228 228 232 234 234 237 237 239 242 243 245 245 248 251
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213
p e n s e r ( B r i n k m a n ) f i t t e d w i t h a 0.1-ml H a m i l t o n s y r i n g e t h a t d e l i v e r s 0.002 m l of s o l u t i o n ; t h e T L C p l a t e w a s p l a c e d 1 m m b e l o w t h e syringe. T h i s p r o c e d u r e p r o v i d e d s p o t s of u n i f o r m size. A s t a n d a r d d e v e l o p i n g t a n k , l i n e d w i t h filter p a p e r , was used for c h r o m a t o g r a p h y , a n d t h e m o b i l e p h a s e w a s e t h e r : a c e t o n e ( 3 : 2 ) . I n t h i s s y s t e m , t h e R I of griseof u l v i n was 0.62 a n d t h a t of g r i s e o f u l v i n - 4 ' - a l c o h o l was 0.50. T h e d r i e d p l a t e s were s c a n n e d a f t e r 12 h r a n d d e t e r m i n a t i o n s were m a d e in duplicate. Q u a n t i t a t i v e e s t i m a t i o n s of t h e a n t i b i o t i c a n d its m e t a b o l i t e were m a d e b y c a l c u l a t i n g t h e a r e a s u n d e r t h e curves p r o d u c e d b y r e c o r d e d s c a n n i n g of t h e fluorescent spots. T h e e s t i m a t e s a g r e e d well w i t h a s s a y s of s i m i l a r l y s p o t t e d T L C p l a t e s t h a t h a d n o t been c h r o m a t o g r a p h e d . T h e p h a r m a c o k i n e t i c conclusion of t h i s s t u d y was t h a t g r i s e o f u l v i n - 4 ' - a l c o h o l , i n a c t i v e in vitro, is c o n v e r t e d r a p i d l y in vivo to b i o l o g i c a l l y a c t i v e griseofulvin.
[9] Gas-Liquid Chromatography of Antibiotics By KIYOSHI T s u J i a n d JOHN H . ROBERTSON I. Introduction . . . . . . . . . . . . . . . . . II. Amino-Cyclitol Antibiotic . . . . . . . . . . . . . Spectinomycin . . . . . . . . . . . . . . . . III. Amino-Glycoside Antibiotics . . . . . . . . . . . . A. Gentamicin . . . . . . . . . . . . . . . . B. Kanamycin . . . . . . . . . . . . . . . . C. Lividomycin . . . . . . . . . . . . . . . . D. Neomycin . . . . . . . . . . . . . . . . . E. Paromomycin . . . . . . . . . . . . . . . IV. Aromatic Antibiotics . . . . . . . . . . . . . . A. Chloramphenicol . . . . . . . . . . . . . . . B. Griseofulvin . . . . . . . . . . . . . . . . V. Glutarimide Antibiotic . . . . . . . . . . . . . . Cycloheximide . . . . . . . . . . . . . . . . VI. Lincomycin-Clindamycin Family . . . . . . . . . . . A. Lincomycin . . . . . . . . . . . . . . . . B. Clindamycin . . . . . . . . . . . . . . . . C. Clindamycin Phosphate . . . . . . . . . . . . . D. Clindamycin Palmitate . . . . . . . . . . . . . VII. Macrolide Antibiotic . . . . . . . . . . . . . . Erythromycin . . . . . . . . . . . . . . . . VIII. Penicillin . . . . . . . . . . . . . . . . . . IX. Tetracycline . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . .
214 215 215 217 217 218 220 220 228 228 228 232 234 234 237 237 239 242 243 245 245 248 251
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METHODS FOR THE STUDY OF ANTIBIOTICS
X. Miscellaneous . . . . . . . . . . . . . . . . . A. Peptolide Antibiotic--Antimycin A . . . . . . . . . . B. Celestosaminide . . . . . . . . . . . . . . . . C. Phosphonomycin . . . . . . . . . . . . . . . . D. Thiamphenicol . . . . . . . . . . . . . . . . E. Validamycin . . . . . . . . . . . . . . . . . F. Summary of GLC Methods for Antibiotic Analysis . . . . .
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253 253 253 254 254 254 255
I. Introduction Antibiotics in general are complex organic molecules, whose commercial preparations usually contain various amounts of isomers, biosynthetic intermediates, and degradation products, each of which m a y possess a different antimicrobial spectrum from its parent compound. I-4 Microbiological methods of analysis,5 the officiallyaccepted method of analysis for the majority of antibiotics,measure the total antimicrobial activity of the various components against a specificmicroorganism. Since the antimicrobial activity of one antibiotic varies from microbial species to species,~,~ the microbiological responses to a given antibiotic compound are not always constant and frequently are influenced by chemical and physical factors.7 During the past decade, gas-liquid chromatography (GLC) has become one of the most important analytical tools for the investigation of various organic compounds, particularly after the development of numerous trimethylsilyl (TMS) reagents to derivatize a compound to give added volatility and thermostability. Several G L C methods have been developed to separate and quantitate various isomers, process intermediates, and degradation products of antibiotics.Drug potency, as calculated from G L C data, generally agrees well with values obtained by microbiological assay methods. Only a handful of the G L C methods have received approval of the Food and Drug Administration as an alternate assay method ~ (lincomycin, clindamycin, spectinomycin, and neomycin) ; however, the listwill certainly increase in the near future. This paper describes the G L C methods for chromatography of intact 1K. Tsuji, J. H. Robertson, R. Bass, and D. J. McInnis, Appl. Microbiol. 18, 396 (1969). K. Tsuji and J. H. Robertson, Anal. Chem. 43, 818 (1971). 8 O. K. Sebek, J. Bacteriol. 75, 199 (1958). ' A. F. Zak, T. I. Navolotskays, G. I. Loseva, N. I. Shukailo, O. B. Ermolova, and L. M. Yacobson, Antibiotiki (Moscow) 18, 324 (1973). Code of Federal Regulations, Title 21, Food and Drugs U.S. Government Printing Office, Washington, D.C., 1974. W. A. Freyburger and L. E. Johnson, Antibiot. Chemother. 6, 586 (1956). W. T. Sokolski, C. G. Chidester, and D. G. Kaiser, J. Pharm. Sc/. 53, 726 (1964).
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215
antibiotic molecules, many by derivatization, and discusses some of the difficulties associated with quantitative derivatization and chromatography and describes precautionary measures for the successful GLC determination of antibiotics. 11. Amino-Cyclitol Antibiotic Spectinomycin 5,s
Spectinomycin is chromatographed intact as the tetrakistrimethylsilyl derivative using an SE-52 column.
Procedure Materials SE-52, 5%, on Diatoport S, 80-100 mesh Column, glass, 3 mm i.d. X 610 mm (2 ft) Vial, glass, 1- or 5-dram size with polyethylene stopper Dimethylformamide (DMF), dry Hexamethyldisilazane (HMDS) Triphenyl antimony Benzyl alcohol Acetone
Solutions Internal standard solution. Prepare a 2 mg/ml solution of triphenylantimony in dry DMF. Apparatus Gas chromatograph: For requirements see the neomycin section. Detector: Flame-ionization detector
Chromatographic Conditions Column: glass, 3 mm i.d. X 610 ram, packed with 5% SE-52 Gas flow rate: hydrogen, 40 ml/min; air, 600 ml/min; and carrier gas (helium), 60 ml/min Oven temperature: 190 ° Detector temperature: 220 ° Flash heater: 200 ° Chart speed: 0.64 cm/min Sample size: 1 ~l at attentuation and range setting of 32 X 10 8 L. W. Brown and P. B. Bowman, J. Chromatog. Sci. 12, 373 (1974).
216
METHODS FOR THE STUDY OF ANTIBIOTICS
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Preparation o] Chromatographic Column Follow the procedure described in the neomycin section. Procedures for the conditioning of the column are listed in the erythromycin section.
Preparation o] Bulk Drug and Re]erence Standard Powder Accurately weigh approximately 50 mg of. spectinomycin bulk powder and spectinomycin dihydrochloride pentahydrate reference standard into separate 5-dram vials.
Sample Preparation Sterile Product. Reconstitute a vial with the labeled volume of sterile water for injection with benzyl alcohol and mix. Withdraw 5.0 ml of the suspension, transfer quantitatively to a 50-ml volumetric flask, dissolve, and dilute to volume with deionized water. Transfer 1.0 ml of this solution to a 5-dram vial. Add 15 ml of acetone and evaporate to dryness with a gentle stream of nitrogen. Silylation Procedure Add 10.0 ml of the internal standard solution and 1.0 ml of H M D S to each vial containing sample and reference standard. Intermittently swirl the vials gently to mix the liquid for 1 hr at room temperature. Centrifuge if necessary and chromatograph.
Calculation Calculate the content of spectinomycin base in micrograms per milligram sample using Eq. (1) described in the kanamycin section.
Comments on the Assay Method Silylation of spectinomycin produces several GLC peaks depending on the silylation conditions and reagents. With HMDS, four TMS groups are added to the molecule while with BSA, a fifth group is added. After silylation, the sample must be chromatographed within approximately 2 hr since the silylated samples are not stable. A decrease in peak area of approximately 10% for the spectinomycin GLC peak is observed after a 12-hr period. The degradation product, actinamine, has a shorter retention time than the internal standard while actinospectinoic acid has a longer reten-
[91
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CHROMATOGRAPHY
OF ANTIBIOTICS
I
I
[
I
0
4
8
12
217
TIME (MINUTES)
FIG. 1. Chromatogram of spectinomycm indicating separation of (1) actinamine, (2) internal standaxd, and (3) spectinomycin [J. Chromatog. Sci., 12, 373 (1974)]. tion time than spectinomycin (Fig. i). Both degradation products usually produce two peaks due to incomplete silylation. A single peak can be produced from actinamine if the silylation time is extended; however, actinospectinoic acid is unstable under such a condition.
III. Amino-Glycoside Antibiotics A. Gentamicin 9
Chromatographic Conditions Detector: Flame-ionization Column: Glass, 3 mm i.d. X 610 mm (2 ft) packed with 3% OV-1 K, Tsuji and J. H. Robertson,unpublishedmethod.
218
METHODS FOR THE STUDY OF ANTIBIOTICS
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on Gas Chrom Q, 100-120 mash (Applied Science Lab, Inc., State College, Pennsylvania) Gas flow rate: Hydrogen, 50 ml/min; air, 600 ml/min; and carrier gas (helium), 70 ml/min Oven temperature: 240 ° Detector temperature: 280 ° Flash temperature: 245 °
Silylation Reagent Add 50 ~l of N-trimethylsilyldiethylamine (TMSDEA, Pierce Chemical Co., Rockford, Illinois) per milliliter of TRI-SIL "Z" (Pierce Chemical Company).
Silylation Procedure Add 1 ml of the silylation reagent in a sealed vial containing approximately 5 mg gentamicin. Heat the vial in a 75 ° oil bath for 45 min. Detailed procedure for the preparation of the column, sample preparation, and silylation procedure may be seen in Section III,D (neomycin). B . K a n a m y c i n 1°
Kanamyein is chromatographed intact as the silyl ether-silyl amine derivative using an OV-1 column.
Procedure Unless otherwise specifically stated, materials, solutions, and apparatus required and preparation of chromatographic column may be referred to in Section III,D.
Materials OV-1, 3% on Gas Crom Q, 100-120 mesh (Applied Science Lab) Column, glass 3 mm i.d. X 1830 mm (6 ft)
Solutions Internal standard--silylation reagent: Add 25 t~l of TMSDEA and 8 mg of trilaurin per ml of TRI-SIL "Z". Cap and mix. Place the vial in an airtight container and store under refrigerator temperature. 1~K. Tsuji and J. H. Robertson, Anal. Chem. 42, 1661 (1970).
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GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS
219
Reference standard:Dry the kanamycin sulfate reference standard at 60 ° in a vacuum oven at less than 5 Inm Hg for 3 hr. Immediately weigh and prepare a water solution containing 10 mg/ml of the standard.
Chromatographic Conditions Column: Glass, 3 mm i.d. X 1830 mm, packed with 3% OV-1 Gas flow rate: Hydrogen 40 ml]min, air 600 ml/min, and carrier gas (helium) 70 ml/min Oven temperature: 300 ° Detector temperature: 315 ° Flash heater: 300 °
Preparation o] the Chromatographic Column Follow the procedure described in Section III,D. The column thus prepared has normally 900 theoretical plates per meter for the silylated kanamycin A.
Sample Preparation Accurately weigh kanamycin bulk sample "as is" and prepare a water solution containing approximately 10 mg/ml of the kanamycin sulfate. Pipette 1.0 ml of the reference standard and sample solution into a vial and freeze dry. After freeze-drying, cap the vial with Teflon closures and metal retainers.
Silylation Procedure Add 1.0 ml of the internal standard-silylation reagent to each vial containing the freeze-dried sample using a 1-ml glass tuberculin syringe. Immerse and heat the vials in a 75 ° silicon oil bath for 35-40 min, swirling occasionally. Store the silylated samples in an airtight container under refrigeration temperature. Warm the sample before chromatography.
Chromatographic Procedure Using a microsyringe remove 1.5-2.0 t~l of sample from the vial and immediately inject it into the chromatograph at an attenuator and range
220
METHODS FOR THE STUDY OF ANTIBIOTICS
[9]
setting of 32 X 10. The sample transferral from the vial to the chromatograph must be made as quickly as possible. Calculation According to Omoto et al. 1~ kanamycin C elutes just prior to the kanamycin B peak. The content of kanamycin in micrograms per milligram of sample is calculated using the following formula: [RI/R~] X [Wr/W~] × F
(1)
where, R1 = (area of sample kanamyein A)/(area of the sample internal standard); R~ = (area of the standard kanamycin A)/(area of the standard internal standard) ; Wr = weight of kanamycin reference standard (mg/ml) ; We = weight of kanamycin sample (mg/ml) ; F = assigned value of kanamycin reference standard expressed in micrograms of anhydrous kanamycin base per milligram of the kanamycin sulfate. Problem Points o] the Assay Method Since the GLC analysis of kanamycin requires silylation of 4 NH~ groups as well as 7-OH groups, problems similar to those of neomycin GLC analysis are commonly encountered. Solutions to some of these problems may be found in Table I. Separation of kanamycin B from kanamycin A requires an extremely efficient column. Strict adherence to the column preconditioning, packing, and conditioning are essential for successful GLC analysis. C. Lividomycin 12 Chromatographic Conditions Column: Glass, 3 mm X 1000 mm, packed with 1% OV-1 on Gas Chrom Q, 100-120 mesh Gas flow rate: Carrier gas (nitrogen), 30 ml/min Oven temperature: 265 ° No silylation condition or other detail in procedure is given. D. Neomycin ~ Neomycin is ehromatographed intact as the silyl ether-silyl amine derivative using an 0V-'I column. ~1S. 0moto, S. Inouye, and T. Miida, J. Antibiol. 24, 430 (1971). 12 T. Mori, Y. Kyotani, I. Watanabe, and T. 0da, J. Antibiot. 25, 149 (1972).
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221
Procedure Materials
OV-1, 3% on Gas Chrom Q, 100-120 mesh (Applied Science Laboratory, Inc., State College, Pennsylvania) Column, glass, 3 mm i.d. X 610 mm (2 ft) Septum, high temperature (No. HT-9, Applied Science Lab.) Ferrule, Teflon front and back ferrules (Swagelok, Crawford Fitting Co., Solon, Ohio) Vial, glass, 2 ml (No. 5080-8712, Hewlett-Packard Co., Avondale, Pennsylvania) with Teflon-coated cap (Disks, No. 5080-8713, Hewlett-Packard) Chloroform, A.R. Hexane Pyridine, A.R. Dimethylchlorosilane iDMCS), (Pierce Chemical Co., Rockford, Illinois) Trimethylsilylimidazole (TSIM) (Pierce Chemical Co.) N-Trimethylsilyldiethylamine (TMSDEA) (Pierce Chemical Co.) TRI-SIL "Z" (Pierce Chemical Co.) or a solution containing approximately 40% TSIM in pyridine may be used Silyl-8 (Pierce Chemical Co.) Trilaurin (Supelco, Inc., Bellefonte, Pennsylvania) Solutions
DMCS 50% in hexane Internal standard--silylation reagent: Add 40 ~1 of TMSDEA and 2 mg of trilaurin per milliliter of TRI-SIL "Z." Cap the vial with a rubber septum and a metal retainer and mix. Place the vial in an airtight container and store under a refrigeration temperature. Reference standard solution: Dry the neomycin reference standard, USP Lot I or equivalent, at 60 ° in a vacuum oven at less than 5 mm Hg pressure for 3 hr. Immediately weigh and prepare a water solution containing 10 mg/ml of the neomycin sulfate standard. The dried neomycin powder is very hygroscopic and, when exposed to an atmosphere of 50% R H on an open surface, the sample can increase in weight by 8% in 3.5 min. In an open weighing bottle, it increases in weight by 1% in the 'first minute and then at a rate of approximately 0.4% per minute. 13 1~W. It. O. International Lab. Biol. Standards, Mill Hill, London January (1970).
222
METHODS FOR THE STUDY OF ANTIBIOTICS
[0]
Apparatus Gas Chromatograph: A Hewlett-Packard 402 or equivalent should be used. The sample injection must be such as to allow on-column sample injection approximately 2 cm into the end of the column. Metal parts and excessive dead space must be excluded from the chromatographic system. 1~,15 Detector: Flame ionization detector Freeze-dryer: A shelf-type freeze-dryer, e.g., No. 10-145MR-BA, VirTis Co., Gardiner, New York, is preferred over a manifold type freeze-dryer. Oil bath: Silicone oil at 75 ° equipped with stirring motor Micropipette: 500-~1 (Eppendorf micropipette, distributed by Brinkman Instruments, Inc., Westbury, New York, or equivalent). Microsyringe: 10 ~l (Hamilton Co., Whittier, California, or equivalent)
Chromatographic Conditions Column: Glass, 3 mm i.d X 610 ram, packed with 3% OV-1 Gas flow rate: Hydrogen, 50 ml/min; air, 600 ml/min; and carrier gas (helium), 70 ml/min. A gas-drying filter trap must be used to remove moisture and organic contaminants, e.g., oil, from the carrier gas, hydrogen, and air. The drying agent, e.g., 4A molecular sieve, must be periodically changed. Oven temperature: 290 ° Detector temperature: 310 ° Flash heater: 290 ° Chart speed: 0.64 cm/min
Column Connection Since the totally silylated neomycin has a molecular weight of 1550, 2 the GLC has to be performed at a high operating temperature (near 300°). Use of Teflon front and back ferrules with a lock nut (Swagelok) and a daily change of the high temperature septum are essential for trouble-free operation.
Preparation o] the Chromatographic Column Precondition an empty glass column, 3 mm i.d. X 610 mm, before packing by filling the column with a 50% solution of DMCS in hexane ~ M. Margosis and K. Tsuji, J. Pharm. Sci. 62, 1836 (1973). ~5G. Belec, W. L. Wilson, and D. W. Hughes, Can. J. Pharm. Sci. 8, 48 (1973).
[91
GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS
223
and allow it to stand for 5 min. Empty the column and wash with 50 ml of hexane followed by 50 ml of chloroform. Dry the column with a stream of dry, clean air. For best results, pack the column 3-4 cm at a time, while gently tapping the side of the column. The column is packed to within 8-10 mm of the outlet end and 15-20 mm of the inlet end of the column. The height of the column packing material at the inlet end should be adjusted to provide "on-column" injection--the tip of a microsyringe should touch the top of the column packing material and a sample should be injected on the column packing material. Fill the remaining portion of the column with a small piece of silylated glass wool. Glass wool is not really necessary at the inlet side of the column. The silylation of glass wool is performed in a similar manner to the preconditioning of an empty column. The theoretical plates of the column thus prepared showed nearly a 20% increase over a column prepared simply by filling the column with OV-1 material all at one time. Heat the column at 350 ° for 30 min with the carrier gas off. Cool the colmnn to room temperature and inspect the column. If a break or settling of the column packing material is observed, add the packing material as needed. Heat the column to 300 ° with gas flow on. When the oven temperature reaches 200% inject two 50-~1 portions of Silyl-8 and when the oven temperature reaches 300 ° continuously inject 2-~1 quantities of silylated neomycin, 15-20 times, into the column. The neomycin peak will be low for the first couple of injections; however, the peak will progressively increase with an increase in the number of injections and stabilize after 10-15 injections. Adjust the oven temperature and gas flow rates to give optimum separation between neomycins B and C, in 10-15 minutes of chromatography. The column is now ready for quantitative use. Prior to the start of the daily routine neomycin analysis, inject a couple of silylated neomycin samples.
Performance of the Column The column thus prepared has normally about 1050 theoretical plates per meter for the silylated neomycin B. The theoretical plates may be calculated using the following formula: Theoretical plates per meter of column = (5.545/*0 X (U/W~/~) 2 (2) where n = length of column in meters; U = distance between the injection point and the peak of the neomycin B; W1/2 = width at half-height of neomycin B peak.
224
METHODS FOR THE STUDY OF ANTIBIOTICS
[0]
Sample Preparation Procedure Neomycin Bulk Powder. Weigh neomycin sulfate bulk powder "as is" and prepare a water solution containing approximately 10 mg/ml of the neomycin sulfate. Pipette 500 ~l each of the reference standard and sample into glass vials, using a 500-~1 micropipette, and freeze-dry. Neomycin in Petrolatum-Based Ointments. 16 Accurately weigh approximately 5 g of ointment or the equivalent of approximately 25 mg neomycin sulfate into a centrifuge tube. Add 25 ml of chloroform and stopper. Chloroform should be washed with water to remove any ethyl alcohol present. Place in a 60 ° water bath for 5 rain and then shake vigorously until the ointment is well dispersed. Centrifuge at 2000 g for 10 min. Remove the chloroform by aspiration; the neomycin remains in the bottom of the tube. Add 15 ml of chloroform and resuspend neomycin. Use of an ultrasonicator is helpful. Centrifuge and remove the chloroform. Add 5.0 ml of water and 15 ml of n-heptane to the tube and shake or sonicate until the neomycin is completely dissolved in the water. Centrifuge at 220 g for 3 rain, and discard the heptane. Pipette 1.0 ml of the water solution into a serum vial and freeze-dry. Neomycin in Cream and Lotion. Prepare product standard solution as follows: Weigh 25 mg of the dried neomycin reference standard into a 50-ml centrifuge tube. Add approximately 5 g of the ointment base into the tube. Melt the contents of the tube using heat and mix thoroughly. Accurately weigh approximately 5 g of the sample containing about 25 mg of neomycin sulfate into a 50-ml centrifuge tube. Melt the contents of the tube as needed with mild heat. Add 40 ml of 2,2-dimethoxypropane to each tube containing sample and the product standard. Stopper and shake vigorously until the sample is well dispersed. To each tube, add 1 drop of 10 N sulfuric acid. Shake vigorously and place the tubes in a water bath at 60 °. Every 2 rain shake the tubes for approximately 10 sec. After 10 min remove the tubes from the bath. Centrifuge the tubes at 1500 g for 10 min. Remove and discard the solvent with suction, taking care not to lose any of the neomycin which is in the bottom of the tube. Add 3.0 ml of water and shake or ul~rasonicate until the neomycin is dissolved. Add 25 ml of 2,2-dimethoxypropane and shake vigorously. Place the tubes in the water bath at 60 ° and every 2 rain shake for 10 sec. After 10 min remove the tubes from the bath. Add 20 ml of heptane and shake or ultrasonicate until the neomycin is well dispersed. Centrifuge at 1500 g for 10 rain and discard the heptane. Add 5.0 ml of water and shake or ultrasonicate until the neomycin is dissolved. ~eB. v a n Giessen and K . Tsuji, J. Pharm. Sci. 60, 1068 (1971).
19]
GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS
225
Centrifuge at 1500 g for 5 rain. Pipette 1.0 ml of the water solution into a glass vial and freeze-dry.
Freeze-Dry Procedure Freeze the vials at or below --40 ° , then freeze-dry overnight at less than 10 tLm Hg pressure. The freeze-drying step eliminates lot-to-lot differences in moisture content and in granule size, which significantly influences the solubility characteristics of neomycin powder in the internal standard silylation reagent, hence, greatly affects the quantitation. During high humidity, high temperature Midwestern summer months, we experienced considerable problems from sample melt-back and losses by flaking using a manifold type freeze-dryer. Use of a shelf type freezedryer eliminated these problems. At the end of the freeze-drying cycle, cap the vial immediately with a Teflon-coated septum and seal tightly with a metal retainer. Natural rubber septums interfere with the silylation reaction and lessen the stability of the silylated sample.
Silylation Procedure Add 1.0 ml of the internal standard silylation reagent to each vial containing the freeze-dried sample using a 1-ml glass tuberculin syringe. Immerse and heat the vials in a 75 ° silicone oil bath for 35-40 min swirling occasionally. The silylated neomycin sample thus prepared is extremely sensitive to moisture and temperature. Better stability can be obtained by storing the vial in an air-tight container and keeping it in a refrigerator until analyzed. If a sample is stored at refrigeration temperatures, warm the sample before chromatography. Using a microsyringe remove a 1.5-2.0 ~1 sample from the vial and immediately inject into the chromatograph at an attenuator and range setting of 64 X 10. Since the silylated neomycin rapidly hydrolyzes by moisture in the air, transfer of the sample from the vial to the chromatograph is critical and must be made as quickly as possible. An automatic GLC sample injector, e.g., Hewlett-Packard Model 7671A, may be used satisfactorily provided that the sample-holding tray is kept cool and the syringe is rinsed with pyridine between the sample injection. The entire injection assembly had to be lowered about 15 mm closer to the GLC column than originally designed so that the sample could be iniected on the column.
226
METHODS FOR THE STUDY OF ANTIBIOTICS
[0]
Calculation A typical chromatogram is shown in Fig. 2. Measure peak areas of the internal standard and neomycins B and C. The content of neomycin, in micrograms per milligram of sample, is calculated using the following formula:
[R1/R2] × [Wr/Ws] X F
(3)
where R1 = (area of the sample neomycin B ~ 1/2 area of the samp]e neomycin C)/(area of the sample internal standard peak); R2 = (area of the standard neomycin peak)/(area of the standard internal standard peak); Wr = weight of neomycin reference standard (mg/ml); Ws = weight of neomycin sample (mg/ml); F = assigned value of neomycin reference standard expressed in micrograms of anhydrous neomycin base
3
I 4
I
I
S 12 TIME (MINUTES)
I 16
FIo. 2. Chromatogram of neomycins indicating separation of (1) neamine, (2) neobiosamine; (3) internal standard, (4) neomycin B, (5) neomycin C, (6) LP,, and (7) LPc.
[9]
227
GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS
HO
~
Neamine
CH2NH2
"" "" "" ". ~
"Zm
~0
R3
)7~--~--~~
"~" "~ .~. 0~/=.= ~ CH2OH I "-~....UH
Neobiosamine
NH2
o,,,4 H
~"
Neomycin B
R1=H
R2=CH2NH2
R3=NH2
Neomycin C
R1 =CH2NH2
R2=H
R3=NH2
Neomycin LPB
RI=H
R2=CH2NH2
R3=NHCOCH3
Neomycin LPC
RI=CH2NH 2
R2=H
R3=NHCOCH 3
FIG. 3. Structure of neomycins [K. Tsuji and J. H. Robertson, Anal. Chem. 42, 1661 (1970). Copyright by the American Chemical Society].
per milligram of the neomycin sulfate (F = 767 pg/mg for the USP Lot I Reference Standard).
Assessment o] Results Mono-N-acetylneomycin C, LPc, (see Fig. 3) elutes after the LPR peak, and paromomycin I, if present, elutes prior to the neomycin B peak. The drug potency of neomycin sample as calculated from the GLC data should be comparable to that obtained by the microbiological cylinder cup agar diffusion assay method using Staphylococcus epidermidis (ATCC 12228) as the assay microorganism. 17 Changes in the assay microorganism require a different response factor for neomycin C. 1
Comments on the Assay Method Each mole of 2,2-dimethoxypropane reacts with 1 mole of water to form 1 mole of acetone and 2 moles of methanol. The reaction requires the use of an acid catalyst. Sulfuric acid was chosen to keep the neomycin in the sulfate form. The concentration of acid is quite important for the ~J. H. Robertson, R. Baas, R. L. Yeager, and K. Tsuji, Appl. Microbiol. 22, 1164 (1971).
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METHODS FOR THE STUDY OF ANTIBIOTICS
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speed of reaction. When the reaction rate is too fast, the neomycin particles occlude with small amounts of excipient in a product, resulting in interference with the analysis. Melt-back and loss by boiling often occurred during freeze-drying, and interference in silylation and chromatography were also experienced when the extraction was not optimal. When the reaction rate is too slow, the neomycin is not completely dehydrated and tends to stick to the side of the tubes. The technique which gives the best results is to use 1 drop of 10 N sulfuric acid at the initial dehydration and add no acid during the clean-up step. The residual sulfuric acid remaining acts as the catalyst. The neomycin is slightly soluble in 2,2-dimethoxypropane-acetonemethanol solution, thereby resulting in a loss of approximately 5% of neomycin. For this reason, the method calls for the use of the product standard as the reference standard. As the sample ages, the neomycin extracted becomes brownish. These neomycins result in a broader neomycin GLC peak than the standard neomycin peak and are low in assay value. The difficulty is not experienced with fresh samples, however. Because of the difficulty in silylating 6 NH2 groups and 7 OH groups in one large molecule, coupled with the extreme sensitivity of the TMS-NH group to moisture, and the requirement of high chromatographic temperature due to the low volatility of the high molecular weight compound (MW 1550), the GLC determination of neomycin may be easily interfered with by numerous factors. Strict adherence to the procedures described above is essential for the successful analysis of neomycin. Table I is provided to assist in identifying problems frequently encountered during the GLC analysis of neomycin and lists solutions to the problems.
E. Paromomycin The procedure for the GLC analysis of paromomycin is analogous to that of neomycin powder} ° Follow the procedure as described under neomycin powder, using paromomycin in place of neomycin. IV. Aromatic Antibiotics
A. Chloramphenico118 Chloramphenicol is chromatographed intact as the bistrimethylsilyl ether derivative using an OV-1 column. ~mM. Margosis, J. Chromatogr. 47, 341 (1970).
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229
TABLE I PROBLEMS AND SOLUTIONS FOR THE ANALYSIS OF NEOMYCIN BY GAs-LIQuID CHROMATOGRAPHY
Problem Very small or fat peak
Indication of a shoulder peak
Lack of quantitation
Short column life
Tailing of peak
Excessive darkening of packing material in inlet Stability of silylated sample
Solution a. Too much moisture in sample. Check freeze-dryer for proper operation. b. Column is not properly conditioned. c. Assure on-column injection. d. Eliminate metal parts from the GLC system. e. Check silylation reagent. f. Sample degradation--high storage temperature or leakage in vial. g. Inject immediately after withdrawing sample from vial. h. Excessive organic materials in sample. Sample clean-up may be required. a. Adjust amount of T M S D E A . b. Check freeze-dryer for proper operation. c. Assure on-column injection. d. Eliminate metal part from the GLC system. e. Excess loss of liquid phase. Change or renew column packing material. f. Sample degradation--high storage temperature or leakage in vial. a. Check freeze-dryer for proper operation. b. Inject immediately after withdrawing. c. Assure on-column injection. d. Remove all metal and/or Teflon in inlet system. e. Check silylation temperature for completion of the reaction. f. Keep samples tightly sealed and store in a cool place. g. Condition column with silylated neomycin. a. Reduce column temperature (less than 300 °) and compensate with increase in carrier gas flow. b. Check drying agent for carrier, hydrogen, and air. c. Check silylation temperature and reagent for complete reaction. d. Incomplete silylation or degradation of silylated sample. a. Assure on-column injection. b. Eliminate metal and/or Teflon in inlet system. c. Check packing material for proper loading of liquid phase. d. Replace column. a. Incomplete silylation, check silylation temperature and reagent. Longer silylation time may be needed. b. Replace first 3 cm of packing material in inlet. Use minimum amount of glass wool and replace as needed. a. Use sealed reaction vial; store at refrigeration temperature. b. Presence of metal particles in reaction vial. Vial should be stoppered tightly with a Teflon-coated stopper.
230
METHODS FOR THE STUDY OF ANTIBIOTICS
[0]
Procedure Materials OV-1, 3-5% on Gas Chrom Q 100-120 mesh (Applied Science Lab., Inc., State College, Pennsylvania) Column, glass, 3 mm i.d. X 1220 mm or 1830 mm (4 ft or 6 ft) Vial, glass, 1-dram size with polyethylene or Teflon cap Ethyl acetate, A.R. Diethyl ether, A.R. Pyridine, A.R. Acetonitrile, glass distilled Celite 545 (acid washed) Cyclohexane N,O-Bis(trimethylsilyl)-acetamide (BSA) (Pierce Chemical Company, Rockford, Illinois) m-Phenylene dibenzoate
Solutions HCI solution, 0.01 N Internal standard-silylation reagent. dibenzoate and dissolve in about of BSA and make the solution pyridine. Phosphate buffer, pH 5.8 Ethyl acetate-diethyl ether mixture
Weigh 200 mg of m-phenylene 6 ml of acetonitrile. Add 1 ml to 10 ml with acetonitrile or
(2:1)
Apparatus Gas Chromatograph: See requirements in Section III,D. Detector: Flame ionization detector
Chromatographic Conditions Column: Glass, 3 mm i.d. X 1220 or 1830 mm, packed with 3 or 5% OV-1 Gas flow rate: Hydrogen, 50 ml/min; air, 600 ml/min; and carrier gas (helium), 60 ml/min. Oven temperature: 240 ° Detector temperature: 255 ° Flash heater temperature: 250 °
Preparation o] Chromatographic Column Follow the procedure described in the neomycin section. The column thus prepared normally has 1500 theoretical plates per meter for silylated chloramphenicol.
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GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS
231
Preparation o] Sample and Re]erence Standard Solution Accurately weigh approximately 10 mg of sample powder and reference standard powder into separate, 1-dram vials, or prepare the reference standard solution by accurately weighing about 125 mg of powder into a 25 ml volumetric flask. Dilute to volume with ethyl acetate. Pipette 2.0 ml of this solution into a glass vial and evaporate to dryness under a stream of nitrogen.
Sample Preparation Hard-Filled Capsules. Empty 20 capsules, collecting the contents quantitatively. Weigh the powder and determine the average capsule fill weight. Mix the powder and accurately weigh a portion equivalent to about 125 mg of chloramphenicol into a 25-ml volumetric flask. Add ethyl acetate to volume and shake vigorously. Allow insoluble material to settle or filter if necessary. Pipette 2.0 ml of the supernatant liquid (or filtrate) into a glass vial. Evaporate to dryness under a stream of nitrogen. Tablets. Accurately weigh l0 tablets and then finely grind, using a Wiley mill with a 60-mesh screen. Accurately weigh a portion of the powder to contain approximately 125 mg of chloramphenicol into a 25-ml volumetric flask. Add ethyl acetate to volume and shake vigorously. Allow insoluble material to settle or filter. Pipette 2.0 ml of the supernarant (or filtrate) into a glass vial. Evaporate to dryness under a stream of nitrogen. Solutions. Pipette a suitable portion of the solution into a separatory funnel and dilute with a 5-fold amount of water. Extract chloramphenicol three times with a mixture of ethyl acetate-diethyl ether. Collect the extracts and wash with water. Evaporate the extract to dryness with a stream of nitrogen. Add ethyl acetate to reconstitute and quantitatively transfer into a volumetric flask of suitable size and add ethyl acetate to have a final concentration of 5-10 mg chloramphenicol per milliliter of solution. Pipette a portion of the solution containing 10 mg of chloramphenicol into a glass vial. Evaporate to dryness under a stream of nitrogen. Ointments. A mixture of about 3 g of Celite 545 and 2 ml of a pH 5.8 phosphate buffer is placed directly into a glass chromatographic column and tamped to a uniform mass. Mix 3 g of celite and 2 g of ointment and place on top of the prepared column and again tamp gently. Add 100 ml of cyclohexane to the column and discard the eluate. Add 100 ml of ethyl acetate to elute chloramphenicol. Collect the eluate and evaporate to dryness. Reconstitute and bring to volume with ethyl acetate to a concentration of 5-10 mg chloramphenicol per milliliter of solution.
232
METHODS FOR THE STUDY OF ANTIBIOTICS
[9]
Pipette a portion of the solution containing approximately 10 mg of chloramphenicol into a glass vial. Evaporate to dryness under a stream of nitrogen. Silylation Procedure
Using a micropipette add 500 ~l of the internal standard-silylation reagent to each vial containing samples and the reference standard. Swirl the vial gently for a few minutes at room temperature. Inject approximately 1 ~l of the silylated chloramphenieol into the gas chromatograph. Calculation
The micrograms of chloramphenicol per milligram of sample may be calculated using Eq. (1) described in Section III,B. Comments on the Assay Method
According to Janssen and Vanderhaeghe,19 silylation with BSA in acetonitrile may form a mixture of mono-, bis-, and tris-TMS derivatives of chloramphenicol. A single bis-TMS derivative may be obtained when the mixture of TMCS and HMDS in pyridine is used. The meta and erythro isomers may be separated from each other; however, the L isomer may not be separated from the chloramphenicol peak. Ch!oramphenicol appears to be stable in ethyl acetate for about 2 weeks, however, the internal standard-silylation reagent with ethyl acetate may not be stable and should therefore be made fresh just prior to silylation. B. Griseofulvin s° Griseofulvin is ehromatographed intact without derivatization using an OV-1 column.
Procedure
Materials
OV-17, 1% on Gas-Chrom Q, 100-120 mesh (Applied Science Lab, State College, Pennsylvania) Column, glass, 4 mm i.d. X 914 mm (3 ft) lo G. Janssen and H. Vanderhaeghe, J. Chromatogr. 82, 297 (1973). 2, M. Margosis, J. Chromatogr. 70, 73 (1972).
[9]
GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS
233
Chloroform, A.R. Tetraphenylcyclopentadienone
Solutions Internal standard solution: tetraphenylcyclopentadienone dissolved in chloroform at a concentration of 5 mg/ml
Apparatus Perkin-Elmer Model 900GC or equivalent. For requirements see Section III, D. Detector: flame-ionization detector
Chromatographic Conditions Gas flow rate: hydrogen and air at the optimum setting; carrier gas (helium), 60 ml/min Oven temperature: 245 ° Detector temperature: 260 ° Injector temperature: 260 °
Preparation o] Chromatographic Column Pack an empty glass column, 4 mm i.d. X 914 mm, with 1% OV-17 column packing material. No-flow condition the column at 340 ° for 1 hour and then bring the column temperature to 250 ° . Maintain the column temperature at 250 ° with carrier gas until a stable baseline is obtained. The column thus prepared has approximately 1350 theoretical plates per meter for both griseofulvin and the internal standard.
Preparation o] Bulk Drug and Re]erence Standard Solution Accurately weigh approximately 125 mg of bulk drug and the reference standard powder into a 25.0-ml volumetric flask. Add chloroform and shake vigorously to dissolve and dilute to volume. Pipette a 2.0-ml aliquot into a glass vial and evaporate to dryness under a stream of dry nitrogen.
Sample Preparation For solid dosage forms (capsules, tablets, boluses) accurately weigh 10 solid dosage units to obtain an average weight of one dosage form. Grind the dosage form, pool the contents of the capsule, and weigh accu-
234
METHODS FOR THE STUDY OF &NTIBIOTICS
[9]
rately a suitable amount of powder into volumetric flasks. Add chloroform and shake with beating if necessary, to dissolve griseofulvin. Dilute to volume with chloroform. Pipette an aliquot of the supernatant containing approximately 10 mg griseofulvin into a glass vial and evaporate to dryness with a stream of dry nitrogen. For suspensions, transfer 5.0 ml of the mixed suspension containing 250 mg of griseofulvin into a separatory funnel and dilute to about 25 ml with water. Extract the mixture with 25 ml of chloroform and collect the chloroform layer. Repeat the extraction twice with 10-ml amounts of chloroform. Pool the chloroform extract and backwash with 5 ml of water, then filter through chloroform-moistened glass wool into a 50 ml volumetric flask and dilute to volume with chloroform. Pipette 2.0 ml of the aliquot into a glass vial and evaporate to dryness with a stream of nitrogen. Chromatographic Procedure Add 1.0 ml of the internal standard solution into each vial containing samples and the reference standard and stir vigorously to obtain a uniform solution. Inject 1 ~l of this solution into the GC. Calculation The content of griseofulvin in micrograms per milligram of sample is calculated by direct comparison of the ratio of the peak area (griseofulvin/tetraphenylcyclopentadienone) with that of the griseofulvin reference standard. Comments on the Assay Method In the preparation of bulk drugs or the reference standard, dissolution in chloroform and evaporation steps are not really necessary but the steps are recommended so that the procedure is consistent with that of sample preparation for the dosage forms. The method resolves dechlorogriseofulvin from griseofulvin but does not resolve dehydrogriseofulvin from isogriseofulvin. V. Glutarimide Antibiotic Cycloheximide ~1 Cycloheximide is derivatised as the monotrimethylsilyl ether by treatment with isopropanol after silylation. =L. W. Brown, Agr. Food Chem. 21, 83 (1973).
[9]
GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS
235
Procedure Materials QF-1, 1% on Gas Chrom Q, 80-100 mesh (Applied Science Lab., Inc., State College, Pennsylvania) Column, glass, 3 mm i.d. X 610 mm (2 ft) Vial, glass, 1-dram size with polyethylene or Teflon cap Isopropyl alcohol, A.R. Chloroform Benzene Pyridine, A.R. Bis (trimethylsilyl) trifluoroacetamide (Regisil) containing 1% trimethylchlorosilane (TMCS). Available as Regisil TMCS from Regis Chemical Co., Chicago, Illinois. Cholesteryl acetate
Solutions Isopropyl alcohol, 3%, in pyridine Internal standard-silylation reagent. Prepare a pyridine solution containing approximately 6 mg of cholesteryl acetate and 0.1 ml of Regisil TMCS per milliliter of solution.
Apparatus Gas chromatograph. See requirements in Section III, D. Detector: flame-ionization detector
Chromatographic Conditions Column: Glass, 3 mm i.d. X 610 ram, packed with 1% QF-1 Gas flow rate: hydrogen, 40 ml/min; air, 600 ml/min; and carrier gas (t~elium), 60 ml/min Oven temperature: 200 ° Detector temperature: 225 ° Flash heater temperature: 200 ° Chart speed: 0.64 cm/min Inject approximately 1 td of sample into the chromatograph.
Preparation o] the Chromatographic Column Follow the procedure described in Section III, D. After no-flow conditioning of the column at 240 ° for 30 min, bring the oven to room temperature. Turn on the oven and carrier gas. Iniect
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METHODS FOR THE STUDY OF ANTIBIOTICS
[9]
a 50-t~l quantity of Silyl-8. Maintain the gas chromatograph under the chromatographic condition overnight. Prior to analysis inject a couple of silylated cycloheximide samples. The column ¢hus prepared normally has 1300 theoretical plates per meter for silylated cycloheximide.
Re]erence Standard Preparation Accurately weigh approximately 5 mg of the cycloheximide reference standard into a 1-dram vial.
Sample Preparation Bulk Drug Sample. Accurately weigh approximately 5 mg of bulk cycloheximide sample in a 1-dram vial. Formulation. For formulations containing materials that are soluble in organic solvents but relatively insoluble in water, a sample equivalent to 10 mg of cycloheximide should be accurately weighed into a 15-ml centrifuge tube and the cycloheximide extracted from the sample with 2 ml of water. A 1.0-ml quantity of this solution is then extracted with chloroform, and the resulting chloroform solution is evaporated to dryness with nitrogen. For formulations containing materials such as ferrous sulfate and sodium alkyl aryl sulfonic acid, which are insoluble in organic solvents, the sample is extracted directly with benzene. A suitable quantity of the benzene solution containing 5 mg of cycloheximide is transferred into a vial and evaporated to dryness. Silylation Procedure Add 1.0 ml of the internal standard-silylation reagent into each vial containing samples and reference standard. Swirl the vial gently to dissolve the solid and place in an oil bath at 50 ° for 2 hr. Remove the vial from the oil bath and add 1.0 ml of the pyridine solution containing 3% isopropyl alcohol. After gently swirling the solution to mix, the vial is allowed to stand for 20 min and then chromatographed.
Calculation The micrograms of cyeloheximide per milligram of sample may be calculated using Eq. (1) of Section III, B.
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Comments on the Assay Method
Normal silylation conditions yield a mixture of mono- and bistrimethylsilyl derivatives of cycloheximide. Since the bistrimethylsilyl derivative could not be formed quantitatively, isopropyl alcohol was added after the silylation reaction to quantitatively convert the bis derivative to the mono derivative, thus yielding a single peak. Addition of alcohol prior to the silylation reaction resulted in no silylation of the cycloheximide. Isocycloheximide and the principal dehydration product, anhydrocycloheximide, two possible impurities in cycloheximide samples, can be determined in one chromatograph. VI. Lincomycin-Clindamycin Family A. Lincomycin 5,2~
Lincomycin is chromatographed intact as the tetrakistrimethylsilyl ether derivative using a 3% SE-30 column. Procedure Materials
SE-30, 3-5% on Gas Chrom Q, 100-120 mesh (Applied Science Lab, Inc., State College, Pennsylvania) Column, glass, 3 mm i.d. X 1830 mm (6 ft) Vial, glass, suitable size with nonreacting airtight cap Pyridine, A.R. N,O-Bis(trimethylsilyl)acetamide (BSA) (Pierce Chemical Co., Rockford, Illinois) Silyl-8 (Pierce Chemical) Tetraphenylcyclopentadienone (Aldrich Chemical Co., Inc., Milwaukee, Wisconsin) Solutions
Internal standard solution. Prepare a pyridine solution containing approximately 2 mg of tetraphenylcyclopentadienone per milliliter of solution. Apparatus
Gas chromatograph. See requirements in Section III, D. Detector: flame-ionization detector ~ R. L. Houtman, D. G. Kaiser, and A. J. Taraszka, J. Pharm. Sci. 57, 693 (1968)
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METHODS FOR THE STUDY OF ANTIBIOTICS
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Chromatographic Conditions Column: glass, 3 mm i.d. X 1830 mm, packed with 3 or 5% SE-30 Gas flow rate: hydrogen, 40 ml/min; air, 600 ml/min; and carrier gas (helium), 60 ml/min Oven temperature: 275° Detector temperature: 300 ° Flash heater temperature: 275 ° Chart speed: 0.64 cm/min
Preparation o] the Chromatographic Column Follow the procedure described in the neomycin section. The column thus prepared has normally 2000 theoretical plates per meter for silylated lincomycin.
Re]erence Standard Preparation Weigh accurately about 20 mg of lincomycin hydrochloride reference standard and transfer into a suitable vial.
Sample Preparation Drug, Tablets, and Capsules. Transfer an accurately weighed sample containing about 20 mg of lincomycin hydrochloride monhydrate into a suitable vial. For tablets, weigh at least 20 tablets to establish the average weight per tablet. For capsules, weigh the contents of at least 10 capsules to establish the average fill weight per capsule. Aqueous Solutions and Syrups. Transfer an accurately measured volume containing 0.9-1.0 g of lincomyein hydrochloride monohydrate to a 100-ml volumetric flask. Dilute to volume with methanol and mix well. Transfer 2.0 ml of this solution to a vial and evaporate to dryness. Silylation Procedure Add 2.0 ml of pyridine into each vial containing lincomycin. Warm the solution on a hot plate for approximately 5 min to completely dissolve lincomycin. Add 1.0 ml of BSA, stopper the vials, and heat at 70-75 ° for 1 hr or allow the vials to stand overnight at room temperature. Pipette 10.0 ml of the internal standard preparation into each vial and mix well. Chromatograph the silylated samples.
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Calculation Calculate the content of lincomycin base "as is" expressed in micrograms per milligram of sample from Eq. (1) described in Section III, B. Comments on the Assay Method The lincomycin tetrakis-TMS derivative is one of the most stable TMS derivatives. For the quantitative derivatization of lincomycin care must be taken to completely dissolve lincomycin in pyridine prior to the addition of BSA. B. Clindamycin T M The clindamycin hydrochloride salt is converted to the free base and extracted with an organic solvent containing the internal standard. The clindamycin base is reacted with trifiuoroacetic anhydride and chromatographed intact using a SE-30 column. Procedure Materials SE-30, 1% on either (a) Gas Chrom Q, 100-120 mesh, (b) Diatoport S, 80-100 mesh, or (c) Chromosorb W, 80-100 mesh Column, glass, 3 mm i.d. X 610 mm (2 ft) Trifluoroacetic anhydride Chloroform A.R. Sodium sulfate, anhydrous Sodium carbonate, anhydrous granules Hexacosane Solutions Sodium carbonate solution, 1% Internal standard solution. Prepare a chloroform solution containing approximately 0.45 mg of hexacosane per milliliter Apparatus Gas chromatograph. See requirements in Section III, D. Detector: flame-ionization detector 23L. W. Brown J. Pharm. Sci. 63, 1597 (1974).
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METHODS FOR THE STUDY OF ANTIBIOTICS
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Chromatographic Conditions Column: Glass, 3 mm i.d. X 610 ram, packed with 1% SE-30 Gas flow rate: Hydrogen, 40 ml/min; air, 600 ml/min; and carrier gas (helium), 60 ml/min Oven temperature: 180 ° Detector temperature: 200 ° Flash heater temperature: 180 ° Chart speed: 0.64 cm/min
Preparation o] the Chromatographic Column Follow the procedure described in Section III, D. The SE-30 column thus prepared has normally 1300 theoretical plates per meter for trifluoroacetylated derivative of clindamycin.
Preparation o] Clindamycin Sample and Re]erence Standard Solution Accurately weigh the bulk powder and reference standard powder, equivalent to approximately 13 mg of clindamycin hydrochloride monohydrate into a 15-ml glass-stoppered centrifuge tube. For hard-filled capsules, empty 20 capsules, collecting the contents quantitatively. Weigh the powder and determine the average capsule fill weight. Mix the powder and dccurately weigh a portion equivalent to about 13 mg of clindamycin into a 15-ml stoppered centrifuge tube. Add 6.0 ml of the Internal Standard solution and 6.0 ml of 1% sodium carbonate solution to each tube. Shake vigorously for 30 min and centrifuge.
Derivatization Procedure Transfer a portion of the chloroform solution into a suitable vial conraining 1 g of anhydrous sodium sulfate to dry the solution. Shake vigorously for 1 min then transfer 2 ml of the dried chloroform solution into a 15-ml centrifuge tube and add 0.50 ml of trifluoroacetic anhydride. Place stoppered centrifuge tubes in a heating block or oil bath to an approximate depth of 5 cm so that the upper part of the centrifuge tubes will act as a reflux condenser. Heat at 45 ° for 30 rain. Chromatograph aliquots of the sample and standard preparations. Sample size: 0.5-2 t~l; range and attenuation 15 X10.
Calculation Measure the area under the peak of clindamycin and the internal standard. Calculate the content of clindamyein base in micrograms per
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GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS
241
milligram of sample from the following formula:
[R1/R2] X [Wr/W.] X [El~F2] X F3
(4)
where R1 = (area of clindamycin sample peak)/(area of the internal standard peak); R2 = (area of the clindamycin standard peak)/(area of the internal standard peak); Wr -- weight of clindamycin hydrochloride hydrate reference standard (rag) ; W8 = sample weight (rag) ; F1 = ml of internal standard solution added to the sample preparation; F2 = ml of internal standard preparation added to the standard preparation; F3 = Assigned potency of clindamycin hydrochloride hydrate reference standard expressed in micrograms of anhydrous clindamycin base per milligram of reference standard.
Comments on the Assay Methods A typical chromatogram may be seen in Fig. 4. The reaction conditions of the trifluoroacetyl procedure are less severe than the acetyl proce-
4
TiM
L 8 (MINUTES)
I 12
FIG. 4. Chromatogram of clindamycin indicating separation of (1) epilincomycin, (2) clindamycin B, (3) clindamycin, and (4) internal standard. (Courtesy of L. W. Brown.)
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METHODS FOR THE STUDY OF ANTIBIOTICS
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dure, 2. and the trifiuoroacetyl procedure is not affected by hydrochloric acid in clindamycin samples, thus eliminating any degradation and shortening the assay time. Also, solvent tailing, a problem with the acetylation is eliminated. Peaks due to incomplete acetylation interfere in the measurement of the impurities, epilincomycin and epiclindamyein, while no interference is experienced with the trifluoroacetyl procedure which separates epilincomycin from epiclindamycin. C. Clindamycin Phosphate 5,23
The GLC method for clindamycin phosphate is based on the hydrolysis of clindamycin phosphate with alkaline phosphatase and chromatography of clindamycin base.
Procedure Materials Intestinal alkaline phosphatase Sodium carbonate, anhydrous
Solutions Borate buffer, pH 9.0. Transfer 3.0 g of boric acid into a l-liter volumetric flask containing 500 ml of water. Mix and add 21 ml of 1.0 N sodium hydroxide and 10 ml of 0.1 M magnesium chloride. Dilute to volume with water and mix well.
Preparation o] Clindamycin Hydrochloride Standard Solution Accurately weigh approximately 9 mg of the clindamycin hydrochloride reference standard into a 35-ml glass-stoppered centrifuge tube and dissolve in 20 ml of pH 9.0 borate buffer.
Preparation o] Sample Solution Accurately weigh approximately 12 mg of the clindamycin phosphate sample into a 50-ml glass-stoppered centrifuge tube. Pipette 25 ml of the pH 9.0 borate buffer into the centrifuge tube. Add 10 ml of chloroform and shake vigorously for 15 rain. Centrifuge the resulting mixture and pipette a 20-ml aliquot of the aqueous phase into a 35-ml centrifuge tube. Add a weighed amount of intestinal alkaline phosphatase equivalent to 24 T. O. Oesterling, ]. Pharm. Sci. 59, 63 (1970).
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GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS
243
50 units of activity and allow the solution to stand until the enzyme has completely dissolved. Place the tube into a water bath at 37 ° for 2.5 hr. After the 2.5-hr hydrolysis, allow the solution to cool.
Extraction Procedure Add 10 ml of the internal standard solution to each sample and standard solution. Shake the centrifuge tubes vigorously for 30 min and centrifuge. Remove the aqueous layer and discard. Shake the tube again and mix in an ultrasonicator for 2 min, then centrifuge. No emulsion should be present at this stage. Remove the remaining aqueous layer by suction and transfer a 3-ml quantity of the chloroform layer to a 1-dram vial containing approximately 1 g of anhydrous sodium sulfate, swirl the vial and allow sodium sulfate to settle. Transfer 1 ml to another 1 dram vial.
Trifluoroacetylation Procedure Add 250 ~l of trifluoroacetic anhydride to each vial and place them into a water bath at 45 ° for 30 rain. Add about 10 granules of anhydrous sodium carbonate to each vial and allow to stand at room temperature for 30 rain. Chromatogram samples under the conditions described in the clindamycin section. D. Clindamycin P a l m i t a t e 5,2~
Procedure Materials SE-54 or UCW-98, 1% on either Diatoport S, 60-80 mesh or Chromosorb WHP 80-100 mesh Column, glass 3 mm i.d. X 610 mm (2 ft) Trilaurin Acetic anhydride Pyridine, A.R. Chloroform, A.R.
Solutions Internal standard solution. Prepare a solution containing 5.0 mg of trilaurin per milliliter in chloroform. ~ K. Tsuji and J. H. Robertson, Anal. Chem. 43, 818 (1971).
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METHODS FOR THE STUDY OF ANTIBIOTICS
[9J
Apparatus Gas chromatograph. For requirements see Section III, D. Detector: flame-ionization detector
Chromatographic Conditions Column: Glass, 3 mm i.d. X 610 mm, packed with 1% SE-54 or UC-W98 Gas flow rate: hydrogen, 40 ml/min; air, 600 ml/min; and carrier gas (helium), 60 ml/min Oven temperature: 275 ° Detector temperature: 290 ° Inj ection port: 280 ° Chart speed: 0.64 cm/min
Preparation o] Chromatographic Column Follow the procedure described in Section III, D.
Preparation o] Sample and Re]erence Standard Solutions Accurately weigh approximately 15 n)g of both the sample and the reference standard into separate glass-stoppered, 15-ml centrifuge tubes. Add 1.0 ml of internal standard solution, 1.0 ml of pyridine, and 0.5 ml of acetic anhydride to each tube. Agitate the tubes to ensure dissolution and complete mixing of the liquids.
Acetylation Cover the top of each centrifuge tube with a plastic cap. Punch a small, 18-gauge needle, hole in the top of each cap to allow vapor to escape. Place the tubes in a 100 ° drying oven for 2.5 hr. Remove the tubes from the oven and allow to cool. Take the plastic cap from each tube and replace with the glass stopper. Centrifuge 10-15 min at 2000-2500 rpm to separate the white solid from the liquid in the tube. Inject 1 ~l of the clear solution into the gas chromatograph.
Calculation The internal standard will elute before the clindamycin palmitate peak. Calculate the clindamycin palmitate in microgram per milligram of sample using Eq. (1) described in Section III, B.
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GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS
245
VII. Macrolide Antibiotic E r y t h r o m y c i n25
Erythromycin is chromatographed intact as the pentakistrimethylsilyl ether derivative using an OV-225 column.
Procedure Materials 0V-225, 3% on Gas Chrom Q, 100-120 mesh (Applied Science Lab., Inc. State College, Pennsylvania) Column, glass, 3 mm i.d. X 1830 mm (6 ft) Septum, high temperature-acid resistant (No. 4971, Anspec Co., Ann Arbor, Michigan) Vial, glass 1-dram size with screw cap (No. 60910, Kimble Glass, Owens-Illinois, Toledo, Ohio) with 26 mil polyethylene liner Pyridine, A.R. Trimethylchlorosilane (TMCS) (Pierce Chemical Co., Rockford,Illinois) N,O-Bis(trimethylsilyl) acetamide (BSA) (Pierce Chemical) N-Trimethylsilylimidazole (TSIM), (Pierce Chemical) 1,3-Dimyristin (Applied Science Lab)
Solutions Internal standard-silylation reagent. Add 5 ml of TMCS to 5 ml of BSA, mix, and add 2 ml of TSIM. Add 12 ml of pyridine containing about 50 mg of 1,3-dimyristin. Cap the vial with a polyethylene or Teflon stopper. Place the vial in an airtight container and store under refrigerated temperature.
Apparatus Gas chromatograph. See requirements in Section III, D. Detector: flame-ionization detector
Chromatographic Conditions Column: Glass, 3 mm i.d. X 1830 mm, packed with 3% 0V-225 Gas flow rate: hydrogen, 40 ml/min; air, 600 ml/min; and carrier gas (helium), 55 ml/min
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METHODS FOR THE STUDY OF ANTIBIOTICS
[0]
Oven temperature: 275 ° Detector temperature: 290 ° Flash heater temperature: 280 ° Chart speed: 0.64 cm/min Chromatograph a 1-~I sample at an attenuation and range setting of 64 X 10.
Preparation o~ Chromatographic Column Follow the procedure described in Section III, D. After the no-flow conditioning at 300 ° for 45 rain, bring the oven temperature to room ternperature. Turn the oven temperature to 275 ° with carrier gas at 55 ml/min. Inject 50 ~1 of Silyl-8 two or three times. When the oven temperature reaches 275 °, inject 2 ~1 of silylated erythromycin sample. Maintain the GC at chromatographic conditions overnight. Prior to the daily analysis, inject a sample of silylated erythromycin. The column thus prepared normally has 1320 theoretical plates per meter for silylated erythromycin A.
Preparation of Bulk Drug and Reference Standard Powder Accurately weigh approximately l0 mg of erythromycin bulk powder and the reference standard powder into a 1-dram screw-cap vial.
Sample Preparation for Enteric Coated Tablet 26 Materials: Methylene chloride, A.R. Apparatus: Wiley mill with a 60-mesh screen. Platform shaker (Eberbach Corp., Ann Arbor, Michigan). Tube rotator (BBL, Cockeysville, Maryland) or equivalent Accurately weigh 10 erythromycin tablets and finely grind using a Wiley mill with a 60-mesh screen. Accurately weigh a portion of the powder containing approximately 500 mg of erythromycin into a roundbottom ground glass-stoppered centrifuge tube. Add 25.0 ml of methylene chloride and stopper tightly. Shake vigorously or rotate continuously for 45 rain. Centrifuge at 10,000 g for 15 rain. Pipette 500 ~1 of methylene chloride extract into a l-dram screw cap vial and evaporate to dryness under a stream of dry nitrogen. To ensure complete dryness, the samples are then dried for an additional 10 rain in a vacuum oven at 60 ° at less than 5 mm Hg pressure. ~J. H. Robertson and K. Tsuji, J. Pharm. Sci. 61, 1633 (1972)
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Silylation Procedure One milliliter of the internal standard silylation reagent is added to the vial containing erythromyein by means of a glass tuberculin syringe. The cap, lined with a 26-ml polyethylene liner, is tightly sealed and placed in an oil bath (silicone) at 75 ° for 24-36 hr. Approximately 1 tA of the silylated sample is injected into the chromatograph at an attenuation and range setting of 64 X 10.
Calculation Since the microbiological response of erythromycins B and C are 50 and 40%, respectively, that of erythromycin A against Staphylococcus aureus H (ATCC 9144), 2~ the following formula was devised to make the GLC data comparable with that of the microbiological assay method: GLC calculated biopotency (ug/mg) EA+0.5EB+0.4EC = E~-~-0~5E~--~4~CJ
] × [Is~I,] × [ W s / W , I × F
(5)
where EA = erythromycin A peak area of a sample; EB = erythromyein B peak area of a sample; EC = erythromycin C peak area of a sample; ESA = erythromycin A peak area of the reference standard; ESB erythromycin B peak area of the reference standard; ESC = erythromycin C peak area of the reference standard; Is = internal standard peak area of the reference standard; It = internal standard peak aiea of the sample; W8 = weight of the erythromycin reference standard (mg) ; Wt = weight of erythromycin sample (rag); F = assigned value of the erythromycin reference standard expressed in micrograms of erythromycin base per milligram of the standard. A typical chromatogram may be seen in Fig. 5.
Comments on the Assay Method Because of the high operating temperature, an 0V-225 column is stable for approximately 3 weeks. Although the use of the 0V-225 column made it possible to separate erythromycin A from various impurities, the use of the PPE-20 or OV-210 column is required for the separation of erythromycin B and anhydroerythromycin A. The GLC method for erythromycin may be used to analyze monoand diacetyl erythromycin A, propionate esters of erythromycin, and the stearate salts of erythromycin.
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METHODS FOR THE STUDY OF ANTIBIOTICS
5
I 4
8
I
[9J
6
I
12 16 TiME (MINUTES)
I 20
24
FIG. 5. Chromatogram of erythromycin indicating separation of (1) internal standard, (2) acid-hydrolyzed erythromycin, (3) erythromycin C, (4) erythromycin A, (5) and (7) isomers of erythromycin A, and (6) erythromycin B and/or anhydroerythromycin A. V I I I . Penicillin 27 Penicillin is chromatographed intact as the silylether derivative using an OV-17 column. Procedure Materials
OV-17, 3% on Gas Chrom Q 10(O120 mesh (Applied Science Lab, Inc.) Column, glass, 3 mm i.d. X 610 mm (2 ft) Chloroform, A.R. 2, C. ttishta, D. L. Mays, and M. Garofalo, Anal. Chem. 43, 1530 (1971).
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249
Pyridine, A. R. Vial, glass 5-a-Cholestane (Mann Research Lab, Inc., New York, New York) Hexamethyldisilazane (HMDS), Pierce Chemical Co.
Solutions pH 2.2 buffer solution. A saturated aqueous solution of ammonium sulfate is adjusted to pH 2.2 with concentrated sulfuric acid. Internal standard-silylation reagent. A 50% (v/v) solution of HMDS in pyridine containing 0.375 mg/ml of 5-a-cholestane was prepared.
Apparatus Gas chromatograph. For requirements, see Section III, D Detector: flame-ionization detector
Chromatographic Conditions Column: glass 3 mm i.d. X 610 mm packed with 3% OV-17 Gas flow rate: hydrogen, 50 ml/min; air, 600 ml/min; and carrier gas (helium), 60 ml/min Oven temperature: 215 ° Detector temperature: 230 ° Flash heater temperature: 215 ° Chart speed: 0.64 cm/min
Preparation o] Chromatographic Column Follow the method described in Section III, D. The column thus prepared normally has 1450 theoretical plates per meter for silylated penicillin G.
Preparation o] Bulk Drug and Re]erence Standard Solutions Penicillin bulk powder and reference standard powder are accurately weighed and dissolved in water at a concentration of 20 mg/ml. Pipette 2.0 ml of this solution into a glass centrifuge tube with ground-glass stoppers and add 8.0 ml of chloroform and 2 ml of pH 2.2 buffer. Shake the mixture vigorously for 1 min and centrifuge. Remove the aqueous layer and pipette 2.0 ml of the chloroform layer to a serum vial. Because of the poor solubility of procaine penicillin G in water, about 5 mg is accurately weighed into a 5-ml serum vial with 2 ml of chloroform.
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METHODS FOR THE STUDY OF ANTIBIOTICS
[9]
Silylation Procedure
Add 2 ml of the internal standard-silylation reagent to each vial. Cap the vial tightly, mix, and then allow to stand at room temperature for 10 min for penicillin G, penicillin V, D- and L-phenethicillin, and methicillin. Oxacillin, eloxacillin, and dicloxacillin require up to 60 min for complete silylation. Chromatograph 2 tJ of the silylated penicillin. Calculation
Calculate the content of penicillin in micrograms per milligram of sample using Eq. (1) described in Section III, B. Comments on the Assay Method
Since penicillin is unstable in the pH 2.2 buffer, the extraction step must be done as quickly as possible. During silylation the procaine cleaves off from procaine penicillin G molecule and chromatographs as procaine and TMS-penicillanic acid. No degradation product was seen
I 4 TiME (MINUTES)
I 12
FIQ. 6. Separation of (1) penicillin G and (2) penicillin V.
[9]
GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS
251
under these chromatographic conditions when penicillin was degraded by either penicillinase or by acid. A typical chromatogram is shown in Fig. 6. The method is not applicable to ampicillin.
I X . T e t r a c y c l i n e 28
Tetracycline is chromatographed intact as the pentatrimethylsilyl ether derivative using a 3% JXR column.
Procedure Materials JXR, 3% on Gas Chrom Q, 100-120 mesh (Applied Science Lab, Inc., State College, Pennsylvania) Column, glass, 3 mm i.d. X 1830 mm (6 ft) Vial, glass 1-dram screw-cap vial (No. 60910, Kimble Glass, OwensIllinois, Toledo, Ohio) with 26 mil polyethylene liner Pyridine, A.R. Trimethylchlorosilane (TMCS), Pierce Chemical Co. N,O-Bis(trimethylsilyl)acetamide (BSA), Pierce Chemical Co. Trioctanoin (Eastman Organic Chemicals, Rochester, New York)
Solutions Internal standard-silylation reagent. Add 5 ml of BSA and 5 ml of TMCS into 10 ml of pyridine containing about 3 ~i trioctanoin per milliliter. Cap tightly with a polyethylene lined stopper and mix well. Prepare the internal standard silylation reagent just prior to the silylation of samples.
Apparatus Gas chromatograph. For requirements, see Section III, D. Detector: flame-ionization detector
Chromatographic Conditions Column: Glass, 3 mm i.d. X 1830 mm packed with 3% JXR Gas flow rate: hydrogen, 40 ml/min; air, 600 ml/min; and carrier gas (helium) 55 ml/min Oven temperature: 260 ° K. Tsuji and J. H. Robertson, Anal. Chem. 45, 2136 (1973).
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METHODS FOR THE STUDY OF ANTIBIOTICS
[9]
Detector temperature: 290 ° Flash heater temperature: 260 ° Chart speed: 0.64 cm/min
Preparation o] Chromatographic Column Follow the procedure described for neomycin powder in Section III, D. After the no-flow conditioning of the column at 310 ° for 30 rain, bring the oven to room temperature. Turn the oven temperature to 260 ° and carrier gas to 55 ml/min. Inject approximately 50 tL1 of Silyl-8 when the oven temperature reaches 200 ° and 2-3 t~l of the silylated tetracycline when the oven temperature reaches 260 °. Maintain the GC under the chromatographic condition overnight. Prior to the analysis, inject two silylated tetracycline samples to stabilize the column. The J X R column thus prepared normally has about 2500 theoretical plates per meter for silylated tetracycline.
Preparation o] Bulk Drug and Re]erence Standard Powder Accurately weigh approximately 10 mg of tetracycline bulk powder and the reference standard powder into a 1-dram vial.
Silylation Procedure One milliliter of the internal standard silylation reagent is added to each vial containing tetracycline by use of a glass tuberculin syringe. Seal the vial tightly and silylate at 20-25 ° for 24 hr. Approximately 2 t~l of the silylated sample is injected into the chromatograph at an attenuation and range setting of 32 X 10.
Calculation Calculate the content of tetracycline in micrograms per milligram of sample using Eq. (1) described in Section III, B.
Comments on the Method Since the separation factors between tetracycline (TC), 4-epitetracycline (ETC), anhydrotetracycline (ATC), and 4-epianhydrotetracycline (EATC) by the J X R column are small, it is essential to have a highly efficient column and operate it at optimum chromatographic conditions. In our hands, the J X R column routinely showed approximately 2500
[9l
GAS-LIQUID CHROMATOGRAPHY OF ANTIBIOTICS
253
theoretical plates per meter for the silylated tetracycline and the JXR column was the only one which made possible the chromatography of TC on a 6 ft-long column. Degradation products of TC may easily be formed during the silylation procedure. Therefore, strict adherence to the silylation procedure described plus the use of fresh silylation reagents for the preparation of the internal standard-silylation reagent and the maintenance of the reaction temperature at 20-25 ° are essential. Even under carefully controlled conditions, a slightly higher ETC in TC samples resulted by the GLC when compared to those obtained by the high-pressure liquid chromatography (HPLC) 29 which requires no derivatization. We prefer the HPLC method for TC analysis because of its superior sensitivity, high speed, and ease of analysis. X. Miscellaneous
The following GLC methods for the analysis of antimyein A, celestosaminide, phosphonomyein, thiamphenicol, and validamycin have not been critically evaluated by the authors. The procedures described in this section are as they appeared in publications. A. Peptolide Antibiotic--Antimyein A 8° Trimethylsilyl ether of antimycin A (blastomycin) was prepared. Five milligrams of antimycin or blastomyein were dissolved in 0.5 ml T H F and heated for 5 min with 0.2 ml HMDS and 0.1 ml TMCS. Then, most of the reagents were removed under a stream of dry nitrogen. A 3 mm i.d. X 1500 mm glass column packed with 1.5% SE-30 on 80-100 mesh diatomaceous earth (Shimalite, Shimazu Seisakusho Ltd., Japan) was used isothermally at 151 ° with a flame ionization detector. He, 57 ml/min. B. C e l e s t o s a m i n i d e 31
Approximately 5 mg of the compound were sealed in a 0.3-ml Reactivial (Pierce Chemical) and was evacuated. Either Supelco silylating reagent or Powersil (DuPont Co.), both having the same composition of BSA, TSIM, and TMCS = 3:3:2, were used for derivatization. The vials wcre heated 5-10 rain at 70 °. A 3 mm i.d. }( 1220 mm glass column packed with 3.8% UCW-98 on 80-100 mesh Diatoports was used isothermally at 280 ° or 290 ° with a flame ionization detector. 59 K. Tsuji, J. H. Robertson and W. F. Beyer, Anal. Chem. 46, 539 (1974). 2oT. Endo and H. Yonehara, J. Antibiot. 23, 91 (1970). 31T. F. Brodasky and A. D. Argoudelis, J. Antibiot. 0.6, 131 (1973).
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METHODS FOR THE STUDY OF ANTIBIOTICS
[9]
C. P h o s p h o n o m y c i n 32
The sample was dissolved in BSA and heated for 5 min at 60 ° or allowed to stand at room temperature for 30 min to form the TMS derivative. Chromatography was carried out with a 1-2 ~l injection of the silylated derivatives using a glass column, 4 mm i.d. X 1830 mm (6 ft), packed with 4% F-60 (DC-560). coated over 1.5% SE-30 on 100-120 mesh Gas Chrom P. The oven temperature was programmed for 65 ° to 220 ° at 3 ° per minute and then held at 2 2 0 ° for 10 min. A hydrogen flame ionization detector was used. D. Thiamphenico183
Samples of 0.5 or 1.0 ml of biological fluid containing thiamphenicol was accurately added to 5.0 ml of ethyl acetate, and the mixture was vigorously shaken for 5 min. Anhydrous Na~S04 was used to dehydrate the sample. Inject 3 ~l of the ethyl acetate solution directly into a glass column, 4 mm i.d. X 1500 mm, packed with 1.5% DEGS (diethylene glycol succinate polyester) on Chromosorb W, 60-80 mesh, and chromatographed under the following conditions: column and detector temperature, 185°; injector port temperature, 220°; nitrogen (carrier gas), 50 ml/min; for quantitative determinations, nitrogen flow rate was increased to 150 ml/min. When using the TMS ester method, 0.5 or 1.0 ml of the ethyl acetate solution was pipetted into a test tube and evaporated in a vacuum desiccator. Two hundred microliters of TMS reagent were added, and the test tube was stoppered and left :at room temperature for 10 min. The solution was evaporated to dryness in a vacuum desiccator. A suitable volume of pyridine was added to the residue, and 2 ~l was injected into a stainless steel column, 4 mm i.d. X 750 mm, packed with 1.5% OV-17 on Shimalite IV, 80-100 mesh (Shimazu, diatomaceous earth) under the following chromatographic conditions: Column and detector temperature, 215°; injection port temperature, 240°; nitrogen flow rate, 130 ml/min. E. Validamycin 34
Approximately 1 mg of sample was weighed into a rubber-capped small tube and dissolved into 100 ~l pyridine. BSA (100 ~l) and TMCS (50 ~l) were then added. The tube was heated for 30 min at 70-80 °. A 32 H. Shafer, W. J. A. Vanderheuyel, R. Ormand, F. A. Kuehl, and F. J. Wolf, I. Chromatogr. 52, 111 (1970). ~ T. Aoyama and S. Iguchi, J. Chromatogr. 43, 253 (1969). 3, S. Horii, Y. Kameda, and K. Kawahara, J. Antibiot. 25, 48 (1972).
[9]
GAS-LIQUID
CHROMATOGRAPHY
OF
ANTIBIOTICS
255
r~
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.~_,
."~
.~_,
.~.
~o
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~
~_~ ~ ,~
~
~
O'©D
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m
m
~
=
~
N
N ~
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.-
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•~ ~1:~,...~
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O
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T
"7 r
~ ' ~
~
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.-
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:~
256
METHODS FOR THE STUDY OF ANTIBIOTICS
[10]
Hitachi 063GC equipped with flame ionization detector was used with the following chromatographic conditions: 1. For TMS validamycins A to F: Glass column, 3 mm i.d. X 2000 mm packed with 1% OV-1 on Chromosorb W AW DMCS. Temperature: column, 280°; injection, 300 °. Carrier gas (helium), 60 ml/min. 2. For separation of TMS-validoxylamines A and B: Glass column, 3 mm i.d. X 2000 mm packed with 3% OV-17 on Chromosorb W AW DMCS. Temperature: oven, 250c; injection, 300 °. Carrier gas (helium), 30 ml/min. 3. For separation of TMS-derivatives of degradation products: Glass column, 3 mm i.d. X 2000 mm packed with 5% OV-17 on chromosorb W AW DMCS. Column temperature: initial, 150°; final, 280 ° (10°/rain). Injection temperature, 300 °. Carrier gas (helium) : 45 ml/min.
Acknowledgment The authors are indebted to their many colleagueswho provided suggestionsand data for this chapter.
[10] I o n - E x c h a n g e C h r o m a t o g r a p h y o f Streptothricin-like Antibiotics
By
DONALD B . BORDERS
I. Introduction . . . . . . . . . . . . . . . . . . II. Ion-Exchange Chromatography of Intact Antibiotics. . . . . . . III. Ion-Exchange Chromatography of Hydrolysis Fragments . . . . .
256 258 260
I. Introduction Members of the streptothricin family of antibiotics are very watersoluble, strongly basic compounds with broad antimicrobial spectra. One of the most effective means of resolving these antibiotics and their hydrolysis products is by ion-exchange chromatography. It is assumed that the ion-exchange chromatographic techniques which apply to streptothricin-type antibiotics would be of general interest since so'me of the techniques might also apply to a number of other structurally different antibiotics having similar chromatographic and ionic properties.
256
METHODS FOR THE STUDY OF ANTIBIOTICS
[10]
Hitachi 063GC equipped with flame ionization detector was used with the following chromatographic conditions: 1. For TMS validamycins A to F: Glass column, 3 mm i.d. X 2000 mm packed with 1% OV-1 on Chromosorb W AW DMCS. Temperature: column, 280°; injection, 300 °. Carrier gas (helium), 60 ml/min. 2. For separation of TMS-validoxylamines A and B: Glass column, 3 mm i.d. X 2000 mm packed with 3% OV-17 on Chromosorb W AW DMCS. Temperature: oven, 250c; injection, 300 °. Carrier gas (helium), 30 ml/min. 3. For separation of TMS-derivatives of degradation products: Glass column, 3 mm i.d. X 2000 mm packed with 5% OV-17 on chromosorb W AW DMCS. Column temperature: initial, 150°; final, 280 ° (10°/rain). Injection temperature, 300 °. Carrier gas (helium) : 45 ml/min.
Acknowledgment The authors are indebted to their many colleagueswho provided suggestionsand data for this chapter.
[10] I o n - E x c h a n g e C h r o m a t o g r a p h y o f Streptothricin-like Antibiotics
By
DONALD B . BORDERS
I. Introduction . . . . . . . . . . . . . . . . . . II. Ion-Exchange Chromatography of Intact Antibiotics. . . . . . . III. Ion-Exchange Chromatography of Hydrolysis Fragments . . . . .
256 258 260
I. Introduction Members of the streptothricin family of antibiotics are very watersoluble, strongly basic compounds with broad antimicrobial spectra. One of the most effective means of resolving these antibiotics and their hydrolysis products is by ion-exchange chromatography. It is assumed that the ion-exchange chromatographic techniques which apply to streptothricin-type antibiotics would be of general interest since so'me of the techniques might also apply to a number of other structurally different antibiotics having similar chromatographic and ionic properties.
[10]
CHROMATOGRAPHYOF STREPTOTHRICIN ANTIBIOTICS
257
The structural relationship of some streptothricin-type antibiotics is indicated in Fig. 1. In general, the antibiotics with known structures vary by the type of amino acid side chain (R~) and methyl or hydrogen substitution (R~ and R~) on the nitrogens of the streptolidine or the aminosugar. Usually gulosamine is the aminosugar moiety. However, fucosRI~ OH ~ H , , ~ O H
streptolidine
N~NH NH
/
Rs
gufosamine
R20"~..,,,,"~OR
3
Substituents Compound
RI
R2, R3
Streptothricin
H
H, CNH2
H
CCH2 CH CH2 CH2 CH2 NH2
Streptolin
H
O H, ~NH2
H
(CCH2 C CH2 CH2 CH2 NH)2 or 3 H
LL-AC541
H
O ii H, CNH2
CH3
C CH2 NH CH
H
O H, CNH2
CH3
O C CH2 NH2
LL-AB664
CH3
O H, CNH2
CH3
O NH C CH2NH CH
Oeformimino LL-AB664
CH3
0 H>CNH2
CH3
0 C CH2NH2
H
O H,(~NH2
CH3
O ~ CH2NH CH3
R4
?
o
Deformimino LL-AC541
LL-BL136
R5
?
NH2 ,NHz
9
N,.
FIC. 1. Structures of streptothricin and some related antibiotics.
258
METHODS FOR THE STUDY OF ANTIBIOTICS
[10l
amine has been proposed as a hydrolysis product from fucothricin I and glucosamine from racemomycin 0. 2 Streptolidine is an amino acid that appears to be unique to this family of antibiotics. The relative stereochemistry of the three asymmetric centers of this moiety was deduced from chemical studies 3 and N M R spectral; subsequent X - r a y analysis of streptolidine dihydrochloride confirmed these results and established the absolute stereochemistry2 As a result of this stereochemistry, the amino and carboxyl groups of the streptolidine moiety are linked together into a strained lactam ring which probably approximates a boat conformation. This strained ring system explains one of the most labile structural features of this family of antibiotics, and ring opening with biological inactivation occurs if the antibiotics are allowed to stand several hours at room temperature in dilute acid or alkali2 ,7 When streptothricin is allowed to stand in water at room temperature for several days inactivation occurs s presumably through this same mechanism. Some of these antibiotics, LL-AC541 and LL-AB664, can also degrade by standing several days in methanol at room temperature due to a very labile formimino group. 4 These stability features should be considered when any chromatographic separations of streptothricin-type antibiotics are undertaken.
II. Ion-Exchange Chromatography of Intact Antibiotics Probably one of the best methods of separating streptothricin-type antibiotics which differby the number of fl-lysinegroups in the side chain has been by salt gradient elution on carboxymethyl cellulose2,I° In our laboratories, this same system has given excellent separation of a mixture of deformimino LL-AC541, LL-AC541, and streptothricin (listedin order 1M. J. Thirumalachar, P. V. Deshmukh, R. S. Sukapure, and P. W. Rahalker, Hindustan Antibiot. Bull. 14, 4, 1971. S. Takemura, Chem. Pharm. Bull. 8, 578 (1960). 3H. E. Carter, C. C. Sweeley, E. E. Daniels, J. E. McNary, C. P. Schaffner, C. A. 83, 4296 (1961). ' D. B. Borders, K. J. Sax, J. E. Lancaster, W. K. Hausmann, L. A. Mitscher, E. R. West, E. E. Van Tamelen, J. R. Dyer, and It. A. Whaley, J. Amer. Chem. Soc. Wetzel, and E. L. Patterson, Tetrahedron 26, 3123 (1970). B. W. Bycroft and T. J. King, Chem. Commun. 1972, 652 (1972). E. E. Van Tamelen, J. R. Dyer, H. A. Whaley, H. E. Carter, and G. B. Whitfield, Jr., J. Amer. Chem. Soc. 83, 4295 (1961). 7It. Taniyama, Y. Sawada, and T. Kitagawa, J. Antibiot. 24, 662 (1971). s A. W. Johnson and J. W. Westley, J. Chem. Soc. (London), p. 1642 (1962). 9A. S. Khokhlov and P. D. Reshetov, J. Chromatogr., 14, 495 (1964). 10p. D Reshetov and A. S. Khokhlov, Khim. Prir. Soedin. 1, 42 (1965).
[101
CHROMATOGRAPHY OF S T R E P T O T H R I C I N ANTIBIOTICS
259
of increasing retention time). This type of chromatography also provides a medium with an optimum pH for antibiotic stability. A very useful variation of this procedure has been elution with a volatile buffer of pyridine-acetic acid in the isolation of the racemomycins. H The procedure described below in the experimental section has been quite successful in our laboratories and involves the use of a KC1 gradient on a weak cationic exchanger such as Amberlite CG-5022 Recovery of the antibiotics from the salt solutions of the column affluents can be accomplished by derivatives or ion-exchange techniques. ~°,1~ Procedure Separation o] LL-AC541 and De]ormimino LL-AC541 by Salt Gradient Elution on a Weak Cationic Exchange Resin. 12 A column is prepared by suspending cycled resin (Amberlite CG-50), K ÷ form, 200-400 mesh, in water and adding with stirring 1 N HC1 until the solution over the resin remains approximately p H 7. A slurry of the resin is then poured into a column, 1.2 X 100 cm, which is rinsed with several bed volumes of water prior to addition of the antibiotic charge. A mixture, 300 mg, of LL-AC541 and deformimino LL-AC541 is dissolved in 1.5 ml of water and adsorbed onto the column. The adsorbed antibiotic is eluted from the column with a gradient formed from water (500 ml) and 2.0 M potassium chloride (500 ml) at a flow of approximately 0.17 m l / m i n while the effluent is collected in approximately 5-ml fractions with the aid of a fraction collector. The gradient is approximately linear and is formed by slow addition of the KC1 solution to water with an apparatus consisting of a reservoir and mixing chamber as described by Parr. 13,14 The deformimino LL-AC541 emerges from the column in 550-580 ml and the LL-AC541 in 610-680 ml of gradient effluent (Fig. 2). The antibiotics in these fractions are readily detected by a disc agar diffusion method against a sensitive organism such as Klebsiella pneumoniae. This is accomplished by dipping paper disks, 13.5 mm diameter, into each tube of eluate and placing the resulting discs on agar seeded with the test organism. Inhibition zones are read as zone diameters after incubation of the test organism at 37 ° overnight. 1~H. Taniyama, Y. Sawada and T. Kitagawa, Chem. Pharm. Bull. 19, 1627 (1971). ,5 V. Zbinovsky, W. K. Hausmann, E. R. Wetzel, D. B. Borders, and E. L. Patterson, Appl. Microbiol. 16, 614 (1968) 1, C. W. Parr, Biochem. J. 56, XXVII (1954); R. M. Block and N.-S. Ling, Anal. Chem. 26, 1543 (1954). 1~j. j. Wren, .l. Chromatogr. 12, 32 (1963).
260
METHODS
FOR
THE
STUDY
[10]
OF ANTIBIOTICS
SO
-g E o U
g
25 20
1.0
L
G Y
I
"5
15.-
>, o
O5 '6
I0 5 029O
0.1 420
550 610 680 ml of Solt Grodient
810
FIG. 2. Resolution of LL-AC541 and deformimino LL-AC541 by chromatography with a KCI gradient on a weak cation exchanger. The two antibiotic components emerging from the column can also be detected by monitoring the column effluent at 215 nm since these compounds have significant end absorption. III. Ion-Exchange Chromatography of Hydrolysis Fragments Quantitative and qualitative studies of hydrolysis fragments have proved to be very valuable in the identification and structure determination of streptothricin-type antibiotics. The most precise procedures for conducting these studies have utilized amino acid autoanalyzers with columns of strong cationic exchangers eluted with a buffer near pH 5. The first reported application of an amino acid autoanalyzer to the study of streptothricin hydrolysates was described by C. A. Egorov et al. 15 They found that the components of various streptothricin-type complexes had the gross structure of streptothricin but differed only by the number of #-lysine groups per molecular ranging from I to 7.16,17 In the course of these studies evidence was presented to suggest that streptolin (Fig. I) might have three #-lysine residues per molecule rather than two as originally proposed2 Subsequent studies extended the analyzer procedures to streptothricintype antibiotics differing by the type of amino acid side chain and to is C. A. Egorov, P. D. Reshetov, and A. S. Khokhlov, J. Chromatogr. 19, 214 (1965). 18A. S. Khokhlov and K. I. Shutova, J Antibiot. 2S, 501 (1972). 17p. D. Reshetov, T. A. Egorov, and A. S. Khokhlov, Khim. Prir. Soedin. I, 117 (1965).
[10]
CHROMATOGRAPHY OF STREPTOTHRICIN ANTIBIOTICS
261
resolution of the different streptolidine-sugar compounds, ls-2° An additional advantage of this modification is that some streptolidine-sugar compounds can be detected by ninhydrin whereas the corresponding sugars do not react because of substitution on the nitrogen of the sugar.4,19 Resolution of the different streptolidine-sugar compounds by paper chromatography or eleetrophoresis has been only partially successful. Although the amino acid autoanalyzer provides excellent reproducibility and separation of the hydrolysis fragments, care should be given to proper controls for each individual analyzer. Variations in resin apparently can cause a reversal of peaks. 21 Procedure Preparation o] Hydrolysates. Hydrolysates are prepared under conditions that favor formation of the streptolidine-sugar compounds. Approximately 1% solutions of the antibiotics in 3 N HC1 are heated in sealed vials at 100-110 ° for 5 hr. The resulting solutions are evaporated under reduced pressure to residues which are dissolved in water to give ,-~ 1% solutions used for autoanalyzer studies. Autoanalyzer Determinations. The compositions of the hydrolysates are determined with a Technicon amino acid autoanalyzer under the conditions described below. The columns are eluted continuously with a pH 5.0 buffer composed of 14.71 g {0.05 mole) of sodium citrate dihydrate, 900 ml of water, 25 ml (0.05 mmole) of 0.002 N sodium hydroxide, 35.07 g (0.60 mole) of sodium chloride, and 10 ml of Brij detergent [polyoxyethylene (23) lauryl ether (10 g) dissolved in 200 ml of water] adjusted to pH 5.0 with 6 N HC1 and diluted to 1 liter with water. Condition A. A column (0.6 cm by 130 cm) of Chromobeads type A (sulfonated polystyrene resin from Technicon Chromatography Corp., Chauncey, New York) is maintained at 60 ° by a water jacket and eluted with pH 5 0 buffer. The column effluent is monitored by a standard automated n!nhydrin-hydrindantin procedure. 2~ The column flow rate is 0.5 ml/min. ~8D. B. Borders, J. P. Kirby, E. R. Wetzel, M. C. Davies, and W. K. Hausmann, Antimicrob. Ag. Chemother. I, 403 (1972). 1, D. B. Borders, W. K. Hausmann, E. R. Wetzel, and E. L. Patterson, Tetrahedron Lett. p. 4187 (1967). :o K. J. Sax, P. Monnikendam, D. B. Borders, P. Shu, L. A. Mitscher, W. K. Hausmann, and E. L. Patterson, Antimicrob. Ag. Chemother. 1967, 442 (1968). ~*J. Shoji, S. Kozuki, M. Ebata, and H. Otsuka, J. Antibiot. 21, 509 (,1968). 2.-Technicon Corporation, Technicon Autoanalyzer Methodology. Section I, pp. 7-8. Technicon Chromatography Corp., Chauncey, New York, 1961.
262
[10]
METHODS FOR THE STUDY OF ANTIBIOTICS TABLE I AUTOANALYZER RETENTION TIMES FOR ANTIBIOTIC HYDROLYSIS FRAGMENTS AND RELATED COMPOUNDS Condition A (14-hr r u n )
Compound Glycine Glucosamine Galactosamine Gulosamine Lysine Streptolidine ~-Lysine Ammonia Methylamine Arginine N-guan-Streptolidyl
Retention time ° (min)
Condition B (5-hr r u n )
Color yield b (area/mmole)
Retention time (Inin)
Color yield (area/mmole)
73 159 ~ 164 ~ 173 c 204 235 258 307 344 437 605
35.3 33.3 32.6 -34.4 29.6 -12.7 10.0 31.6 --
35 61 -69 -93 99 113 --236
19.1 15.7 -14.3 -8.3 15.4 6.4 --9.8
767
31.9
302
9.9
gulosaminide N-guan-Streptolidyl
N~-methylgulosaminide a R e t e n t i o n t i m e averages for a p a r t i c u l a r c o m p o u n d were u s u a l l y + 2 m i n f r o m given value. b Area in a r b i t r a r y units. c S e p a r a t i o n of s u g a r s confirmed b y a m i x e d r u n . TABLE II AUTOANALYZER COMPARISONS OF NINHYDRIN-PosITIVE FRAGMENTS FROM HYDROLYSATES OF VARIOUS STREPTOTHR1CIN-TYPE ANTIBIOTICS a O b s e r v e d mole ratios of f r a g m e n t s Antibiotic fragment
Deformimino LL-AC541 LL-AB664 b
Streptothricin
LL-AC541
Glycine Gulosamine Streptolidine B-Lysine Ammonia
-0.2 0.6 1.0 1.1
1.0 -0.2 -2.3
1.0 -0.1 -1.3
1.0 ---2.4
N-guan-Streptolidyl
O. 4
--
--
--
--
O .8
O.6
--
gulosaminide N-guan-Streptolidyt
NP-methylgulosaminide Values o b s e r v e d f r o m h y d r o l y s e s w i t h 3 N HC1 at 100-110 °. b T h e N - I n e t h y l s t r e p t o l i d i n e a n d s t r e p t o l i d i n e - s u g a r c o m p o u n d f r o m LL-AB664 were essentially n i n h y d r i n - n e g a t i v e .
[11]
CHROMATOGRAPHY OF AMINOGLYCOSIDES
263
Condition B. Condition B is the same as condition A except that the column is 0.6 cm by 75 cm of Chromobeads type C2, and the column effluent is monitored by an automated ninhydrin-hydrazine procedure. 2:~ The prepared reagent for this modified detection system is more stable than that used in condition A. Column flow rate is 0.9 ml/min. The retention times and color yields for various hydrolysis products and related compounds are given in Table I, and a comparison of quantitative results for different streptothricin-type antibiotics is given in Table II. 18 :~Technicon Corporation, Res. Bull. No. 20, Technicon Chromatography Corp., Chauncey, New York, 1968.
[11] I o n - E x c h a n g e C h r o m a t o g r a p h y o f Aminoglycoside Antibiotics
By HAMAO UMEZAWA and SHINICHI KO•DO I. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . II. P r e p a r a t i o n of Resin Columns . . . . . . . . . . . . . III. Extraction and Purification b y Using Carboxylic Acid Resins . . . . A. Isolation of K a n a m y c i n . . . . . . . . . . . . . . B. Isolation of 4-Amino-4-deoxy-a,a-trehalose . . . . . . . . C. Separation of Antibiotics b y Elution with a G r a d i e n t of A m m o n i a . D. Separation of N e b r a m y c i n s on a Large Scale . . . . . . . . E. Separation of Derivatives of 3 ' , ¥ - D i d e o x y k a n a m y c i n B . . . . . IV. Extraction and Purification Using Sulfonic Acid Resins and Phosphonic Acid Resins . . . . . . . . . . . . . . . . . . A. Isolation of K a s u g a m y c i n by Amberlite IR-120 . . . . . . . B. Separation of Validamycins by Dowex 50 . . . . . . . . . V. Extraction a n d Purification Using Cellulose and Sephadex Exchangers A. Separation of Gentamicin C Complex b y CM-Sephadex . . . . . B. Separation of Lividomycins b y CM-Sephadex . . . . . . . . VI. Nonionic Adsorption C h r o m a t o g r a p h y b y Anion Exchange Resins. A. P r e p a r a t i o n of Nonionic Columns . . . . . . . . . . . B. C h r o m a t o g r a p h i c Procedure . . . . . . . . . . . . . C. Application to Separation of K a n a m y c i n s . . . . . . . . . 1). Application to Separation of Neomycins . . . . . . . . . E. Application to Separation of Destomyeins . . . . . . . . . F. High-Pressure Liquid C h r o m a t o g r a p h y . . . . . . . . . .
263 267 268 268 269 270 271 271 272 273 273 273 274 275 275 276 276 276 277 277 278
I. I n t r o d u c t i o n A m i n o g l y c o s i d e a n t i b i o t i c s ( T a b l e I ) p r o d u c e d b y Streptomyces, Micromonospora, a n d Bacillus h a v e b e e n e x t r a c t e d a n d p u r i f i e d b y a p p l i c a -
[11]
CHROMATOGRAPHY OF AMINOGLYCOSIDES
263
Condition B. Condition B is the same as condition A except that the column is 0.6 cm by 75 cm of Chromobeads type C2, and the column effluent is monitored by an automated ninhydrin-hydrazine procedure. 2:~ The prepared reagent for this modified detection system is more stable than that used in condition A. Column flow rate is 0.9 ml/min. The retention times and color yields for various hydrolysis products and related compounds are given in Table I, and a comparison of quantitative results for different streptothricin-type antibiotics is given in Table II. 18 :~Technicon Corporation, Res. Bull. No. 20, Technicon Chromatography Corp., Chauncey, New York, 1968.
[11] I o n - E x c h a n g e C h r o m a t o g r a p h y o f Aminoglycoside Antibiotics
By HAMAO UMEZAWA and SHINICHI KO•DO I. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . II. P r e p a r a t i o n of Resin Columns . . . . . . . . . . . . . III. Extraction and Purification b y Using Carboxylic Acid Resins . . . . A. Isolation of K a n a m y c i n . . . . . . . . . . . . . . B. Isolation of 4-Amino-4-deoxy-a,a-trehalose . . . . . . . . C. Separation of Antibiotics b y Elution with a G r a d i e n t of A m m o n i a . D. Separation of N e b r a m y c i n s on a Large Scale . . . . . . . . E. Separation of Derivatives of 3 ' , ¥ - D i d e o x y k a n a m y c i n B . . . . . IV. Extraction and Purification Using Sulfonic Acid Resins and Phosphonic Acid Resins . . . . . . . . . . . . . . . . . . A. Isolation of K a s u g a m y c i n by Amberlite IR-120 . . . . . . . B. Separation of Validamycins by Dowex 50 . . . . . . . . . V. Extraction a n d Purification Using Cellulose and Sephadex Exchangers A. Separation of Gentamicin C Complex b y CM-Sephadex . . . . . B. Separation of Lividomycins b y CM-Sephadex . . . . . . . . VI. Nonionic Adsorption C h r o m a t o g r a p h y b y Anion Exchange Resins. A. P r e p a r a t i o n of Nonionic Columns . . . . . . . . . . . B. C h r o m a t o g r a p h i c Procedure . . . . . . . . . . . . . C. Application to Separation of K a n a m y c i n s . . . . . . . . . 1). Application to Separation of Neomycins . . . . . . . . . E. Application to Separation of Destomyeins . . . . . . . . . F. High-Pressure Liquid C h r o m a t o g r a p h y . . . . . . . . . .
263 267 268 268 269 270 271 271 272 273 273 273 274 275 275 276 276 276 277 277 278
I. I n t r o d u c t i o n A m i n o g l y c o s i d e a n t i b i o t i c s ( T a b l e I ) p r o d u c e d b y Streptomyces, Micromonospora, a n d Bacillus h a v e b e e n e x t r a c t e d a n d p u r i f i e d b y a p p l i c a -
264
METHODS FOR THE STUDY OF ANTIBIOTICS
[11]
TABLE I AMINOGLYCOSIDE ANTIBIOTICSa
1. Monosaccharide antibiotics: nojirimycin, 3-amino-3-deoxy-D-glucose, b streptozotocin, prumycinc 2. Simple disaccharide antibiotics: trehalosamine, mannosyl glucosaminide,d 4-amino4-deoxy-a,a-trehalose" 3. Inositol-containing antibiotics: kasugamycin, myomycin/ 4. Inosamine-inosadiamine-containingantibiotics: (1) Inosamine group: minosaminomycing (2) Streptomycin group: streptomycin, mannosidostreptomycin (streptomycin B), hydroxystreptomycin (reticulin), dihydrostreptomycin, glebomycin (bluensomycin) (3) Actinospectacin (spectinomycin) (4) Hybrimycin group: hybrimycin A1, A2, Aa, BI, B~, Bah (5) Neamine group: neamine (neomycin A), paromamine (6) Kanamycin group; kanamycin (A), B, C, NK-1001, NK-1012-1, NK-1012-2, NK-1013-1, NK-1013-2, NK-10O3,i tobramycinJ (nebramycin factor 6), apramycink (factor 2), 6"-0-carbamoylkanamycin Bk (factor 4), 6"-0carbamoyltobramycin~ (factor 5'), gentamicin A, C~, C~, C2, sisomicin,t verdamicin,~ G-418~ (7) Neomycin group: neomycin B, C (streptothricin BII, BI), neomycin Lps, Lpc, paromomycin I, II, lividomycin A,° B,p mannosyl paromomycin,q ribostamycinr (SF-733), butirosin A, B,* Bu-17O9 E~, E.~t (8) Destomycin group: hygromycin B, destomycin A, B, A-396-I,~ SS-56C ~ 5. Other cyclitol-containing antibiotics: v'alidamycin A,~ B, • C, D, E, F~ Most antibiotics were described in H. Umezawa, "Index of Antibiotics from Actinomycetes." University of Tokyo Press, Tokyo, 1967. b S. Umezawa, K. Umino, S. Shibahara, M. Hamada, and S. Omoto, J. Antibiot. Ser. A 20, 355 (1967). c S. Omura, M. Tishler, M. Katagiri, and T. Hata, Chem. Commun., p. 633 (1972). d M. Uramoto, N. Otake, and H. Yonehar~, J. Antibiot. Ser. A 20, 236 (1967). • It. Naganawa, N, Usui, T. Takita, M. Hamada, K. Maeda, and H. Umezawa, J. Antibiot. 27, 145 (1974). I j. C. French, Q. R. Bartz, and H. W. Dion, J. Antibiot. 26, 272 (1973). a M. Hamada, S. Kondo, T. Yokoyama, K. Miura, K. Iinuma, H. Yamamoto, K. Maeda, T. Takeuchi, and H. Umezawa, J . Antibiot. 27, 81 (1974). h W. T. Shier, K. L. Rinehart, Jr., and D. Gottlieb, J. Antibiot. 23, 51 (1970). i M. Murase, T. Ito, S. Fukatsu, and H. Umezawa, "Progress in Antimicrobial and Anticancer Chemotherapy," Vol. II, p. 1098. Univ. of Tokyo Press, Tokyo, 1970. K. F. Koch and J. A. Rhoades, Antimicrob. Ag. Chemother. 1970 309 (1971). k K. F. Koch, F. A. Davis, and J. A. Rhoades, J. Antibiot. 26, 745 (1973). z D. J. Cooper, R. S. Jaret, and H. Reimann, Chem. Commun., p. 285 (1971). " P. J. L. Daniels and A. S. Yehaskel, 13th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., September, 1973. n p. j. L. Daniels, A. S. Yehaskel, and J. Morton, 13th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., September, 1973. o T. Oda, T. Mori, Y. Kyotani, and M. Nakayama, J. Antibiot. 24, 511 (1971).
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CHROMATOGRAPHY OF AMINOGLYCOSIDES
265
P T. Mori, Y. Kyotani, I. Wutanabe, and T. Oda, J. Antibiot. 25, 149 (1972). q T. Mori, Y. Kyotani, I. Watanabe, and T. Oda, J. Antibiot. 25, 317 (1972). E. Akita, T. Tsuruoka, N. Ezaki, and T. Niida, J. Antibiot. 28, 175 (1970). P. W. K. Woo, H. W. Dion, and Q. R. Bartz, Tetrahedron Left., p. 2125 (1971). t H. Tsukiura, K. Saito, S. Kobaru, M. Konishi, and H. Kawaguchi, J. Antibiot. 26, 386 (1973). J. Shoji ~nd Y. Nakaguwa, J. Antibiot. 23, 569 (1970). " S. Inouye, T. Shomura, H. Watanabe, K. Totsugawa, and T. Niida, J. Antibiot. 26, 374 (1973). S. Horii and Y. Kameda, Chem. Commun., p. 747 (1972). T. Iwasa, Y. K~meda, M. Asai, S. Horii, and K. Mizuno, J. Antibiot. 24, 119 (1971). YS. Horii, Y. Kameda, and K. Kawahara, J. Antibiot. 25, 48 (1972). tion of an ion-exchange chromatography. Among these antibiotics, streptomycin, dihydrostreptomycin, k a n a m y c i n (which can be also called k a n a m y c i n A), k a n a m y c i n B, a mixture of gentamicins C1, C2 and Cla, a mixture of neomycins B and C, a mixture of p a r o m o m y c i n s I and I I , and ribostamycin are commercially available as chemotherapeutic agents useful in treating infections. H y g r o m y c i n B and destomycin A are used as animal anthelminties. K a s u g a m y c i n and validamycin are used for prevention of plant diseases. More than one aminoglycoside antibiotic is generally produced by the same strain as in cases of k a n a m y c i n s A, B, and C by S t r e p t o m y c e s k a n a m y c e t i c u s , 1 gentamicins C1, C,_,, and C ~ by M i c r o m o n o s p o r a purpurea"- and butirosins A and B by Bacillus circulans, a Therefore, complete separation of the analogous antibiotics which are produced in a same culture filtrate has been studied. These antibiotics are adsorbed by cation exchangers such as resin, cellulose, and Sephadex ion-exchangers, and separated. Commercially available cation exchangers used for their separation are shown in T a b l e II. A weak cation exchange resin possessing carboxylic acid as the functional group is most useful not only in extraction of these antibiotics from culture liquids, but also in separation of a mixture of these antibiotics into each components. For the first time in order to extract streptomycin ill about 1949, tile resin process was introduced into industrial extraction of natural products. This resin was a polyacrylic acid resin, Amberlite IRC-50, manufactured by R o h m and H a a s Co., Philadelphia. 1K. Maeda, M. Ueda, K. Yagishita, S. Kawaji, S. Kondo, M. Murase, T. Takeuchi, Y. Okami, and H. Umezawa, J. Antibiot. Set. A 10, 228 (1957). 2j. p. Rosselet, J. Marquez, E. Meseck, A. Murawski, A. Hamdan, C. Joyner, R. Schmidt, D. Migliore, and H. L. Herzog, Anlimicrob. Ag. Chemother. 1963, 14 (1964). H. W. Dion, P. W. K. Woo, N. E. Willmer, D. L. Kern, J. Onaga, and S. A. Fusari, Antimicrob. Ag. Chemother. 2, 84 (1972).
266
[11]
METHODS FOR T H E STUDY OF ANTIBIOTICS
TABLE II COMMERCIAL CATION EXCHANGERS FOR SEPARATION OF AMINOGLYCOSIDE ANTIBIOTICS
Functional group Matrix Resin
Sephadex Cellulose
Carboxylic acid
Phosphonic acid
Sulfonic acid
Amberlite IRC-50" Amberlite CG-50~ Amberlite IRC-84" Duolite CC-3b Lewatit CNP ~ Bio-Rex 70d CM-Sephadex C-25I CM-celluloseg
Duolite ES-63b Bio-Rex 63d
Amberlite IR-120~ Dowex 50~ Duolite C-20b Lewatit SP-120° SE-Sephadex C-25s
P-cellulosea
Manufactured by Rohm and Haas Co., Philadelphia, Pennsylvania. b Diamond Shamrock Chemical Co., Resinous Products Div., Cleveland, Ohio. c Naftone, Inc., Park Ave., New York. d Bio-Rad Laboratories, Richmond, California. DoT Chemical Co., Midland, Michigan. I Pharmacia Fine Chemicals, Division of AB Pharmacia, Uppsala, Sweden, CM-: carboxymethyl group, SE-: sulfoethyl group. g Carboxymethyl celluloses and cellulose phosphates are supplied by many different manufacturers. Even now it is widely used in antibiotic industries. Streptomycin adsorbed on a column of this resin in the Na* form was eluted with aqueous mineral acid in a good yield. Extraction of neomycins and kanamycins which are stable in alkaline solution is more efficiently accomplished by adsorption of NH4 * form of this resin and by elution with aqueous ammonia. A mixture of m a n y kinds of aminoglycoside antibiotics are efficiently separated into each components by a linear gradient elution of ammonia from a column of Amberlite CG-50 (chromatographic grade, a polyacrylic acid resin). 4 Processes using strong cation exchange resins possessing sulfonic acid as the functional group were introduced into extraction of weakly basic aminoglycoside antibiotics. In this case also, Amberlite CG-50 resin was utilized for further purification, eluting the weakly adsorbed antibiotics with water. Adsorption of aminoglycoside antibiotics on a resin is often interfered with by various kinds of cations in culture filtrates. Multivalent inorganic cations such as Ca 2÷ and Fe 3÷ are tightly bound to cation exchange resins. Therefore, these cations are first removed from culture liquids before application of the resin processes. 4H. Yamamoto, Y. Ikeda, S. Kondo, and H. Umezawa, unpublished data, 1973.
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C H R O M A T O G R A P HOF Y AMINOGLYCOSIDES
267
For separation and purification of aminoglycoside antibiotics, cellulose and Sephadex ion-exchangers are also useful. The most efficient chromatography for separation of analogous aminoglycoside antibiotics can be accomplished by the use of anion exchange resin, Dowex l-X2 (Dow Chemical Co., Midland, Michigan). This technique by nonionic adsorption chromatography was first employed for separation of kanamycins (Rothrock et al.5). Many of the other antibiotics can be purified by this technique. II. Preparation of Resin Columns Ion-exchange resin columns can be made in any desired size, from a few milliliters up to many liters. Height of exchanger beds for a good separation is usually 10-20 times longer than a column diameter. In a case of difficult separation, a much longer column can be used. In a conventional method of packing the column, ~ the resin is stirred in an excess of the first eluent to be used, and after most of the resin is settled, small particles are decanted off. The remaining resin is slurried into the column and allowed to settle by gravity to form the packed bed. It is often disadvantageous to use too large a volume of resin. Larger exchanger beds need more flow time and more rinsing, and yield inconveniently large solution volumes. An efficient system employed in antibiotic industries uses a minimum amount of the resin which is divided into more than one column. The culture liquid is continuously charged on the first column which is connected to the second column. From each column on which the antibiotic is fully adsorbed, it is eluted. By continuous repetition, the antibiotic can be extracted in excellent yield. The adsorption and elution are carried out usually in laboratories by downflow, but the upflow (backflow) technique is frequently used in large-scale operations. Before adsorption of an antibiotic, commercial ion-exchange resins are treated by repeated cycles of washing with 1-2 N hydrochloric acid and sodium hydroxide to remove imourities, and converted to a desired form by treatment with 1 M solution of an appropriate salt, acid or base. After the conversion is completed, the resin is washed with distilled water. Resins shrink and swell considerably and therefore the conversion is handled most easily in a large diameter column. Then, the resin is packed into the desired size column. J. W. Rothrock, R. T. Goegelman, and F. J. Wolf, Antibiot. Annu. 1958--1959, 796 (1959). G. Zweig and J. Sherma, eds., "Handbook of Chromatography," Vol. II, p. 61. Chem. Rubber Publ. Co., Cleveland, Ohio, 1972.
268
METHODS FOR THE STUDY OF ANTIBIOTICS
[11]
Weak cation exchange resins possessing carboxylic acid as the functional group are usually converted to H ÷ form by 1 N hydrochloric acid and to Na ÷ or NH4 + form by 1-2 N sodium hydroxide or ammonia. If a partially regenerated form is necessary, H + form and alkaline form of resins are well mixed, or the resin is washed with phosphate buffer of the proper pH. III. Extraction and Purificationby Using Carboxylic Acid Resins
Since carboxylic acid resins bind very strongly with H ÷ and weakly with N a +, K +, and N H 4 +, m a n y aminoglycoside antibiotics in aqueous solution are efficiently adsorbed by ion-exchange on a column of N a +, K +, or N H 4 + form of the resins. And these antibiotics can be eluted from the column with aqueous mineral acids such as hydrochloric acid and sulfuric acid. Alkaline-stable aminoglycoside antibiotics, for example, neomycins and kanamycins, are isolated by adsorption on N H 4 + form of this resin and by elution with aqueous ammonia. A m o n g the aminoglycoside antibiotics shown in Table I, those except weakly basic antibiotics are well adsorbed on Amberlite IRC-50 resin (Na + or N H , ÷ form) from culture filtrates.The weakly basic antibiotics, for example, nojirimycin, 4-amino-4-deoxy-a,a-trehalose, kasugamycin, and validamycins, are not adsorbed on carboxylic acid resin from culture filtrates. However, there is some retention of them on Amberlite CG-50 (NH4 + form) column, and they can be purified by elution with water from the resin. Strongly basic antibiotics, adsorbed on Amberlite IRC-50 columns, are usually eluted with less than I N hydrochloric acid or aqueous ammonia in good yields. The use of ammonia for their purification is preferred, because in this case a salt-free eluate is obtained. However, alkaline-unstable antibiotics, for example, streptomycins and streptothricins, must be eluted with a proper ion strength of salts or mineral acids. Efficient separation of m a n y alkaline-stable aminoglycoside antibiotics can be accomplished by elution with a linear gradient of ammonia from a column of Amberlite CG-50 resin,4 as shown in Figs. 1 and 2. A. Isolation o f K a n a m y c i n 7
Kanamycin in a culture filtrate (more than 500 ~g/ml) of Streptomyces kanamyceticus s can be purified by a single resin process followed 7K. Maeda, "Streptomyces Products Inhibiting Mycobacteria," p. 60. Wiley, New York, 1965. s H. Umezawa, M. Ueda, K. Maeda, K. Yagishita, S. Kondo, Y. 0kami, R. Utahara, Y. Osato, K. Nitta, and T. Takeuchi, J. Antibiot. Set. A 10, 181 (1957).
[lll
CHROMATOGRAPHY
OF
AMINOGLYCOSIDES
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60 70 8b 90 160 Fraction number FIG. 1. Separation of neomycin B (sulfate, 6.8 mg), paromomycin I (sulfate, 5.8 mg), lividomycin A (5.4 mg), and lividomycin B (5.2 mg) by a column (0.8 X 20 cm) of Amberlite CG-50 (type II, NH4÷ form, 10 ml). The column which adsorbed the antibiotics mixture in water (0.5 ml) was washed with 0.1 N NH40H (40 ml) and then eluted with a linear gradient of 0.1 N to 0.8 N NH4OH (gradient rate: 0.5%/min, flow rate: 21 ml/hr). These antibiotics in fractions (1 ml each) were detected by Rr values of thin-layer chromatography (TLC) of silica gel (Merck, Art 5721) using butanol-ethanol-chloroform-28% ammonia (4:5:2:8 in volume) as developing solvent, followed by coloration with ninhydrin reagent. Each antibiotic in fractions was assayed by agar plate method using Bacillus subtilis PCI 219 as a test organism.
by crystallization. A column of Amberlite I R C - 5 0 resin (70% Na + or NH4 ÷ form) on which kanamycin is adsorbed is eluted with 1 N aqueous ammonia. The eluate is concentrated by evaporation to remove ammonia, acidified with sulfuric acid to p H 3, and decolorized with active charcoal. The pH of the solution is then adjusted to 8.0-8.2, that is, the p H of kanamycin monosulfate solution. To this concentrate containing about 100 mg of kanamycin base per milliliter, the same volume of methanol is added with stirring and cooling. After crystallization starts, more methanol is added up to 60% methanol concentration. With continued stirring, crystals of kanamycin monosulfate are obtained in 80% yield from the culture filtrate.
B. Isolation of 4-Amino-4-deoxy-(~,~-trehalose A streptomyces antibiotic, 4-amino-4-deoxy-~,a-trehalose9 has been purified by Amberlite CG-50 column chromatography by the authors. "H. Naganawa, N. Usui, T. Takita, M. Hamada, K. Maeda, and H. Umezawa, J. Antibiot. 27, 145 (1974).
270
METHODS FOR THE STUDY OF ANTIBIOTICS Paromam i ne Destomycin A Lividomycin A Kanamycin A Paromomycin l Kanamycin ]3 Ribostamycin Lividomyci n B Tobrarnycin Neamine DKI3a Gentamicin Cb Neomycin B Butirosins c
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[11]
0.2
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Norrna[ity of ammonia
Fie. 2. The concentrations of ammonia for elution of aminoglycoside antibiotics from a column (0.8 × 20 cm) of Amberlite CG-50 (type II, NH~÷ form, 10 ml). The column which adsorbed antibiotics (approximately 5 mg each) was washed with 0.1 N NH,OH (40 ml) and then eluted with a linear gradient of 0.1 N to 0.8 N NH40H (gradient rate : 0.5%/min, flow rate : 21 ml/hr). These antibiotics in fractions (1 ml each) were detected by thin-layer chromatography of silica gel (Merck, Art 5721) using butanol-ethanol-chloroform-28% ammonia (4:5:2:8 in volume) as a developing solvent, high-voltage paper electrophoresis under 3300 V for 15 min using formic acid-acetic acid-water (25:75:900 in volume) as an electrolyte solution, and agar plate method using Bacillus subtilis PCI 219 as a test organism. (a) 3',4'dideoxykanamycin B; (b) gentamicin C complex; (c) butirosins A and B.
An aqueous solution (3 ml) of the crude powder of the antibiotic extracted from a culture filtrate by a carbon adsorption process is charged to a column (1.25 X 31 cm) of Amberlite CG-50 resin (type I, NH~ + form, 40 ml) and the c h r o m a t o g r a m is developed with water. The eluate in fractions 22-56 (2 ml each) showing bioaetivity against Escherichia coli N I H J or Bacillus subtilis P C I 219 are combined and lyophilized to yield a white powder (391 mg) of the pure antibiotic.
C. Separation of Antibiotics b y Elution with a G r a d i e n t of A m m o n i a 4 An aqueous solution (0.5 ml) containing neomycin B sulfate (6.8 mg), p a r o m o m y c i n I sulfate (5.8 mg), lividomycin A (5.4 rag), and lividomycin B (5 2 rag) is charged to a column (0.8 X 20 era) of Amberlite CG-50 resin (type I I , NH4 ÷ form, 10 ml). After the column is washed with 0.1 N aqueous ammonia (40 ml), the antibiotics are eluted with a linear gradient of aqueous ammonia from 0.1 to 0.8 N for 200 min (a gradient rate of 0.5% per minute) at a flow rate of 21 ml per hour using an Aerograph LC-4200 gradient system (Varian Instruments, California). With
[11]
CHROMATOGRAPHY OF AMINOGLYCOSIDES
271
the aid of a fraction collector approximately 1-ml fractions of the efituent are collected. Each antibiotic in the fractions is determined by thin-layer chromatography on silica gel (Merck, Art 5721), for instance, using butanol-ethanol-chloroform-28% ammonia (4:5:2:8 in volume) as a developing solvent, followed by treatment with ninhydrin reagent. Each antibiotic in the fractions can be also assayed by an agar plate method using Bacillus subtilis PCI 219 as a test organism. These four antibiotics are completely separated by this technique as shown in Fig. 1. This technique has been applied to the other strongly basic antibiotics and the concentrations of ammonia in eluent for each antibiotic are summarized in Fig. 2.
D. Separation of Nebramycins on a Large Scale I° A solution of nebramycin complex has been obtained from culture filtrates of Streptomyces tenebrarius by adsorption on Amberlite IRC-50 resin, elution with 1 N aqueous ammonia and concentration to remove excess ammonia. The following example was reported. The solution (20 liters, 82 X 106 units of the activity against Klebsiella pneumoniae FDA KL4) was applied to a 10 cm diameter column which contained 20 liters of Bio-Rex 70 resin (NH4 * form). The column was washed with 12 liters of water and then eluted with a gradient prepared by adding 0.2 N aqueous ammonia to a 50-liter constant-volume reservoir which was charged with 0.05 N aqueous ammonia. The flow rate was 20 ml per minute and fractions of 500 ml were collected. The following fractions were combined, concentrated, and freeze-dried: fractions 39-100, 102 g (35 X 10~ units) of factor 2 (apramycin); fractions 115-154, 14 g (8.1 X 10~ units) of factor 4 (6"-O-carbamoylkanamycin B) ; fractions 188-240, 24 g (30 X 10~ units) of factor 5' (6"-O-carbamoyltobramycin). Eighty-nine percent of the activity was recovered.
E. Separation of Derivatives of 3',4'-Dideoxykanamycin B In the course of chemical derivation of kanamycins based on mechanism of resistance to aminoglycoside antibiotics, 11 3',4'-dideoxykanamycin B (DKB), which inhibits sensitive and resistant organisms including ~*K. F. Koch, F. A. Davis, and J. A. Rhoades, d. Antibiot. 26, 745 (1973). 11H. Umezawa, "Progress in Antimicrobial and Anticancer Chemotherapy," Vol. II, p. 567. Univ. of Tokyo Press, Tokyo, 1970. Published also in Advan. Carbohyd. Chem. 30, 183-225 (1975).
272
METHODS FOR THE STUDY OF ANTIBIOTICS
[lll
Pseudomonas aeruginosa, was synthesized, 12 and thereafter, N-(S)-4amino-2-hydroxybutyryl (AHB) derivatives were prepared. 13,~4 These products were purified by the following resin processes. 6'-N-t-Butyloxycarbonyl DKB (1109 rag) was acylated with (S)-4-amino-2-hydroxybutyric acid and a mixture of positional isomers of AHB-DKB was obtained after removal of protecting groups. The mixture (trifluoroacetate, 3067 mg in 5 ml water) was charged to a column (14 mm diameter) of Ambertire CG-50 resin (type I, NH4 ÷ form, 50 ml). After washing with water (250 ml), the column was eluted by stepwise elution with 500 ml each of 0.5, 0.75, and 1.0 N ammonia. The unreacted DKB (297 mg, 32% yield) and 2'-AHB-DKB (238 mg, 21%) were eluted with 0.5 N ammonia, 3-AHB-DKB (72 mg, 6%), 1-AHB-DKB (131 mg, 12%) and 3"-AHB-DKB (53 mg, 4%) with 0.75 N ammonia, and 3,2'-diAHB-DKB (46 rag, 3%), 1,2'-diAHB-DKB (47 rag, 3%) and the other diacyl derivatives with 1.0 N ammonia. By this chromatography, these positional isomers 14 of AHB-DKB, were completely separated. 1-AHB-DKB and 1,2'-diAHB-DKB show strong activity against sensitive and resistant organisms. The latter organisms produced kanamycin phosphotransferases and kanamycin nucleotidyltransferase. IV. Extraction and Purification Using Sulfonic Acid Resins and Phosphonic Acid Resins Weakly basic aminoglycoside antibiotics, for example, nojirimycin, 3-amino-3-deoxy-D-glucose, trehalosamine, mannosyl glucosaminide, 4-amino-4-deoxy-a,a-trehalose, kasugamycin, and validamycins can be extracted from culture filtrates by adsorption on strong cation exchange resins possessing sulfonic acid groups, such as Amberlite IR-120, Dowex 50, and Lewatit SP-120, and elution with dilute aqueous ammonia. Since sulfonie acid resins hold H + more weakly than Na ÷ and NH4 +, these resins are generally used as H + form. From a column of Amberlite IR-120 resin kanamycin is efficiently eluted with 1 N aqueous ammonia, but not with t N hydrochloric acid. Therefore washing the column with the acid and elution of kanamycin with ammonia gives a highly purified kanamycin. 1 Phosphonic acid resins have not been used widely. However, extraction of kanamycin from culture filtrates by adsorption on Duolite C-62, 12H. Umezawa, S. Umezawa, T. Tsuchiya, and Y. Okazaki, J. Antibiot. 24, 485 (1971). 1aS. Kondo, K. Iinuma, It. Yamamoto, K. Maeda, and H. Umezawa, J. Antibiot. 26, ¢12 (1973). ~4S. Kondo, K. Iinuma, H. Yamamoto, Y. Ikeda, K. Maeda, and H. Umezawa, J. Antibiot. 26, 705 (1973).
[11]
CHROMATOGR/kPHY OF AMINOGLYCOSIDES
273
a phosphonic acid resin formerly supplied by Diamond Shamrock Chemical Co., Resinous Products Division, Ohio, and elution with 5% aqueous ammonia has been reported? Most of aminoglycoside antibiotics can be adsorbed on phosphonic acid resins.
A. Isolation of Kasugamycin by Amberlite IR-120 Kasugamycin15 in culture filtrate of Streptomyces kasugaensis has been successfully extracted by Amberlite IR-120 resin as follows. The filtrate (1570 liters, 530 ~g/ml) was charged on Amberlite IR-120 column (H ÷ form, 300 liters). The column was washed with water and then eluted with 0.5 N aqueous ammonia. The first (53 liters), the second (200 liters), and the third (200 liters) eluates contained 28.5 g, 738 g, and 44.2 g of kasugamycin, respectively. The second eluate was adjusted to pH 6.6 with hydrochloric acid and concentrated to 6.32 liters under reduced pressure. After addition of ethanol (60 liters) to this concentrate, the crude crystals of kasugamycin hydrochloride (850 g, 90% purity) were obtained.
B. Separation of Validamycins by Dowex 5016 Validamycin E-rich fractions and F-rich fractions obtained by Dowex l-X2 resin chromatography (see Section VI) were further chromatographed on a column of Dowex 50-W X2 resin by eluting with a pyridineacetic acid buffer (pH 6.0). In this chromatography, validamycin F was eluted first and thereafter validamycin E.
V. Extraction and Purification Using Cellulose and Sephadex Exchangers Various kinds of cation exchangers having hydrophilic supporting matrix are commercially available. Cellulose and Sephadex ion-exchangers are used generally for separation of large molecules. Among these exchangers, carboxymethylcellulose, cellulose phosphate, CM-Sephadex C-25, and SE-Sephadex C-25 can be used for separation of basic antibiotics. Separation of streptothricin components by column chromatography 15H. Umezawa, Y. Okami, T. Hashimoto, Y. Suhara, M. Hamada, and T. Takeuchi, J. Antibiot. Ser. A 18, 101 (1965). S. Horii, Y. Kameda, and K. Kawahara,J. Antibiot. 25, 48 (1972).
274
METHODS FOR THE STUDY OF ANTIBIOTICS
[11]
of carboxymethyl cellulose has been reported27 Many phleomycinTM and bleomycinTM components are efficiently separated by column chromatography of CM-Sephadex C-25 with a gradient of ammonium formate. Most of the aminoglycoside antibiotics can be purified by column chromatography on carboxymethyl cellulose, cellulose phosphate, CMSephadex C-25, and SE-Sephadex C-25 using various ion strengths of salt solutions, such as sodium chloride and ammonium formate. Streptomycin is adsorbed on a column of CM-Sephadex C-25 equilibrated with 0.1 M sodium chloride or 1.0 M ammonium formate and eluted with 0.55 M sodium chloride or 2.0 M ammonium formate, s° The antibiotic, adsorbed on CM-cellulose (Brown Co., Fifth Ave., New York, 0.63 mEq/g, equilibrated with 0.1 M ammonium formate), is eluted with 0.6 M ammonium formate, s° A. Separation of Gentamicin C Complex by CM-Sephadex Maehr and Schaffneff1 have described the separation of gentamicin components by Dowex 1-X2 resin chromatography (see Section VI). The authors 2s found another powerful method using CM-Sephadex column as follows. Gentamicin C complex (800 rag) in 0.1 M ammonium formate (400 ml) was adsorbed on a column (35 mm diameter) of CM-Sephadex C-25 (800 ml, equilibrated with 0.1 M ammonium formate). The column was washed successively with 450 ml of 0.1 M, 1275 ml of 0.5 M, 4590 ml of 1.0 M and 1860 ml of 1.5 M ammonium formate and then eluted with 1.8 M ammonium formate. The effluent was cut into each approximately 15-ml fractions, and the fractions were assayed by agar plate method using Bacillus subtilis PCI 219 as a test organism and by silica gel (Merck, Art. 5721) thin-layer chromatography using butanol-ethanol-chloroform-28% ammonia (4:5:2:5 in volume) as a developing solvent followed by coloration with ninhydrin reagent. The three main peaks of gentamicins C1, C~, and C1~ appeared in fractions 569, 594, and 613, respectively. The eluates in fractions 557-575 were combined and diluted with water (6 liters). Gentamicin C~ in the diluted solution was adsorbed on a column of Amberlite CG-50 resin (type I, NH4 ÷ form, 50 ml). Elution with 0.5% aqueous ammonia gave 172 mg of pure gentamicin C1. 1~A. S. Khokhlov and P. D. Reshetov, J. Chromatogr. 14, 495 (1964). T. Ikekawa, F. Iwami, H. Hiranaka, and H. Umezawa, Y. Antibiot. Set. A 17, 194 (1964). 1~H. Umezawa, Y. Suhara, T. Takita, and K. Maeda, J. Antibiot. Ser. A 19, 210 (1966). 2oK. Yokose, Y. Suhara, M. Miyamoto, S. Kondo, and H. Umezawa, unpublished data, 1973. ~ It. Maehr and C. P. Schaffner,J. Chromatogr. 30, 572 (1967). 22M. Yagisawa,S. Kondo, and H. Umezawa,unpublished data, 1973.
[11]
CHROMATOGRAPHY OF AMINOGLYCOSIDES
275
B. Separation of Lividomycins by CM-Sephadex 2~ The separation of each lividomycin has been accomplished by the following processes. A crude powder of lividomycins (24 mg) which was obtained by adsorption on Amberlite IRC-84 resin (NH4 + form) from a culture filtrate of S t r e p t o m y c e s lividus and elution with 1 N aqueous ammonia was dissolved in water (5 ml) and charged to a column (1 }( 40 cm) of CM-Sephadex C-25 (NH4 ÷ form). After washing with water, the column was eluted by a gradient between 0.12 N (200 ml) and 0.35 N aqueous ammonia (25 ml) at a flow rate of 25 ml per hour at 27% All fractions (3 ml each) were assayed by the paper disk method using Bacillus subtilis ATCC 6633 as a test organism. Four peaks of active fractions appeared at fractions 30, 44, 64, and 72, which were designated as No. 2230-C (mannosyl paromomycin), lividomycin A, No. 2230-D (paromomycin), and lividomycin B, respectively.
VI. Nonionic Adsorption Chromatography by Anion Exchange Resins In using anion exchange resins for the removal of colored impurities from crude kanamyein aqueous solutions, Rothrock et al. ~ observed some retention of the antibiotic by strong anion exchange resins. Further investigations revealed that porous, strongly basic anion exchange resins with quaternary amine functional groups could be used at low loadings to separate kanamyein A from kanamycin B. Of these, Dowex 1-X2 resin (Dow Chemical Co., Midland, Michigan, 50-100 mesh) in OH- form is satisfactory for the chromatographic analysis of kanamycin mixtures. This technique by ion exclusion chromatography can be most efficiently used for separation of analogous aminoglycoside antibiotics. Complete separations of neomycins,24 paromomycins,~4 destomycins,"-5 gentamicins,21 lividomycins,2~ butirosins, 33 and validamycins16 have been reported by many researchers, and this technique can be applied £o largescale preparative operations. Recently, high-pressure liquid chromatography using a column of Aminex A-2726 or A-28 =7 resin (a quaternary amine resin, Bio-Rad Laboratories, California) was introduced into analysis or purification of aminoglycoside antibiotics. =3T. Mori, T. Ichiyanagi, H. Kondo, K. Tokunaga, T. Oda, and K. Munakata, J. Antibiot. 24, 339 (1971). ~4H. Maehr and C. P. Schaffner,Anal. Chem. 36, 104 (1964). 25S. Kondo, M. Sezaki, M. Koike, M. Shimura, E. Akita, K. Satoh, and T. Hats, I. Antibiot. Ser. A 18, 38 (1965). 2~T. Ohtake and M. Yaguchi, "Liquid Chromatography at Work," No. 5. Nippon Electric Varian Ltd., 1973. ~7H. Umezawa, H. Yamamoto, M. Yagisawa, S. Kondo, and T. Takeuchi, J. Antibiot. 26, 407 (1973).
276
METHODS FOR THE STUDY OF ANTIBIOTICS
[11]
A. Preparation of Nonionic Columns T M The anion exchange resin, D0wex I-X2 (CI- form, 50-100 mesh) is backwashed free of fines before being converted to the O H - form by columnwise washing with 2 resin volumes of I % sodium sulfate solution and 2 resin volumes of 10% sodium hydroxide solution. The resin shrinks and swells considerably and therefore these operations can be done most easily in a large-diameter column. The resin is washed thoroughly with C02-free distilled water to remove excess alkali. The chromatographic tube is filled up to two-thirds capacity with C02-free distilled water, and the resin in the O H - form is slurried directly into the column with C02free distilled water, so that the resin does not entrap air bubbles. The water is drained from the bottom of the column to the resin bed level. A column prepared in this manner conveniently can hold 400 ml of resin in a bed depth of I00 cm which gives efficient chromatography to handle I0 g of crude kanamycin. For smaller samples a proportionately smaller column can be employed. B. Chromatographic Procedure TM
An approximately 25% aqueous solution of aminoglycoside antibiotics is introduced to the top of the resin bed. Although hydrochloride or sulfate salts of antibiotics are usually used for the chromatography, free bases of antibiotics give more satisfactory separation. The column is developed with C02-free distilled water at the effluent flow rate of one resin volume per 2-4 hr. With the aid of an automatic fraction collector, the effluent is fractionated (usually one-tenth volume of a resin volume for each fraction). Antibiotics in the effluent are determined quantitatively by bioactivity, conductivity, refractive index, and colorations with reagents, such as ninhydrin. Antibiotics in the fractions can be also detected by paper and thin-layer chromatography, and high-voltage paper electrophoresis. C. Application to Separation of Kanamycins
Rothrock et al2 achieved a complete separation of kanamycins A and B by the technique described above. By application of this technique, crystalline kanamycin C was isolated from a mixture of kanamycins. 2s An example of quantitative separation of kanamycins is as follows. 29 A ~sM. Murase, T. Wakazawa, M. Abe, and S. Kawaji, J. Antibiot. Set. A 14, 156 (1961). 29S. Inouye and H. Ogawa, J. Chromatogr. 13, 536 (1964).
[lll
CHROMATOGRAPHY OF AMINOGLYCOSIDES
277
mixture of kanamycin A (2.08 mg), B (0.207 rag), and C (0.143 rag) is chromatographed on a column (0.9 X 39 cm) of Dowex l-X2 resin (OH- form, 200-400 mesh) with an effluent flow rate of 30 ml per hour. The antibiotics in the effluent are automatically determined by color development of ninhydrin reaction using an amino acid analy~,er (Hitachi Type KLA-2, Hitachi Co., Tokyo). The elution peaks of these antibiotics have retention times as follows, kanamycin B: 85 min, C: 108 min, and A: 187 min.
D. Application to Separation of Neomycins Neomycins were completely separated into three components by Maehr and Schaffner.2. An approximately 25% aqueous solution of a commercial neomycin sulfate sample (2 g) containing about 30% neomycin C, 70% neomycin B, and a trace of neomycin A (neamine) is charged to a column (2.5 X 100 cm) of Dowex l-X2 resin (OH- form, 50-100 mesh) and the column is developed with distilled water at a linear flow rate of 0.4-0.6 cm per minute. The effluent is cut into fractions, approximately 40 ml each, and the antibiotics in these fractions are analyzed by a quantitative ninhydrin procedure. Three elution peaks of neomycins A, C, and B appear in fractions 11, 22, and 41, respectively.
E. Application to Separation of Destomycins 25 By application of this technique, a crude powder of destomycins which was obtained by adsorption on Amberlite IRC-50 resin (Na ÷ form) from a culture filtrate of Streptomyces rimofac~ens and elution with 2% aqueous ammonia has been purified. A crude powder (175 g) of destomycins is dissolved in 200 ml of water and charged to a column of Dowex I-X2 resin (OH- form, 50-100 mesh, 1600 ml). The column is developed with water at a flow rate of 4 ml/min. The eluate is cut into 20-ml fractions each, and the bioactivities of all fractions are determined using Bacillus subtilis ATCC 6633 and Mycobacterium smegmatis ATCC 607 as the test organisms. Destomycin B is eluted in fractions 64--72, and destomycin A starts to appear in fraction 73. Fractions 76-200 are combined and lyophilized to yield a white powder (69.5 g) of pure destomycin A. The eluate in fractions 64-75 gives a white powder (13.7 g of a mixture of destomycins) on lyophilization. The mixture is dissolved in 50 ml of water and rechromatographed on a column of the resin (OH- form, 400 ml). Destomycin B is eluted in fractions 15-26 (20 ml each), which contains 4.0 g of pure destomycin B.
278
METHODS
FOR
THE
STUDY
OF ANTIBIOTICS
[11]
F. High-Pressure Liquid Chromatography High-pressure liquid chromatography using a column of Aminex A-2726 or A-2827 resin (a quaternary amine resin, Bio-Rad Laboratories,
California) is a powerful technique for analysis and purification of aminoglycoside antibiotics. The differential quantitative determination of kanamycin, lividomycin A, and 3 ' , 4 ' - d i d e o x y k a n a ~ c i n B TM has been accomplished by the following method? ~ A commercial ~pparatus, Aerograph LC-4200 system of Varian Instruments, Californi~ was used. A mixture of 40 ~g each of kanamycin A, 3',4'-dideoxykanafa~rcin B, and lividomycin A in water (12 ~l) is injected in the top of ~ e column (0.2 X 100 cm) of Aminex A-28 resin (8-12 ~m, OH- form)~and the column is developed with distilled water, operating at 50 °, 15 ml/hr, and 3400 psi. The amounts of these antibiotics in the effluent are automatically recorded by a refractive index detector. As shown in Fig. 3, these antibiotics can be completely separated in a short time and can be quantitatively determined by measuring the height of each peak. Such a differential assay is useful in studies on kinetics of an enzyme which phosphorylates or acetylates these antibiotics. High-pressure liquid chromatography is described in detail elsewhere. 3°
J
"N
.r I
0
:5 I
i
5 10 Minutes
I
15
FIG. 3. Separation of kanamycin, lividomycin A, and 3',4'-dideoxykanamycin B by high-pressure liquid chromatography on a column (0.2 X 100 cm) of Aminex A-28 (8-12 /zin, OH- form). A mixture of each 40 ~g of antibiotics in water (12 /~l) was injected to the top of the column connected with Varian Aerograph LC-4200 system and the column was developed with distilled water, operating at 50°, 15 ml/hour, 3400 psi. The amounts of these antibiotics in effluent were automatically recorded by the RI detector (4 × 10-5 refractive index units in full scale).
~°K. TsuJi, this volume [15].
[12]
E L E C T R O P H O R E SOF I S ANTIBIOTICS
[12] E l e c t r o p h o r e s i s
279
of Antibiotics
B y HAMAO UMEZAWA and SHINICHI KONDO
I. II. III. IV. V.
Introduction . . . . . . . . . . . . . . . Paper Electrophoresis . . . . . . . . . . . . . Thin-Layer Electrophoresis . . . . . . . . . . . Agar Gel Electrophoresis . . . . . . . . . . . . Carrier-Free Continuous Electrophoresis . . . . . .
. . . . . . . . . . . . . . .
279 280 286 287 289
I. I n t r o d u c t i o n A basic or acidic antibiotic moves in an electrical field at a rate directly related to the number of its net charges, t h a t is, its mobility is an expression of its ionic character. In studies of antibiotics, electrophoresis has been mainly employed for the identification and confirmation of the purity, the same as paper c h r o m a t o g r a p h y and thin-layer chromatography. Among various techniques of electrophoresis, 1-7 paper electrophoresis, especially high-voltage paper electrophoresis, has been most widely used for studies on antibiotics. Application of high-voltage gives a good separation of various kinds of antibiotics, especially watersoluble antibiotics, even with crude preparations, by a short-time operation. s Though not yet widely applied, a high-voltage thin-layer electrophoresis will become a powerful method of identification and separation of antibiotics. Agar gel electrophoresis and carrier-free continuous electrophoresis have been also applied to some antibiotic studies, but from viewpoints of efficiency and simplicity of the techniques, high-voltage 1R. Audubert and S. de Mende, "The Principles of Electrophoresis." Hutchinson, London, 1959. M. Bier, "Electrophoresis. Theory, Methods, and Applications." Academic Press, New York, 1959. 3L. P. Ribeiro, E. Mitidieri, and 0. R. Affonso, "Paper Electrophoresis." Elsevier, Amsterdam, 1961. 4 C. Wunderly, "Principles and Application of Paper Electrophoresis." Elsevier, Amsterdam, 1961. 5 M. Lederer, "Introduction to Paper Electrophoresis and Related Methods." Elsevier, Amsterdam, 1962. 6j. R. Whitaker, "Paper Chromatography and Electrophoresis," Vol. I. "Electrophoresis in Stabilizing Media." Academic Press, New York, 1967. E. Heftmann, "Chromatography," 2nd ed. Van Nostrand-Reinhold, Princeton, New Jersey, 1967. 8 K. Maeda, A. Yagi, H. Naganawa, S. Kondo, and H. Umezawa, J. Antibiot. 22, 635 (1969).
280
~ETHODS FOR THE STUDY OF ANTIBIOTICS
[12]
paper or thin-layer electrophoresis seems to be more useful. In this paper, the results obtained by electrophoreses described above are reviewed.
II. Paper Electrophoresis The technique of electrophoresis using a filter paper as a carrier is now most commonly used for analytical purpose. In 1951, an application of paper electrophoresis to separation of antibiotics was reported by Hosoya et al. 9,1° Roseothricins (H-277) were separated into two components by eleetrophoresis on a filter paper (1 X 20 cm) in 0.1 M acetate buffer (pH 5.0) or 0.1 M phosphate buffer (pH 7.0) for 4-5 hr, and streptomycin showed one component. Using paper electrophoresis under 200 V for 2.5 hr in six buffer solutions (pH 4.53, 5.59, 6.47, 6.98, 7.38, and 8.30), Takahashi and Amano 11 classified antibiotics into eight following groups. 1. Streptomycin type (toward cathode at every pH) : (a) streptomycin, dihydrostreptomycin, reticulin, neomycin, streptothricins I, II, III, IV, polymyxin, (b) flaveolin, luteomycin, xanthomycin, (c) erythromycin, albomycetin 2. Chlortetracycline type (toward cathode at neutral and acid but not at alkaline pH) : chlortetracycline, oxytetracyeline, grisein 3. Chloramphenicol type (toward cathode at alkaline pH, slight at neutral and acid pH) : chloramphenicol 4. Actinomycin type (slightly toward cathode at every pH): actinomycin, aureothricin, vinacetin, vinactin 5. Trichonin type (more toward anode at alkaline than at acid pH) : trichonin, N-187 6. Penicillin type (more toward anode at acid than at alkaline pH) : penicillin, acidomycin 7. Griseoflavin type (slightly toward anode at every pH): griseoflavin 8. Orientomycin type (toward anode at alkaline, and cathode at acid pH) orientomycin By long-run electrophoresis under 200 V for 15 hr, ~ it is possible to differentiate streptothricins I, II, and III, flaveolin and luteomycin, and erythromycin and albomycetin from one another. But it is difficult to differentiate streptomycin, dihydrostreptomycin, reticulin, neomycin A, and streptothricins I and IV from one another. S. Hosoya, M. Soeda, N. Komatsu, N. Hara, Y. Sonoda, and R. Arai, J. Antlblot. 4, 314 (1951). l°S. Hosoya, M. Soeda, N. Komatsu, Y. Sonoda, and R. Arai, J. Antibiot. 4, 317
(1951). 11B. Takahashi and Y. Amano, J. Antibiot. Ser. A 7, 104 (1954) ; Ser. B 7, 81 (1954).
[12]
ELECTROPHORESIS
OF ANTIBIOTICS
281
Low-voltage (less t h a n 300 V) paper electrophoretic data on chloramphenicol, 12 xanthomycin A, ~3 streptomycins, ~4-16 streptothricins, ~4 neomycins, 16 kanamycin, ~ viomycin, ~6 ferrimycins, ~ ferrioxamines, ~v ristocetins, 's cephalosporins, 19--~ and penicillins 2~-23 have been reported by m a n y researchers. High-voltage electrophoresis which can be completed in a short-time operation is now most widely used. With the increase in potential gradient, the mobility of ions increases linearly, but the heat generated increased quadratically. Following the developments of effective heat exchangers, a number of safe high-voltage apparatuses with liquid heat exchanger or with solid heat exchanger became commercially available. I n 1969, M a e d a et al. s reported the relative mobilities of 92 antibiotics on high-voltage paper electrophoresis, similar to the technique described by Atfield and Morris 24 for the separation of amino acids. These antibiotics can be classified into two groups according to their extraction properties: group I, antibiotics which can be extracted with organic solvents from the aqueous solutions; group II, antibiotics which are difficult to be extracted with the solvents, but which can be obtained by carbon adsorption processes. These authors found t h a t extraction procedures using ion-exchange resins were influenced by the relative mobilities of the antibiotics. Conditions ]or H i g h - V o l t a g e E l e c t r o p h o r e s i s s
An example using a commercial a p p a r a t u s with liquid heat exchanger, Model LT-48A with HV-5,000-3TC of Savant Instruments, Inc., Hicks12N. K. King and H. M. Doery, Nature (London) 171, 878 (,1953). 1~D. Dougall and E. P. Abraham, Nature (London) 176, 256 (1955). ,4 M. C. Foster and G. C. Ashton, Nature (London) 172, 958 (1953). ,5 M. Yamagishi, M. Yokoo, T. Masuda, and M. Asai, Nippon Yakugaku Zasshi 74, 283 (1954). '~ H. Bickel, E. Giiumann, G. Nussberger, P. Reusser, E. Vischer, W. Voser, A. Wettstein, and H. Z[ihner, Hclv. Chim. Acta 43, 2105 (1960). '~H. Bickel, R. Bosshardt, E. Giiumann, P. Reusser, E. Vischer, W. Voser, A. Wettstein, and H. Ziihner, Helv. Chim. Acta 43, 2118 (1960). ~ J. E. Philip, J. R. Scbenck, and M. P. Hargie, Antibiotics Annu. 1956/1957, 699 (1957). 1, G. G. F. Newton and E. P. Abraham, Nature (London) 175, 548 (1955). 20C. W. Hale, G. G. F. Newton, and E P. Abraham, Biochem. J. 79, 403 (1961). 5, B. Loder, G. G. F. Newton, and E. P. Abraham, Biochem. J. 79, 408 (1961). 2~G. G. F. Newton and E. P. Abraham, Biochem. J. 58, 103 (1954). ~ B. H. Olson, D. M. Schuurmans, M. W. Fisher, and S. A. Fusari, Nature (London) 176, 551 (1955). 54G. N. Atfield and C. J. O. R. Morris, Biochem. J. 81, 606 (1961).
282
METHODS FOR THE STUDY OF ANTIBIOTICS
[12]
ville, New York, is described as follows. A 2-~1 portion of a sample solution (1-5 mg/ml) is spotted on a strip (60 X 15 cm) of T o y o No. 51 filter paper on a transverse line 16 cm from one end. The paper strip is uniformly sprayed with an electrolyte solution of formic acid-acetic acid-water (25:75:900 in volume, pH 1.8). A constant current under 3300 V (about 40 mA/15 cm) is then applied to the paper strip for 15 min with cooling less than 20 ° by use of Isopal L (a light petroleum fraction, Esso-Standard Co.).
Detection and Results s After electrophoresis, the paper strip is dried under flowing warm dry air to remove the electrolyte solution. Antibiotics on the paper strip are detected by bioautography using test organisms, such as Staphylococcus aureus, Micrococcus flavus, Bacillus subtilis, or Escherichia coli. In some cases, color reactions and ultraviolet illumination are applied for detection of antibiotics. Under the same conditions, alanine moves toward the cathode about 10 cm. The relative mobilities (R~) of antibiotics to alanine are shown in Tables I and II. Most antibiotics assigned to group II (Table II) are adsorbed on cation-exchange resins. Interestingly, most antibiotics having an R~ value greater than 1.0 (including glebomycin) are efficiently adsorbed on weak cation exchange resins such as Amberlite IRC-50 and most antibiotics with R~ of less than 1.0 can be extracted with strong cation-exchange resins, such as Amberlite IR-120 (see "Ion-Exchange Chromatography of Aminoglycoside Antibiotics"25). The following methods of detections for antibiotics are employed frequently. Bioautography. A 120-ml portion of nutrient agar containing 1.0% (w/v) meat extract, 1.0% (w/v) peptone, 0.2% (w/v) sodium chloride, and 1.2% (w/v) agar in water (adjusted to pH 7.0) is poured at 60 ° and distributed over a sterilized glass plate with a stainless steel frame (46 X 16 cm). After solidification of the agar layer for 30 min, a second layer (60 ml) of nutrient agar seeded with a 0.5% (v/v) suspension of a test organism (Bacillus subtilis PCI 219: a suspension of about 50 million spores per milliliter; Staphylococcus aureus 209P and Escherichia coli NIHJ: an overnight culture in a nutrient medium at 37°; Micrococcus flavus: a overnight culture in a nutrient medium at 27 °) is poured at 40 ° on the agar layer. The paper strip is superimposed on the upper layer of the agar for 20 rain. After removal of the paper strip, the double :5This volume [11].
[12]
283
ELECTROPHORESIS OF ANTIBIOTICS
TABLE I RELATIVE MOBILITIES OF ANTIBIOTICSEXTRACTEDBY SOLVENT PROCESS (GRouP I) ~
Antibioticb
R,, ~
Detection
Spiramycins I, II, III Pluramycin A Bacitracin" Amicetin Lincomycin Pyridomycin Methymycin Mitomycin C Narbomycin Picromycin Tetracycline Oxytetracycline Oleandomycin Chlortetracyeline Tertiomycins A, B Erythromycin Carbomycin Leucomycins Josamycin ~ Tylosin Azomycin Noboviocin Coumermycin A, Trichomycin Pentamycin Nystatin Amphotericin B Actinomycin C Chromomycin A~
0.88 0.84 0.83 0.77 0.77 0.71 0.63 0.63 0.59 0.59 0.58 0.58 0.57 0.55 0.51 0.50 0.50 0.46 0.46 0.45 0.11 (} 0 0 0 0 0 0 0
Staphylococcus aureus S. aureus
Ninhydrin UV S. aureus
UV S. aureus
UV S. aureus S. aureus
UV UV S. aureus
UV S. S. S. S. S. S. M
aureus aureus aureus aureus aureus aureus icrococcus fiavus
UV UV UV UV UV UV UV UV
The data were cited from K. Maeda, A. Yagi, H. Naganawa, S. Kondo, and H. Umezawa, J . Antibiot. 22, 635 (1969). b Most antibiotics were described in H. Umezawa, "Index of Antibiotics from Actinomycetes," Univ. of Tokyo Press, Tokyo, 1967. c B. A. Johnson, H. S. Anker, and F. L. Meleney, Science 102, 376 (1945). T. Osono, Y. Oka, S. Watanabe, Y. Numazaki, K. Moriyama, H. Ishida, K. Suzaki, Y. Okami, and H. Umezawa, J . Antibiol. Ser. A 20, 174 (1967). e Relative mobility to alanine as 1.0. l a y e r of the agar m e d i u m is i n c u b a t e d a t 37 ° for 18 hr ( M i c r o c o c c u s at 2 7 - 3 0 ° ) . After i n c u b a t i o n , the positions of the a n t i b i o t i c s are detected b y a t r a n s p a r e n t i n h i b i t i o n zone. U l t r a v i o l e t L i g h t D e t e c t i o n . T h e p a p e r strip is i l l u m i n a t e d b y a u l t r a flavus
284
[12]
METHODS FOR T H E STUDY OF ANTIBIOTICS
TABLE II RELATIVE MOBILIT1ES Of ANTIBIOTICS EXTRACTED BY ADSORPTION PROCESS ( G R o u P II) a
Antibioticb Neamine 3',4'-Dideoxykanamycin B c Tobramycin d Gentamicin C complex Kanamycin B Neomycins B, C Lividomycin B e Noformicin Paromomycins I, II Paromamine Kanamycin A RibostamycinS Kanamycin C Butirosins A, Ba Lividomycin A • Capreomycins I, I I Streptothricin (A-249) Hygromycin B Negamycin h Phleomycins Destomycin A Blasticidin S Dihydrostreptomycin Streptomycin Destomycin B Cycloserine Hydroxystreptomycin Actinospectacin Cyclamidomycin~ (Desdanine) Viomycin Tuberactinomycinsi Alboverticillin Mannosidostreptomycin Gougerotin Polymyxin B Colistin Angustmycin B (adenine) SF-701 k BD-12~ BY-8V Kikumycin B Bleomycins 3-Amino-3-deoxy-D-glucose'~ Kikumycin A Nojirimycin Glebomycin Cordycepinn Tubercidin Aristeromycino
R~ ~ 2.05 1.95 1.93 1.91 1.89 1.87 1.85 1.82 1.82 1.76 1.74 1.72 1.70 1.63 1.58 1.54 1.50 1.50 1.50 0.69-1.50 ° 1.49 1.47 1.47' 1.45* 1.40 1.39 1.38" 1.38 1.37 1.37 1.35 1.33' 1.30 1.30 1.30 1.30 1.30 1.28 1.27 1.27 1.15 0.53-1.10' 1.09 1. O0 1.00
0.97 0.90 0.90 0.90
Detection Ninhydrin, Ninhydrin, Ninhydrin, Ninhydrin, Ninhydrin, Ninhydrin, Ninhydrin, Ninhydrin, Ninhydrin, Ninhydrin, Ninhydrin, Ninhydrin, Ninhydrin, Ninhydrin, Ninhydrin, Ninhydrin Ninhydrin Ninhydrin Ninhydrin
Rydon-Smith Rydon-Smith Rydon-Smith Rydon-Smith Rydon-Smith Rydon-Smith Rydon-Smith Rydon-Smith Rydon-Smith Rydon-Smith Rydon-Smith Rydon-Smith Rydon-Smith Rydon-Smith Rydon-Smith
Bacillus subtilis
Ninhydrin UV Sakaguchi Sakaguchi Ninhydrin Ninhydrin Sakaguchi Ninhydrin Rydon-Smith Ninhydrin Ninhydrin Ninhydrin B. subtilis, Sakaguchi UV Ninhydrin Ninhydrin UV Ninhydrin B. B. B. B.
subtilis subtilis subtilis, Eschcrichia coli subt ilis
Ninhydrin B. subtilis, E. coli S. aureus B. subtilis
UV UV UV
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285
ELECTROPHORESIS OF ANTIBIOTICS TABLE II (Conlinued) Antibioticb
R,, ~
Angustmycin A (decoyinin) Netropsin Actinobolin Angustmycin C (psicofuranine) Formycin Sangivamycin Toyocamycin Trehalosamine Kasugamycin Puromycin Ferrimycin A HON Danomycin Polyoxin A O-Carbamyl-D-serine Gramicidin SP Formycin B Oxoformycin B q
0.82 0.79 0.79 0.79 0.79 0.78 0.75 0.74 0.74 O. 72 0.71 0.58 0.49 0.45 0.43 0.37 0.04 0
Detection UV E. coli
UV UV UV UV UV Ninhydrin Ninhydrin UV B. subtilis
Ninhydrin Barton UV Ninhydrin Ninhydrin UV UV
a Most data were cited from K. Maeda, A. Yagi, H. Naganawa, S. Kondo, and H. Umezawa, J. Antibiot. 22, 635 (1969). b Most antibiotics were described in H. Umezawa, "Index of Antibiotics from Actinomycetes." Univ. of Tokyo Press, Tokyo, 1967. c H. Umezawa, S. Umezawa, T. Tsuchiya, and Y. Okazaki, J. Antibiot. 24, 485 (1971). d K. F. Koch and J. A. Rhoades, Antimicrob. Ag. Chemother. 1970, 309 (1971). e T. Mori, T. Ichiyanagi, H. Kondo, K. Tokunaga, T. Oda, and K. Munakata, J. Antibiot. 24, 339 (1971). / E. Akita, T. Tsuruoka, N. Ezaki, and T. Niida, J. Antibiot. 23, 175 (1970). g P. W. K. Woo, H. W. Dion, and Q. R. Bartz, Tetrahedron Lett., p. 2125 (1971). h S. Kondo, S. Shibahara, S. Takahashi, K. Maeda, H. Umezawa, and M. Ohno, J. Amer. Chem. Soc. 93, 6305 (1971). iS. Takahashi, M. Nakajima, Y. Ikeda, S. Kondo, M. Hamada, K. Maeda, and H. Umezawa, J. Antibiot. 24, 902 (1971). J H. Yoshioka, T. Aoki, H. Goko, K. Nakatsu, T. Noda, H. Sakakibara, T. Take, A. Nagata, J. Abe, T. Wakamiya, T. Shiba, and T. Kaneko, Tetrahedron Lea.. p. 2043 (1971). k T. Tsuruoka, T. Shomura, N. Ezaki, T. Niwa, and T. Niida, J. Antibiot. 21, 237 (1968). Y. Ito, Y. Ohashi, Y. Sakurai, M. Sakurazawa, H. Yoshida, S. Awataguchi, and T. Okuda, J. Antibiot. 21, 307 (1968). " S. Umezawa, K. Umino, S. Shibahara, M. Hamada, and S. Omoto, J. Antibiot. Set. A 20, 355 (1967). H. R. Bentley, K. G. Cunningham, and F. S. Spring, J. Chem. Soc. (London), p. 2301 (1951). ° T. Kishi, M. Muroi, T. Kusaka, M. Nishikawa, and K. Mizuno, Chem. Commun., 852 (1967). p R. L. M. Synge, Biochem. J. 99, 363 (1945). q M. Ishizuka, T. Sawa, G. Koyama, T. Takeuchi, and H. Umezawa, J. Antibiot. 21, 1 (1968). TRelative mobility to alanine as 1.0. Tailing.
286
METHODS FOR THE STUDY OF ANTIBIOTICS
[12]
violet lamp (254 nm), and the positions of antibiotics are detected as absorption zones or fluorescent zones. Ninhydrin Reagent ]or Amines. The paper strip is uniformly sprayed with a 0.2% (w/v) solution of ninhydrin in a mixture of acetone and pyridine (20:1 in volume). It is placed in a dry oven at 105 ° for 5-10 rain. Barton Reagent 28/or Reducing Properties. The paper strip is sprayed with the reagent solution prepared just before use by mixing equal volumes of a 1% (w/v) aqueous potassium ferricyanide solution and a 2% (w/v) aqueous ferric chloride solution. The colors are intensified by subsequent spraying with 2 N hydrochloric acid. Rydon-Smith Reagent 27,2s ]or Amides. The paper strip is sprayed with 0.5% (w/v) aqueous sodium hypochlorite. When dry, it is sprayed with 95% ethyl alcohol. Finally, after the ethyl alcohol has evaporated, it is sprayed with starch-iodide reagent prepared just before use by mixing equal volumes of a 1% (w/v) soluble starch solution and a 1% (w/v) potassium iodide solution.
III. Thin-Layer Electrophoresis
The techniques of thin-layer electrophoresis is now easily carried out by the use of commercially available electrophoretic apparatus with solid heat exchangers. Especially, the use of cellulose powder plate gives a good rapid separation, similar to high-voltage paper electrophoresis. Fujii et al. 29 applied this technique to separation of bleomycins A2, A~, and demethyl A~,3° as described below.
Conditions o/Electrophoresis 2~') An example using a commercial apparatus with solid heat exchanger, Model FP-18 with HV-1,000B of Savant Instruments, Inc., Hicksville, New York, is described as follows. Each 5 t~g of bleomycins A2, A~, and demethyl A2 in aqueous solution is spotted on a Avicel SF glass plate (10 X 5 cm, a cellulose plate for thin-layer chromatography, Funakoshi Yakuhin Co., Tokyo) on a origin line 1.5 cm from one end. The thin~ G. M. Barton, R. S. Evans, and J. A. F. Gardner, Nature (London) 170, 249 (1952). 2~H. N. Rydon and P. W. G. Smith, Nature (London) 169, 922 (1952). S. C. Pan and J. D. Dutcher, Anal. Chem. 28, 836 (1956). A. Fuiii, Y. Muraoka, T. Takita, and I-I. Umezawa, unpublished data, 1973. a°A. Fujii, T. Takita, K. Maeda, and H. Umezawa, J. Antibiot. 26, 396 (1973); 26, 398 (1973).
[12l
ELECTROPHORESIS OF ANTIBIOTICS
287
layer plate is uniformly sprayed with an electrolyte solution of formic acid-acetic acid-water (25:75:900 in volume, pH 1.8). The wet thin-layer plate is placed on an aluminum cooling plate of the apparatus and is connected to the electrode vessels with a filter paper soaked in the electrolyte solution. Electrophoresis is carried out under 810 V, 4 mA for 10 min. Detection and Results ~9
After the electrophoresis, the thin-layer plate is dried under flowing warm dry air to remove the electrolyte solution. Bleomycins on the plate are detected by ultraviolet light (254 nm), and the alanine used as the standard is detected by ninhydrin reagent. Under this condition, alanine moves toward the cathode 2.8 cm. The mobilities of bleomycins relative to alanine are shown as follows: A~, 1.02; A.,, 0.86; and demethyl A2, 0.63.
IV. Agar Gel ]~lectrophoresis In 1965, Lightbown and de Ross[ ~1 reported in detail a unique electrophoretic method in agar gel. This method permitted separation, identification, and quantitative determination of many antibiotics. The antibi•otics studied were classified into 11 groups and it was possible to separate antibiotics belonging to different groups, but not those within a group. The groups and relative mobilities of some antibiotics are shown in Table III. Preparation o] Bu]]ered Agar Gel Slab 31
A 100-ml portion of a buffer solution consisting of 75 mM tris(hydroxymethyl)aminomethane, 75 mM maleic acid, and 23 mM sodium hydroxide (final pH 5.60) is mixed with an equal volume of a 2% (w/v) solution of agar (Difco Noble Agar) in water, previously liquefied by heating at 100°. The buffered agar is poured at 80 ° and distributed over a leveled plate glass with a stainless steel frame (internal dimensions, 91.5 X 17 cm; previously sealed to the glass sheet by a small amount of buffered agar) to produce a layer of uniform depth (1.27 ram). Electrophoresis in Agar Gel 3l
Small volumes (2-20 t~l) of solutions of antibiotics (0.125-50 ~g) in water are applied to small holes, approximately 1 mm in diameter, pre21j . W. Lightbown and P. de Rossi, Analyst 90, 89 (1965).
288
METHODS FOR THE STUDY OF ANTIBIOTICS
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TABLE I I I RELATIVE MOBILITIES OF ANTIBIOTICS ON AGAR GEL ELECTROPHORESIS~ Group I
II III IV V
Antibiotic
R,, b
Streptomycin Dihydrostreptomy cin Viomycin Spiramy cin Erythromycin Oleandomycin Oleandomycin Triacetyloleandomycin Ristocetin Bacitracin Streptozotocin Vancomycin
- 3.6 - 3.7 ~ - 4.0 - 3.3 - 3.2 -3.2 - 3.2 -2.2 - 2.5 - 1.7 - 1.7
Group VI
VII VIII IX X XI
Antibiotic
R,~b
Ampicillin Tetracycline Oxytetracycline Chlortetracy cline Rifamycin S Polymyxin Gramicidin Novobiocin Neomycin B Paromomycin Cephalosporin C Penicillin G Cloxacillin
- 1.4 - 1.3
- 1.0 _+0 ~ - 0.1 c -k-0.1 +0.9 ~ + 1.0 c + 1.2 + 1.1 + 1.3
a j. W. Lightbown and P. de Rossi, Analyst 90, 89 (1965). Eleetrophoresis in the buffered agar gel (pH 5.60) was carried out under 2000 V, 120 mA for 3 hr by circulating a coolant ( - 4 ° ) . Relative mobilities (R,~) to rifamycin S were calculated from figures of electropherograms shown b y J. W. Lightbown and P. de Rossi. ~ When rifamycin B or SV was applied, most of the antibiotic was converted to rifamycin S during the electrophoresis. -k, migration toward the anode; - , toward the cathode from origin. Tailing. v i o u s l y p u n c h e d in t h e a g a r b y a glass c a p i l l a r y p i p e t t e . T h e a g a r gel s l a b is p l a c e d on a a l u m i n u m p l a t e in t h e e l e c t r o p h o r e s i s a p p a r a t u s . T h e s l a b is c o n n e c t e d to t h e e l e c t r o d e vessels c o n t a i n i n g a n e l e c t r o l y t e solut i o n ( p r e p a r e d b y a n e q u a l v o l u m e of d i l u t i o n of t h e a b o v e - m e n t i o n e d buffer w i t h w a t e r ) w i t h a d o u b l e t h i c k n e s s of A b s o r b e n t L i n t B. P. C. filter p a p e r s o a k e d in t h e e l e c t r o l y t e solution. E l e c t r o p h o r e s i s is c a r r i e d o u t u n d e r 2000 V for 3 h r w i t h cooling a t - - 4 ° b y c i r c u l a t i n g a 2 0 % ( w / v ) e t h y l e n e g l y c o l a q u e o u s solution.
Detection ~1 A second l a y e r (400 ml) of a n u t r i e n t a g a r c o n t a i n i n g 0.6% ( w / v ) p e p t o n e ( E v a n s M e d i c a l L t d . ) , 0.15% ( w / v ) beef e x t r a c t ( L e m c o ; Oxoid, Ox L t d . ) , 0.3% ( w / v ) y e a s t e x t r a c t ( Y e a s t r e l ; B r e w e r s F o o d S u p p l y Co. L t d . ) a n d 1.0% ( w / v ) a g a r ( D a v i d G e l a t i n e Co., N e w Z e a l a n d ) in w a t e r ( p H 7.9 ~ 0.1 a f t e r s t e r i l i z a t i o n ) is seeded w i t h 2 % ( w / v ) of a s u s p e n s i o n of a t e s t o r g a n i s m (Bacillus subtilis N C T C 8241; 2 0 - 3 0
[12]
ELECTROPHORESIS OF ANTIBIOTICS
289
million spores per milliliter) and is poured at 70 ° on another sheet of the leveled plate glass with the stainless steel frame. When the agar layer solidifies for 5 min, it is superimposed on top of the buffered agar gel slab by gently sliding it from the plate glass. The double layer of agar is incubated at 39 ° for 18 hr and the positions of the antibiotics are evident as zones of inhibition. By the measurement of the diameters, the antibiotics can be assayed quantitatively.
V. Carrier-Free Continuous Electrophoresis By combination of a horizontal electric field with a vertically flowing electrolyte, continuous separations may be accomplished. In the continuous electrophoretic method, a sample is continuously added slowly to the electrolyte and separated into its various components. By applying an electrical potential at right angles to the direction of electrolyte flow, the ionized components are caused to deviate from the main course by an amount proportional to the charge density of the molecule. Usually, a vertically suspended sheet of filter paper is irrigated from top to bottom with an electrolyte. Another possibility for the separation of large amounts of material is continuous-flow electrophoresis without employing filter paper as carrier material. In 1964, this method was first described by Hannig, 32 and an apparatus of this type is commercially available from Bender and Hobein G. m. b. H., Munich. In Elphor VaP2-apparatus, the sample to be separated is continuously supplied to an electrolyte film flowing constantly between two cooled vertical glass plates (distance; 0.5 mm) through one of nine feed boreholes on the top edge of the separation chamber (48 X 48 cm). The effluent electrolyte solution through 50 outlet boreholes at the bottom of the separation chamber is collected into 100-ml glass tubes. In high-molecular substances, such as enzymes, the application of carrier-free continuous electrophoresis gives a good separation but has not been applied to antibiotics except for a trial to separate kanamycin and 6'-N-t-butyloxycarbonylkanamycin.33 In this case a good separation was obtained as shown in Fig. 1. This derivative of kanamycin is a useful intermediate for chemical derivation of new antibiotics, a~ A mixture of kanamycin ( l l rag) and 6 ' - N - t - b u t y l o x y c a r b o n y l k a n a mycin (10 rag) in 5 ml of 0.5 M acetic acid is continuously supplied ~ K. Hannig, Hoppe-Seyler's Z. Physiol. Chem. 338, 211 (1964). ~ T. Hara, S. :Kondo,and H. Umezawa,unpublished data, 1973. a4S. Kondo, K. Iinuma, M. Hamada, K. Maeda, and H. Umezawa, J. Antibiot. 27, 90 (1974).
290
[12]
METHODS FOR T H E STUDY OF ANTIBIOTICS
200
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Tube number Fio. I. Carrier-freecontinuouselectrophoresisof kanamycin and 6"-N-t-butylo~earbonylkanamycin (6'-N-BOC-kanamycin) by Elphor VaP2-apparatus (Bender and Hobein, G.m.b.H., Munich). A mixture of kanamycin (11 rag) and 6'-N-BOC-kanamycin (10 mg) in 5 ml of 0.5 M acetic acid was charged for 140 min. Electrolyte solution, 0.5 M acetic acid, flowed at the rate of 2 ml/min. Electrophoresis was operated under 1800 V, 70 mA for 210 min at 10.5°. Kanamycin was assayed by disk plate method using Bacillus subtilis PCI 219 as a test organism. 6'-N-BOC-kanamycin was determined by the assay of the regenerated kanamycin on heating at 100° for 1 hr. Kanamycin and 6'-N-BOC-kanamycin in tubes were detected by relative mobility (R~) to alanine on high-voltage paper electrophoresis (ttVPE) under 3000 V (2.9 mA/cm) for 9 min. They were colored by ninhydrin and Rydon-Smith reagents. (T. Hara, S. Kondo, and H. Umezawa, unpublished results.) through the first feed borehole from the left by use of a small proportioning p u m p operating on a flexible tube for 140 min. An electrolyte (0.5 M acetic acid) film constantly flows from the top of the separation chamber at the rate of 2 m l / m i n by means of multiple flexible-tube type pumps. The electrophoresis (anode, the left side; cathode, the right) is operated under 1800 V, 70 mA for 210 min at 10.5 °. The effluent electrolyte solution is collected into 50 fraction tubes (approximately 8 ml each). E a c h fraction obtained b y the electrophoresis is applied to highvoltage p a p e r electrophoresis, and k a n a m y c i n and 6"-N-t-butyloxycarb o n y l k a n a m y c i n are detected by ninhydrin reaction (see the preceding section), as shown in Fig. 1. K a n a m y c i n in the fractions is assayed by disc plate method using Bacillus subtilis P C I 219 as a test organism. The fractions containing 6 ' - N - t - b u t y l o x y c a r b o n y l k a n a m y c i n are heated at 100 ° for 1 hr. The k a n a m y c i n yielded by the hydrolysis of 6"-N-tb u t y l o x y c a r b o n y l k a n a m y c i n is assayed by the method described above.
[13]
SILICA GEL CHROMATOGRAPHY OF ANTIBIOTICS
291
[13] Silica Gel Chromatography of Antibiotics By
I. II. IlI. IV. V. VI. VII.
GARY G. MARCONI
Introduction . . . . . . The Adsorbent . . . . . Selecting the Solvent System Preparing the Column . . . Applying the Sample . . . . Colurnn Elution . . . . . Monitoring the Column . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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291 291 292 293 295 295 296
I. Introduction Silica gel column c h r o m a t o g r a p h y is one of the most commonly used methods for the purification of antibiotics. Adsorption onto silica gel is governed by v a n der Waals forces and hydrogen bonding. Adsorbed solutes are eluted with organic solvents of various polarities and the competition between the solvent and adsorbent for the solute determines the rate of migration through the column. Silica gel is most effectively employed with antibiotics t h a t are soluble in organic solvents (e.g., monensin) and is less applicable to water-soluble antibiotics, such as aminoglycosides. Antibiotics comprise a wide v a r i e t y of "structural types," and as a result they exhibit vastly different physical and chemical properties. This diversity makes it impossible to describe a single system for the purification of antibiotics. This report is intended to outline general principles t h a t can be applied to the selection of a solvent system and to illustrate some basic methodology which one can use to optimize solute resolution, More detailed information on adsorption chromatography and the use of silica gel can be obtained from the books by Bobbitt e t a l . , 1 Mikes,'-' Snyder, 3 and W a g m a n and Weinstein. ~ II. T h e Adsorbent Silica gel (SiO._,-XH~O) is a polar, acidic adsorbent t h a t binds polar solutes more strongly t h a n nonpolar solutes and binds basic compounds ' J . M. Bobbitt, A. E. Schwarting, and R. J. Gritter, "Introduction to Chromatography." Van Nostrand-Reinhold, Princeton, New Jersey, 1968. 0. Mik6s, "Laboratory Handbook of Chromatographic Methods." Van NostrandReinhold, Princeton, New Jersey, 1970. 3L. R. Snyder, "Principles of Adsorption Chromatography." Dekker, New York, 1968. * G. H. Wagman and M. J. Weinstein, "Chromatography of Antibiotics." Elsevier, Amsterdam, 1973.
292
METHODS FOR THE STUDY OF ANTIBIOTICS
[13]
TABLE I SOURCES OF SILICA GEL FOR ADSORPTION CHROMATOGRAPHY
Brand
Particle size
Distributor
Fisher Mallinckrodt Merck ( D a r m s t a d t , G e r m a n y )
28-200 mesh 100-200 mesh 28-200 mesh 100 mesh < 0.08 mm 0.02-0.05 mm 0.2-0.5 mm
W . R . Grace & Co. Davison Chemical Division Fisher Mallinckrodt Chemical Works Brinkmann
Davison
Woelm
Alupharm
more strongly than those that are neutral or acidic. The activity of silica gel can be modified by heating it at 150-200 ° for several hours. This treatment removes surface water, thereby increasing the binding strength of the adsorbent. The silica gel should not be overheated because at higher temperatures the surface hydroxyl groups condense to form water, leaving relatively inactive siloxane groups. For most applications, silica gel containing 10-20% water can be used. This is the form in which most commercially available material is supplied. Several suppliers are listed in Table I.
III. Selecting the Solvent System A nonpolar solvent such as cyclohexane will move a solute of moderate polarity through a column slowly or not at all while a polar solvent, such as ethanol, will rapidly move all but the most polar solutes. Organic solvents can be arranged in order of increasing (or decreasing) eluting strength. This ranking, which is known as the eluotropic series, is reproduced in Table II. Polarities other than those of members of the series can be obtained by preparing a mixture of two solvents. A solvent system for silica gel column chromatography can be selected by determining the mobility of the antibiotic on silica gel thin-layer chromatography (TLC) plates using various solvent systems. The transfer from TLC to column will be more successful if the adsorbents for both techniques come from the same manufacturer. To be useful in a column separation, a solvent system must be found in which the majority of components, including the antibiotic, have an R~ value on TLC of less than 0.4. In general, solutes that move with an RI greater than 0.5 will move with the solvent front on a column. A series of TLC plates can be developed in solvents of different polarities, such as cyclohexane, benzene, chloroform, ethyl acetate, acetone, and ethanol. In most cases the antibiotic will move with a high RI value in some systems, and not at all
[13]
SILICA GEL CHROMATOGRAPHY OF ANTIBIOTICS
293
TABLE II ELUOTROPIC SERIES a'b
Cyclohexane Carbon tetrachloride Trichloroethylene Toluene Benzene Dichloromethane Chloroform Ethyl ether Ethyl acetate Acetone n-Propanol Ethanol Methanol Water W. Trappe, Biochem. Z. 305, 150 (1940). bListed in order of increasing eluting power. in others. One can then attempt to use mixtures of the polar and nonpolar solvents to obtain the desired Rs value. Care must be exercised in mixing solvents because the polarity of the solvent system is not a simple linear function of solvent concentration. For example, the addition of a small amount of methanol (1-2%) to chloroform increases polarity greatly; whereas, an increase from 40% methanol in chloroform to 50% changes the polarity very little. Small changes in solvent concentration are not critical when the components of the solvent system are close together in the eluotropic series. Given a choice between two solvent mixtures of similar polarity, such as benzene/ethyl acetate (50:50) and ether/methanol (98:2), the former is usually preferred because small errors in preparing the mixture do not significantly alter the polarity. Neher 5 prepared a diagram that equates the polarities of different solvent mixtures (Fig. 1). The diagram can be used to prepare solvent systems that contain different solvents but exhibit similar polarity. Solvent systems of similar polarities may differ in their resolving power even though they move solutes at about the same rate. Thus, Fig. 1 can be used to vary solute resolution without altering the RI value on TLC or the rate of migration through a column. IV. Preparing the Column
A silica gel column can be prepared by partially filling the empty column with solvent and adding the adsorbent continuously as a thin R. Neher, "Steroid Chromatography." American Elsevier, New York, 1964.
294
METHODS FOR THE STUDY OF ANTIBIOTICS
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;100 I
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100
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90:10 5 0 : 5 0
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II
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I
90:10
,
80:20
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50:50 100 I
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90:10
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100 I. BuAc OR ETHER
I
I
50:50
100
I
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90:!0
.
80:20 1100
I 90:10 '
I
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50:50
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100
,,
I
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90:10 ll 100
An
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80:2o I
90:10
,
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x 90:10 Fro. 1. Solvents and solvent mixtures arranged in an "equi-eluotropic" series. A vertical line will connect solvent mixtures having approximately equal eluting power. Pure methanol is off the chart. Cy, cyclohexane; Be, benzene; BuAc, butyl acetate; EtAc, ethyl acetate; An, acetone. Reproduced through the courtesy of Elsevier Publishing Co. from R. Neher, "Steroid Chromatography," American Elsevier, New York, 1964.
[13]
SILICA GEL CHROMATOGRAPHY OF ANTIBIOTICS
295
slurry in the same solvent. The stopcock is then opened to allow the silica gel to settle, after which the bed is washed with 2-3 column volumes of solvent. An alternative method is to fill the column with solvent, and then add dry silica gel continuously to avoid layering. The stopcock is opened, and the bed is allowed to settle. After packing is completed, the column is washed with several column volumes of solvent and a small amount of sand or glass wool is placed on top of the adsorbent bed so that subsequent operations will not disturb it. The greater the length-to-diameter ratio of a column, the greater the resolution. A length-to-diameter ratio of at least 10 is desirable. Difficult separations require longer columns, and a length-to-diameter ratio of 100 is not unusual. The amount of adsorbent used is determined by the weight of sample applied. Although the amount of sample applied to a given amount of silica gel varies with the sample composition, 25 mg of sample per gram of silica gel may be used as a guideline. V. Applying the Sample
The antibiotic samples that are purified on silica gel are usually concentrated organic extracts of fermentation broth filtrates or extracts of the mycelia. It is desirable to partially purify an antibiotic extract prior to silica gel chromatography. Partial purification may be achieved by methods such as back-extraction and/or precipitation of the active components. The sample to be applied is dissolved in a small volume of the eluting solvent. The amount of solvent used should be less than 10% of the column bed volume. The solution should be carefully applied to the column in small portions with the stopcock open to permit the sample to enter the adsorbent bed. Often the sample to be applied to a column is insoluble in the eluting solvent. These samples can be applied after they are adsorbed onto dry silica gel. A sample is adsorbed by dissolving it in a solvent in which it is soluble and then adding dry silica gel to the resultant solution. Enough silica gel is used so that no free liquid remains. The silica is dried under vacuum and added directly to the top of the column bed. VI. Column Elution
A column may be eluted with a single solvent system or by making stepwise changes in the polarity of the solvent mixture. A single solvent system has the advantages of simplicity of operation and reproducibility
296
METHODS FOR THE STUDY OF ANTIBIOTICS
[14]
of results; however, this method is more time consuming than gradient elution and m a y lead to less solute resolution due to tailing. Stepwise gradient elution is initiated with a less polar solvent and, as elution progresses, the polarity of the solvent system is increased by the stepwise addition of small amounts of a more polar solvent. For example, elution can begin with chloroform, and after several column volumes have been used, the eluting solvent can be changed to chloroform containing 1% methanol. After several additional column volumes, the solvent can be changed to chloroform containing 2% methanol, and so on. A useful sequence for the addition of the more polar solvent is 1%, 2%, 5%, 10%, 25%, and 50%. The solvents used for the gradient are selected using T L C as described earlier. If benzene/ethyl acetate (75:25) is selected, elution could begin with several column volumes of benzene followed by similar amounts of benzene with increasing amounts of ethyl acetate. VII. Monitoring the Column The eluates from a column are collected manually or by an automatic fraction collector. Solute elution can be monitored by conventional techniques, such as color reactions, UV, and refractive index. Antibiotic activity is easily detected by dipping paper pads in each fraction, air-drying them, and then placing the pads on agar plates seeded with an organism sensitive to the antibiotic. The size of the zone of inhibition is proportional to antibiotic concentration in the fraction. The separation can also be monitored by the use of paper or thin-layer bioautograms.
[14] M a c r o r e t i c u l a r
Resin Chromatography
By
of Antibiotics
SEAN C. O'CONNOR
I. Introduction . . . . . . . . . . . . . . . . . . II. Macroreticular Resins . . . . . . . . . . . . . . . . A. Choice of Conditions for Optimum Adsorption . . . . . . . B. Desorption of Compounds from the Resin . . . . . . . . . C. Clean-up of New Resin Prior to Use . . . . . . . . . . D. Regeneration of Resin . . . . . . . . . . . . . . E. Rehydration of Resin . . . . . . . . . . . . . . . III. Isolation of Cephalosporin C from Fermentation Broth . . . . . .
296 297 298 298 298 298 299 299
I. Introduction Ion-exchange resins have been widely used in the isolation and purification of antibiotics. Their major use has been with those compounds
296
METHODS FOR THE STUDY OF ANTIBIOTICS
[14]
of results; however, this method is more time consuming than gradient elution and m a y lead to less solute resolution due to tailing. Stepwise gradient elution is initiated with a less polar solvent and, as elution progresses, the polarity of the solvent system is increased by the stepwise addition of small amounts of a more polar solvent. For example, elution can begin with chloroform, and after several column volumes have been used, the eluting solvent can be changed to chloroform containing 1% methanol. After several additional column volumes, the solvent can be changed to chloroform containing 2% methanol, and so on. A useful sequence for the addition of the more polar solvent is 1%, 2%, 5%, 10%, 25%, and 50%. The solvents used for the gradient are selected using T L C as described earlier. If benzene/ethyl acetate (75:25) is selected, elution could begin with several column volumes of benzene followed by similar amounts of benzene with increasing amounts of ethyl acetate. VII. Monitoring the Column The eluates from a column are collected manually or by an automatic fraction collector. Solute elution can be monitored by conventional techniques, such as color reactions, UV, and refractive index. Antibiotic activity is easily detected by dipping paper pads in each fraction, air-drying them, and then placing the pads on agar plates seeded with an organism sensitive to the antibiotic. The size of the zone of inhibition is proportional to antibiotic concentration in the fraction. The separation can also be monitored by the use of paper or thin-layer bioautograms.
[14] M a c r o r e t i c u l a r
Resin Chromatography
By
of Antibiotics
SEAN C. O'CONNOR
I. Introduction . . . . . . . . . . . . . . . . . . II. Macroreticular Resins . . . . . . . . . . . . . . . . A. Choice of Conditions for Optimum Adsorption . . . . . . . B. Desorption of Compounds from the Resin . . . . . . . . . C. Clean-up of New Resin Prior to Use . . . . . . . . . . D. Regeneration of Resin . . . . . . . . . . . . . . E. Rehydration of Resin . . . . . . . . . . . . . . . III. Isolation of Cephalosporin C from Fermentation Broth . . . . . .
296 297 298 298 298 298 299 299
I. Introduction Ion-exchange resins have been widely used in the isolation and purification of antibiotics. Their major use has been with those compounds
[14]
297
MACRORETICULAR R E S I N CHROMATOGRAPHY OF ANTIBIOTICS
commonly regarded as nonextractable with organic solvents, although this restriction is more a matter of convenience than of applicability. Most ion-exchange resins in use today are of the gel type. In recent years, however, resins of another type have become available in a number of forms. These are the macroreticular (macroporous) resins. We are concerned here with the use of one of these in the isolation of both extractable and nonextractable antibiotics from fermentation broth. The resin in question is an un]unctionalized divinylbenzene-cross-linked polystyrene marketed as XAD-2 by Rohm and Haas Co. l II. Macroreticular Resins This class of resins is characterized by a well-developed system of pores, a large surface area, and a high degree of cross-linking. This degree of cross-linking leads to outstanding abrasion-resistance and mechanical strength. Handling difficulties due to swelling are almost nonexistent with this kind of resin. Typical properties of XAD-2 as determined by the manufacturer are given in the table. 2 The large surface area confers on the resin some of the absorption characteristics of activated carbon. The purely hydrocarbon skeleton favors hydrophobic bonding, and the porous structure is potentially capable of discrimination based on molecular size. We have not found a clearcut example of this latter behavior. The manufacturer claims, however, that a comparison of XAD-2 with the very similar X A D - 4 reveals that the former has the higher capacity for alkylbenzene sulfonates while the latter shows the higher capacity for phenol. 2 I t is suggested that this is a reflection of the difference in average pore size between the resins (90 X 10 -s cm for XAD-2, 50 X 10-s cm for XAD-4). This may not be the whole explanation since the resins also have different surface areas. PROPERTIES OF X A D - 2 RESIN
Chemical n a t u r e
Helium porosity (vol. %)
Surface area (m 2 g-i)
Average pore diameter (cm X 10-s)
Polystyrene-divinylbenzene
40
300
90
' See M. L. Bastos, D. Jukofsky, E. Saffer, M. Chedekel, and S. J. Mul~, J. Chromatog. 71, 549 (1972) for a comparison of XAD~2 with a number of other unfunctionalized resins Richard M. Simpson, "The Separation of Organic Chemicals from Water" presented at the Third Symposium of the Institute of Advanced Sanitation Research, April 1972.
298
METHODS FOR THE STUDY OF ANTIBIOTICS
[14]
A. Choice of Conditions for Optimum Adsorption
Since the major factor governing adsorption to the resin appears to be hydrophobic bonding, the simplest means of maximizing adsorption is to suppress ionization in the compound to be adsorbed. Thus, organic acids are most efficiently recovered from water at low pH whereas amines show their highest affinities for the resin at high pH. As an example of the above phenomenon, consider cephalosporin C which contains two carboxyl groups and one amino .group. The lowest pKa lies between 2 and 3. By adjusting the pH of a cephalosporin C solution to 2.5, the ionization of all but one of these groups, the amine, is suppressed. In agreement with this, it appears that the capacity of the resin for this compound is greater at pH 2.5 than at 7.0. Another factor influencing uptake of compounds is the salt concentration of the medium. The affinity of many compounds for the resin is increased in the presence of inorganic salt. This behavior appears to be strictly analogous to the commonly used "salting-out" technique in twophase liquid extractions. As is the case with other procedures where either batch or column operation is possible, the efficiency is higher using column procedures. B. Desorption of Compounds from the Resin
Most compounds are removed from the resin by methanol. A convenient method is to elute the resin in a column with water containing increasing amounts of the organic solvent. Solvents other than methanol, such as acetone or acetonitrile, can be used, but it appears to be more difficult to remove the last traces of these solvents from the resin during regeneration. C. Clean-up of New Resin prior to Use
As supplied by the manufacturer, the resin is only marginally useful. It must first be stripped of solvent and of low molecular weight polymer. This may be accomplished by washing the resin with water, then with methanol (2 bed volumes X 3), and finally with water to remove all the methanol. We have found it advantageous to degas the resin prior to packing it into a column. Holding the resin, in two volumes of water, under a vacuum of 20-50 mm Hg for 15 min is adequate for degassing. D. Regeneration of Resin s
After elution of the material of interest from the column, there may remain a band of color on the resin. This and other less visible contamis D. N. Osborne and B. H. Gore, J. Chromatogr. 77, 233 (1973).
[14]
MACRORETICULAR RESIN CHROMATOGRAPHY OF ANTIBIOTICS
299
nants are removed during regeneration. It is recommended that some such procedure always be used between runs: (a) 3-4 bed volumes of 1% cone. hydrochloric acid in methanol; (b) 3-4 bed volumes of acetone--methanol-water 10:45:45 (v/v) ; (c) 10 bed volumes of water. The resin should then be reclassified by either repacking or backflushing. E. Rehydration of Resin Resin which has become dried out cannot be directly hydrated with water. Dehydrated resin is suspended in methanol; after 15 min, the methanol is replaced with water either by packing in a column and flushing with water or by dilution with water followed by careful decantation. III. Isolation of Cephalosporin C from Fermentation Broth Cephalosporin C is a nonextractable antibiotic which is usually recovered from the fermentation broth by the use of ion-exchange resins. The following procedure is fast and yields material of greater than 50% purity. Filtered broth, i liter, containing 4 mg of eephalosporin C per milliliter was adjusted to pH 2.5 and passed, at 10 ml/min, through a column containing 250 ml of XAD-2 resin. The column was then washed with 250 ml of water and eluted with 500 ml of water-methanol 3:1 (v/). Reduction of the eluate to dryness yielded 5.3 g of residue containing, by UV, 80% of the cephalosporin C present in the original broth. The material was approximately 60% pure. (It may be further purified by conversion to the zinc salt as outlined in British Patent 1,928,142 assigned to Ciba, A. G.) In our experience any compound extractable from aqueous solution with an organic solvent may be adsorbed from water onto XAD-2 under appropriate conditions. Thus, the patent literature reveals that lincomycin may be recovered from whole broth using XAD-2 resin, from which it is eluted with 97% MEK. 4 Since the completion of this chapter many further applications of the type cited above have appeared in the scientific and patent literature. Particularly worthy of note are a separation of polymixin factors, 5 a separation of the spiramycins and leucomycins,~ and a very elegant separation of two enduracidinsJ 4 U.S. P a t e n t 3,515,717 (1970).
Japanese Patent 7329151 (1973). 'Japanese Patent 7376880 (1973). 7M. Hori. Chem. Pharm. Bull. 21, 1171 (1973).
300
METHODS FOR THE STUDY OF ANTIBIOTICS
[15]
[15] High-Pressure Liquid Chromatography of Antibiotics By
KIYOSHI T s u J I
I. Introduction . . . . . . . . . . . . . . II. Aromatic Antibiotics with Nitrogen . . . . . . A. Novobiocin . . . . . . . . . . . . . B. Rifampin . . . . . . . . . . . . . . C. Virginiamycin . . . . . . . . . . . . III. Clindamycin Phosphate . . . . . . . . . . . IV. Griseofulvin . . . . . . . . . . . . . . V. Kanamycin . . . . . . . . . . . . . . VI. Macrolide Antibiotics . . . . . . . . . . . A. Erythromycin . . . . . . . . . . . . B. Leucomycin . . . . . . . . . . . . . VII. Penicillins . . . . . . . . . . . . . . . A. Ampicillin . . . . . . . . . . . . . B. Cephalosporin C . . . . . . . . . . . . C. Penicillin G . . . . . . . . . . . . . D. Penicillin V . . . . . . . . . . . . . VIII. Polypeptide Antibiotics . . . . . . . . . . . A. Actinomycin . . . . . . . . . . . . . B. Bacitracin . . . . . . . . . . . . . IX. Tetracyclines and Related Antibiotics . . . . . A. Tetracycline . . . . . . . . . . . . . B. Oxytetracycline . . . . . . . . . . . . C. Daunomycin . . . . . . . . . . . . . X. Summary of HPLC Methods for Antibiotic Analysis
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
300 301 301 304 305 306 307 307 308 308 309 309 309 311 312 313 313 313 314 315 315 318 318 319
I. Introduction G a s - l i q u i d c h r o m a t o g r a p h y ( G L C ) has been blessed w i t h f a s t speed of a n a l y s i s , good precision, r e p r o d u c i b i l i t y , a n d s p e c i f i c i t y a t m o d e s t ins t r u m e n t a t i o n costs. I n c o n t r a s t , l i q u i d c h r o m a t o g r a p h y ( L C ) has t r a d i t i o n a l l y been a slow t e c h n i q u e r e q u i r i n g hours a n d even d a y s to c o m p l e t e an analysis. W i t h i n t h e l a s t five y e a r s , however, L C h a s been d e v e l o p e d into an a n a l y t i c a l tool w i t h h i g h l y sensitive, low d e a d v o l u m e d e t e c t o r s a n d now r i v a l s G L C in speed of a n a l y s i s a n d c o l u m n efficiency. T h e m o d e r n L C uses a s m a l l - b o r e column, a b o u t 2 m m i.d., p a c k e d w i t h s m a l l - d i a m e t e r c o l u m n p a c k i n g p a r t i c l e s , 30 ~m or less, to increase c o l u m n efficiency. T h i s r e q u i r e s high p r e s s u r e in o r d e r to o b t a i n a d e q u a t e flow r a t e to s h o r t e n a n a l y t i c a l time. L C e q u i p p e d w i t h a h i g h l y s e n s i t i v e d e t e c t o r a n d a high efficiency c o l u m n a t high o p e r a t i n g p r e s s u r e is u s u a l l y c a l l e d h i g h - p r e s s u r e , high-speed, or h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y (HPLC).
[15]
HIGH-PRESSURE LIQUID CHROMATOGRAPHY
301
M a n y review articles on H P L C have recently appeared in the literature. 1 '~ The specific information on the equipment, detectors, column packing materials, operating conditions, and calculation of resolutions m a y be obtained from several references2 -n Papers published to date on the application of H P L C for the analysis of antibiotics are rather limited. I n this article as m a n y available methodologies as possible are listed. Since the H P L C instruments have not been standardized as GLC, some modification of the methods m a y be required in each laboratory. The methods thus far reported do not require derivatization of antibiotics, as required by m a n y of the G L C methods. I n m a n y applications the H P L C assay procedure is simple, and formation of degradation products is minimal.
I I . A r o m a t i c Antibiotics with N i t r o g e n A. N o v o b i o c i n w" Procedure Materials
Methanol, absolute, A.R. Sodium phosphate, monobasic Sodium phosphate, dibasic Phosphoric acid Prednisolone Z I P A X H y d r o c a r b o n polymer Delaware)
(HCP,
DuPont,
Wilmington,
Solutions
Phosphate buffer, p H 7.0 at 0.1 M. Prepare 0.1 M solution of monobasic and dibasic sodium phosphate. Mix the solutions to p H 7.0. G. W. Ewing, J. Chem. Educ. ~0, A429 (1973). 2E. Heftmann, J .Chromatogr. Sci. 11,295 (1973). M. Martin and G. Guiochon, Bull. Soc. Chim. Fr. No. 1,161 (1973). 4 A. F. Michaelis, D. W. Cornish, and R. Vivilecchia, J. Pharm. Sci. 62, 1399 (1973). Y. I. Yashin and I. I. Frolov, Zh. Anal. Khim. 27, 923 (1972). C. D. Chandler, J. Chromatogr. Sci. 11,468 (1973). R. D. Conlon, Anal. Chem. 41, 107A (1969). s R. E. Majors, J. Chromatogr. Sci. 11, 88 (1973). 8 G. J. Fallick and J. L. Waters, Amer. Lab. 4, (8), 21 (1972). 1. L. R. Snyder, J. Chromatogr. Sci. 10, 200 (1972). 1~L. R. Snyder, J. Chromatogr. Sci. 10, 369 (1972). 12K. Tsuji and J. H. Robertson, J. Chromatogr. 94, 245 (1974).
302
METHODS FOR T H E STUDY OF ANTIBIOTICS
[15]
Phosphate buffer, pH 2.5. Prepare 0.2 M solution of dibasic sodium phosphate and add phosphoric acid to pH 2.5. Mobile phase. Add 150 ml of methanol and 200 ml of pH 7.0, 0.1 M phosphate buffer into a 1000-ml graduated cylinder. Add water to volume. Internal standard solution. Weigh about 87 mg of prednisolone into a 250-ml graduated cylinder. Add 150 ml of absolute methanol and shake to dissolve prednisolone. Add 50 ml of pH 7.0, 0.1 M phosphate buffer followed by water to volume. Column Rinse Solution A. 40% methanol in 0.02 M, pH 7.0 phosphate buffer B. 20% methanol in 0.02 M, pH 2.5 phosphate buffer
Chromatographic Conditions Detector: 254 nm UV at 0.04 absorbance unit full scale Column: Stainless steel, 2.1 mm i.d. X 1000 mm (DuPont) packed with Z I P A X - - H C P (DuPont) Column temperature: ambient Column pressure: 68 atm (1000 psi) Mobile phase: 15% methanol, 0.02 M, pH 7.0 phosphate buffer Flow rate: 0.85 ml/min Chart speed: 0.64 cm/min
Preparation of the Chromatographic Column Pretreat an empty stainless steel column, 2 mm i.d. X 1000 mm as follows: Rinse the column with tetrahydrofuran (THF) followed by vigorous cleaning of the inside of the column with a cotton string, presoaked with T H F to remove loose metal particles. Rinse the column with chloroform and dry with a stream of dry nitrogen. Insert a 10-~m pore size stainless steel frit into the inlet end of the column and attach a hex nut (DuPont, No. 820349) to the column with stainless steel front and back lock ferrules and a cap (DuPont, No. 201724). Dry-pack ZIPAXH P C column packing material into the open end of the column by adding a small amount of H P C at a time and tap lightly on the floor. After the column is tightly packed, insert a 2 ~m pore size stainless steel frit into the outlet end of the column. The chromatographic column thus prepared is attached to the injector port and to a 254 nm UV monitor. The mobile phase is pumped into the column under the chromatographic conditions listed above until the baseline stabilizes. The efficiency
[15]
HIGH-PRESSURE LIQUID CHROMATOGRAPHY
303
of the column thus prepared is approximately 430 theoretical plates per meter for the novobiocin peak.
Preparation ]or Bulk Drug and Re]erence Standard Powder Accurately weigh approximately 10 mg of bulk drug powder and the novobiocin acid reference standard into a 10-ml volumetric flask. Prior to analysis, each sample and standard is dissolved and diluted to volume with the internal standard solution. An ultrasonicator (Ultrasonics, Inc., Plainview, New York) is used to quickly dissolve samples and reference standard. Inject approximately 1 td of sample and reference standard into the chromatograph.
Calculation Measure the area under the novobiocin and internal standard peak. Determine the peak area ratio of novobiocin and the internal standard. The content of novobiocin in micrograms per milligram of sample is calculated using the formula:
[R~/Rt] X [Wt/W~] × F where Rs -- peak area ratio of sample novobiocin to the internal standard p e a k s ; Rt = peak area ratio of the reference standard novobiocin to the internal standard peaks; Wt = weight of the reference standard (mg); W~ = weight of sample novobiocin (mg); F = assigned potency if the reference standard (ug/mg)
Rinsing and Cleaning o] the Column After the end of each day's analysis, the column should be rinsed with the column rinse solution A for at least 30 min followed by rinse solution B for 1 hr. Prior to the start of each day's analysis the mobile phase should be pumped under the chromatographic condition until a stable baseline is obtained.
Comments on the Assay Method A typical chromatogram of calcium novobiocin, indicating the separation of various isomers and degradation compounds, is shown in Fig. 1. Daily column rinsing and the cleaning operation are an essential feature of the operation to maintain the separation of isonovobiocin from novobiocin.
304
METHODS
FOR
THE
STUDY
O F ANTIBIOTICS
[15]
6
I 4
I 8
12
I 16
I 20
TIME (MINUTES)
Fro. 1. Chromatogram of novobiocins indicating the separation of (1) novobiocic acid, (2) novenamine, (3) demethyldeearbamylnovobiocin, (4) decarbamylnovobiocin, (5) isonovobiocin, (6) novobiocin, and (7) dihydronovobiocin.
Phosphate buffer of pH 7.0 is used in the mobile phase and in the internal standard solution for the stability and solubility of novobioein.13,14 B. Rifampin 15
Chromatographic Conditions Instrument: high-pressure liquid chromatograph with linear gradient capi~bility Detector: 254 nm UV Column: stainless steel, 2.1 mm i.d. X 1000 mm (DuPont) packed with ODS-Permaphase (DuPont) Column temperature: 50 ° Column pressure: 68 arm (1000 psi) Mobile phase: water to methanol, linear gradient of 8 ~ change per minute Flow rate: 1 ml/min la G. S. Libinson and K. I. Surkova, Antibiotiki (Moscow) 12, 8 (1967). 1, M. J. Busse, K. A. Lees, and V. J. Vergine, J. Pharm. Pharmacol. 11, 250T (1959). 15j . A. Schmit, R. A. Henry, R. C. Williams, and J. F. Dieckman, J. Chromatogr. Sci. 9, 645 (1971).
[15]
HIGH-PRESSURE LIQUID CHROMATOGRAPHY
305
RETENTION TIME (MINUTES)
Fro. 2. Separation of rifampin antibiotic and rifampin derivatives. (1) 3-Formylrifampin, (2) deacetylrifampin, (3) rifampin, and (4) rifampin quinone [J. A. Schmit, R. A. Henry, R. C. Williams, and J. F. Dieckman, I. Cromatogr. Sci. 9, 645 (1971)]. A chromatogram indicating separation of rifamycin from its derivatives is shown in Fig. 2. No information regarding the assay variation under the conditions of linear gradient, without internal standard, is available. C. V i r g i n i a m y c i n 16
Chromatographic Conditions
Detector: refractive index (RI) Column: Corasil/Cl~ (Waters Associates, Milford, Massachusetts) Column temperature: ambient Column pressure: information not given Mobile phase: acetonitrile/water ( l / l ) Flow rate: 0.45 ml/min le C. Pidacks, Waters Associates, Milford, Massachusetts, personal communication.
306
METHODS FOR THE STUDY OF ANTIBIOTICS
[15]
III. Clindamycin Phosphate 17
Chromatographic Conditions Detector: 254 nm UV Column: stainless steel, 2.1 mm i.d. X 1000 mm dry-packed with TEAE-cellulose (Cellex-T, Bio-Rad, Richmond, California) Column temperature: 60 ° Column pressure: 28 atm (410 psi) Mobile phase: 0.25 M boric acid, adjusted to pH 8.8 with NaOH Flow rate: 0.23 ml/min
Preparation of Chromatographic Column A stainless steel column, 2.1 mm X 1 meter, is packed by attaching a funnel to one end of the column. TEAE-cellulose, contained in a 15-ml vial fitted with a 50-mesh screen, is added to the funnel in about 20-mg portions by brief vibration. The bed is compacted three times with a loose-fitting stainless steel rod (1.8 mm o.d.) while turning the column 120 ° after each addition. A fully packed column contains 1.4-2 g of support. The column is attached to the injector system and water (5-50 ml) is pumped through the column at 136 atmospheric pressure (2,000 psi) in an attempt to dislodge traces'of the support from the detector end of the column. After connecting the detector housing, the column is washed with 10-50 ml quantities of methanol, 0.5 M sodium nitrate and finally 0.5 M sodium acetate.
Sample Preparation Solutions of clindamycin phosphate are prepared at a concentration of 2-15% in water with the addition of NaOH to a pH of 8.8. Inject 7 ~l of the sample solution.
Comments on the Assay Method Borate was chosen as the mobile phase for HPLC since reversible interaction occurs with clindamycin 2-phosphate to form clindamycin 2-phosphate borate complex, but not with its 3- or 4-phosphate esters to obtain separation (Fig. 3). The molar absorptivity (E) of clindamycin phosphate at 254 nm is only 16.8 in water at pH 8.9. Detection of clindamycin phosphate is possible only by use of the high capacity support, TEAE-cellulose. The exchange capacity of a 2.1 mm X 1 m column is 810-1160 microequivalents as calculated from the column weight and the exchange capacity of the support. 17 W. Morozowich and R. G. Williams, J. Pharm. Sci. 64 (2) (1975).
[15l
HIGH-PRESSURE LIQUID CHROMATOGRAPHY
307
1
I 0
I 5
I I 10 15 TIME (MINUTES)
I 20
l 25
Fro. 3. Separation of clindamycin-2-phosphate (3) from the corresponding 3- and 4-phosphate esters (2,1). By courtesy of W. Morozowich.
IV. Griseofulvin 18
Chromatographic Conditions Detector: 254 nm UV Column: stainless steel, 2.1 mm i.d. X 1000 mm (DuPont) packed with Permaphase-ETH (DuPont) Column temperature: ambient Column pressure: 34 atm (500 psi) Mobile phase: 5% chloroform in hexane Flow rate: information not given A chromatogram indicating separation of griseofulvin from its related impurities is shown in Fig. 4. V. K a n a m y c i n ~9
Chromatographic Conditions Detector: RI Column: stainless Steel, 1.8 mm i.d. X 1000 mm packed with Aminex A-27-OH (Bio-Rad, Richmond, California) as F. Bailey and P. N. Brittain, J. Chromatogr. 83, 431 (1973). 19T. Ottake, Nippon Electric Varian, L.C. at Work, p. 5.
308
METHODS FOR THE STUDY OF ANTIBIOTICS
[15]
2
5
FIG. 4. Chromatogram of the separation of griseofulvin (4) from its related impurities (1, 2, 3, 5) IF. Bailey and P. N. Brittain, J. Chromatogr. 83, 431 (1973)]. Column temperature: 40 ° Column pressure: 136 atm (2000 psi) Mobile phase: water Flow rate: 0.5 ml/min Sample size: 4 ~l containing about 200/~g of kanamycin Separatio n of kanamycins A and B may be made in 13 min of chromatographic time VI. Macrolide Antibiotics A. Erythromycin 1~
Chromatographic Conditions Detector: RI Column: 2.3 mm i.d. X 1830 mm packed with Corasil II (Silica gel, Waters Associates, Milford, Massachusetts) Column temperature: ambient Column pressure: information not available Mobile phase: chloroform (alcohol free) Flow rate: 0.5 ml/min A chromatogram indicating separation of anhydroerythromycin from erythromycin is shown in Fig. 5.
[15]
HIGH-PRESSURE LIQUID CHROMATOGRAPHY
309
2
1
Fro. 5. Separation of (1) anhydroerythromycin from (2) erythromycim By courtesy of C. Pidacks.
B. L e u c o m y c i n 2°
Chromatographic Conditions Detector: 232.5 nm UV spectrophotometer Column: JASCOPACK SV-02-500 (chemically bonded reverse phase, JASCO Co., Ltd., Japan) Column temperature: ambient Column pressure: 63 arm (925 psi) Mobile phase: MeOH: M/15 acetate buffer (pH 4.9): acetonitrile (35:60:5) Flow rate: 1.0 ml/min The maximum UV absorption of leucomycin is at 232.5 nm. The use of 254 nm, the wavelength generally applied, gave poor sensitivity,
V I I . Penicillins A. Ampicillin 21
Chromatographic Conditions Detector: 254 nm UV Column: Stainless steel, 2.1 mm i.d. X 1000 mm packed with s0 S. Omura, Y. Suzuki, A. Nakagawa, and T. Hara, J. Antibiot. 26, 794 (1973). 51K. Tsuji and J. H, Robertson, J. Pharm. Sci. (in press).
310
METHODS FOR THE STUDY OF ANTIBIOTICS
[15]
VYDAC P150 Anion Exchange (Applied Science Lab., Inc., State College, Pennsylvania) Column temperature: ambient Column pressure: 34 atm (500 psi) Mobile phase: 20 mM NAN03 in 10 mM Na-borate, pH 9.1 solution Flow rate: 0.85 ml/min Sample size: 2 ~l containing 1.0 mg/ml Electrometer range setting: 0.02 full scale
Preparation o] Bulk Drug and Reference Standard Powder Approximately l0 mg of ampicillin bulk powder and ampicillin reference standard are accurately weighed using an electrobalance (e.g., Cahn Electrobalance, model G, Cahn Instrument Corp., Paramount, California), and placed in a 10-ml volumetric flask. Prior to analysis, the standard and samples are dissolved and diluted to volume with the mobile phase.
Sample Preparation Hard-Filled Capsules. To minimize capsule weight variation, ten capsules are emptied and their contents accurately weighed. A portion of the powder equivalent in weight to contain 1000 mg ampicillin is accurately weighed and placed in a 1000 ml volumetric flask. The contents are then diluted to volume with double distilled water and shaken for 5 min to dissolve the ampicillin. About 25 ml of the suspension is placed into a 50-ml conical-bottom centrifuge tube and centrifuged at 2000 rpm for 20 min. Sterile Injectable. Each vial containing 250 mg, 500 rag, and 1 g of product is reconstituted with the recommended volume of double distilled water. An ultrasonicator is used to quickly dissolve each sample. From each of four 250 mg vials, two 500 mg vials, and one 1-gram vial, 0.1 ml is accurately removed and placed in a 100 ml volumetric flask. The sample is then diluted to volume with the mobile phase. Oral Formulation. From one 125-mg bottle or 250-mg bottle, 5 ml is pipetted into a 50-ml round-bottom centrifuge tube. A 0.1 ml quantity of concentrated HC1 is added and mixed, followed by 30 ml of chloroform. The centrifuge tube is then capped and shaken vigorously for 5 min using an Eberbach reciprocal shaker. The tube is then centrifuged for 2 min at 2000 rpm. Four milliliters of the upper aqueous layer from 125 mg dosage form or 2 ml from 250 mg form is pipetted into a 100 ml
[15]
HIGH-PRESSURE LIQUID CHROMATOGRAPHY
311
volumetric flask and is diluted to volume with 0.02 M phosphate buffer pH 6.0.
Comments on the Assay Method Since a slight degradation can occur when ampicillin is left standing in the mobile phase, the ampicillin standard and sample should be diluted in the mobile phase just prior to the analysis. The relative standard deviation of the HPLC method is less than 1% with quantitative injection, without an internal standard, using a septumless injector valve (e.g., Micromeritics, Inc., Norcross, Georgia). The minimum quantity of ampicillin detection is approximately 20 ng per sample injected at an electrometer range setting of 0.01 full scale. The method is capable of detecting penicillenic and penicilloic acids of ampicillin.
B. Cephalosporin (222
Chromatographic Conditions Detector: 254 nm UV Column: stainless steel, 2.1 mm i.d. X 125 mm packed with SAXZIPAX (Strong anionic resin, DuPont) Column temperature: ambient Column pressure: 55 atm (800 psi) Mobile phase: 0.35 M boric acid adjusted to pH 9.6 with NaOH Flow rate: 0.45 ml/min Sample size: 5 t~l containing 0.5-5.0 tLg
Sample and Re]erence Standard Preparation The sodium salt of cephalosporin C (NaC~6H~oN3OsS.2H~O) and the sodium salt of deacetyl cephalosporin (NaCl~H1.~N~07S) used as the reference standard were chromatographically pure and their molar extinction coefficients were 8470 and 7900, respectively, at 260 nm. The cephalosporin bulk powder and the reference standard powder were dissolved in pH 7.0 phosphate buffer and were analyzed immediately or stored overnight at --20 ° prior to analysis. The cephalosporin is stable between the pH range of 3 and 9. For the analysis of fermentation broth samples, aliquots of the samples were generally diluted to approximately 0.01% active substance to 22j . Konecny, E. Felber, and J. Gruner, J. Antibiot. 26, 135 (1973).
312
METHODS FOR THE STUDY OF ANTIBIOTICS
[15]
4
2
I
[
I
o
2'o
TIME (MINUTES)
Fro. 6. Chromatogram of cephalosporin C: (1, 2) unknown, (3) deacetyl cephalosporin C, and (4) cephalosporin C [J. Konecny, E. Felber, and J. Gruner, J. Antibiot. 26, 135 (1973)].
prolong column life. Five microliters, corresponding to 0.5-5.0 ~g of cephalosporin (optimum concentration range) were injected. A typical chromatogram of cephalosporin C is shown in Fig. 6. C. Penicillin (~21
Chromatographic Conditions Detector: 254 nm UV Column: stainless steel, 2.1 mm i.d. X 1000 mm packed with VYDAC P150 Anion Exchange (Applied Science Lab., Inc., State College, Pennsylvania) Column temperature: ambient Column pressure: 34 atm (500 psi) Mobile phase: 30 mM NaNO~ in 10 mM Nu borate, pH 9.1 solution Flow rate: 0.85 ml/min Sample size: 2 ~l containing 1 mg/ml Alternative Method
TM
Chromatographic Conditions Detector: 254 nm UV Column: stainless steel, 2.1 mm i.d. X 610 mm (2 ft) packed with Porasil A (Waters Associates, Milford, Massachusetts) Column temperature: ambient Column pressure: information not available Mobile phase: chloroform:methanol: pH 6.7, 0.1 M phosphate buffer (400:79:9)
[151
HIGH-PRESSURE LIQUID CHROMATOGRAPHY
313
Flow rate: 1.5 ml/min Sample concentration: 2 mg/ml Prior to analysis, pump pH 6.7, 0.1 M phosphate buffer for about 30 min followed by the mobile phase until the baseline stabilizes. D.
P e n i c i l l i n V ~1
Chromatographic Conditions Detector: 254 nm UV Column: stainless steel, 2.1 mm i.d. X 1000 mm packed with VYDAC P150 Anion Exchange (Applied Science Lab., Inc. State College, Pennsylvania) Column temperature: ambient Column pressure: 34 atm (500 psi) Mobile phase: 10% methanol in 30 mM NaNO:~ and 10 mM Naborate, pH 9.1 solution Flow rate: 0.85 ml/min Sample size: 2 ~1 containing I mg/ml
VIII. Polypeptide Antibiotics A. Actinomycin 23
Chromatographic Conditions Detector: 254 nm UV Column: stainless steel, 2.3 mm i.d. X 1830 mm (6 ft), packed with BONDAPAK Cl~/Corasil (Waters Associates, Milford, Massachusetts) Column temperature: 22 ° Column pressure: 68 atm (1000 psi) Mobile phase: acetonitrile :water (1 : 1) Flow rate: 1.0 ml/min Sample size: 1 td containing 1% actinomycin in methanol Electrometer range setting: 0.08 full scale.
Comments on the Assay Method For better stability, act:nomycin sample is dissolved in methanol and stored in the dark at --4 ° until analysis. Actinomycins C1 (D), C2, and C3 can be separated in less than 40 rain of chromatographic time. 53 W. J. Rzeszotarski and A. B. Mauger, J, Chromatogr. 86, 246 (1973).
314
[15]
METHODS FOR THE STUDY OF ANTIBIOTICS
I
i
I
I
I
i
0
4
8
12
16
20
i
I
24 28 MINUTES
J
i
J
i
i
32
36
40
44
48
FIG. 7. C h r o m a t o g r a m of bacitracin.
B, Bacitracin ~
Chromatographic Conditions Detector: 254 nm UV Column: stainless steel, 2.1 mm i.d. X 1000 mm packed with BONDAPAK C18/Corasil (Waters Associates, Milford, Massachusetts) Column temperature: ambient Column pressure: 68 arm (1000 psi) Mobile phases: (A) 50 ml of ~bsolute methanol and 750 ml water are added into a 1000-ml graduated cylinder; 200 ml of 0.1 M, pH 4.5 potassium phosphate buffer is then added and mixed. (B) 400 ml of absolute methanol, 200 ml of acetonitrile, and 200 ml of water are added into 1000-ml graduated cylinder. After mixing, 200 ml of 0.1 M, pH 4.5 phosphate buffer are added and mixed. Flow rate: 0.90 ml/min Sample: 2 ~l containing 10 mg/ml in O.02N HC1 in 80% methanol Electrometer range setting: 0.08 full scale An Ultragrad gradient mixer (LKB model 11330, Stockholm, Sweden) with a 0.25 ml mixing chamber (model 11361-1) was used. A programmed convex gradient elution rate followed an exponential equation y = [191/(1-[-e°'~38-°.°67~)] --78.5 where x equals time in minutes and y equals percent of the mobile phase B t o A. A chromatogram indicating separation of bacitracin is shown in Fig. 7. The peaks eluting after the major peak (retention time of 34 min) ~4K, Tsuji and J. YI. Robertson, J. Chromatogr. 99, 597 (1974).
[15]
HIGH-PRESSURE LIQUID CHROMATOGRAPHY
315
are bacitracin F. The major peak is presumed to be bacitracin A and four peaks eluting ahead of bactracin A may be bacitracins B and C. These five peaks have about equal antimicrobial activity. No other peak has antimicrobial activity.
IX. Tetracyclines and Related Antibiotics A. Tetracycline 25 Procedure Solutions. Phosphate buffer, pH 2.0. Prepare 0.2 M solution of dibasic sodium phosphate and adjust pH to 2.0 with phosphoric acid. Phosphate buffer, pH 4.5, 10 mM. Mobile Phases A: 50 ml of acetonitrile and 850 ml water are added into a 1000 ml graduated cylinder. 100 ml of pH 2.0 phosphate buffer is then added and mixed B: 500 ml of acetonitrile and 400 ml water are added into a 1000 ml graduated cylinder. 100 ml of pH 2.0 phosphate buffer is then added and mixed Column Rinse Solution. An aqueous solution containing 80% acetonitrile is prepared. The column is cleaned as needed. Chromatographic Conditions Detector: 280 nm UV Column: Stainless steel, 2.1 mm i.d. X 1000 mm (DuPont) packed with BONDAPAK C18/CORASIL (Waters Associates, Milford, Massachusetts). A 5 ~m pore size Teflon frit is inserted into the inlet and the outlet ends of the column Column temperature: ambient Column pressure: 78 atm (1000 psi) Mobile phases: (A) 5% acetonitrile and (B) 50% acetonitrile, both in pH 2.0, 20 mM phosphate buffer Flow rate: 0.85 ml/min Sample size: 2.0 ~l containing 0.4 mg/ml Electrometer range setting: 0.08 full scale An Ultragrad gradient mixer (LKB model 11300, Stockhohn, Sweden) with a 0.25 ml mixing chamber (model 11361-1) was used. A programmed convex gradient elution rate followed an exponential equation
y = 1 3 9 . 3 - 147.8 e-°'l°x :~K. Tsuji and J. H. Robertson, J. Pharm. Sci. (in press).
316
METHODS FOR THE STUDY OF ANTIBIOTICS
[15]
where x equals time in minutes and y equals percent of the mobile phase B to A, for 11 min.
Preparation o] TC Standard TC.HC1 reference standard is dried at 60 ° under less than 5mm Hg vacuum for 3 hr. After drying, approximately 4 mg of the reference standard is accurately weighed using an electrobalance (e.g., Cahn Electrobalance model G, Cahn Instrument Corp., Paramount, California) and placed in a 10 ml volumetric flask. Just prior to the analysis, each standard is dissolved with 1 ml of absolute methanol and then diluted to volume with 10 mM, pH 4.5 phosphate buffer.
Preparation o] Sample Bulk Powder. Approximately 4 mg of the TC bulk powder is accurarely weighed into a 10 ml volumetric flask. Just prior to the analysis, 1 ml of absolute methanol is added and then diluted to volume with 10 mM, pH 4.5 phosphate buffer. HARD-FILLED CAFSULES.Ten capsules containing 250 mg TC. HC1 are carefully emptied and weighed. The equivalent weight of two capsules are then weighed and placed in a 50 ml round-bottom, glass-stoppered centrifuge tube; 25 ml of absolute methanol is then added and the contents shaken for 5 min. The tubes are then centrifuged for 10 min at 1800 rpm. After centrifugation, 2 ml is removed and placed in a 100 ml volumetric flask. Just prior to the analysis, the flask is diluted to volume with 10 mM phosphate buffer, pH 4.5. SYavP. Three milliliter of thoroughly mixed syrup containing 125 mg TC base per 5 ml is transferred into a 200 ml volumetric flask and diluted to volume with absolute methanol. The contents are then thoroughly shaken and then about 30 ml is decanted into a 50 ml glass-stoppered, round-bottom centrifuge tube and centrifuged at 1800 rpm for 5 min. After centrifugation, 2 ml of the supernatant is pipetted into a 10 ml volumetric flask. The flask is diluted to volume with 10 mM, pH 4.5 phosphate buffer. Calculation The microbiological equivalence of TC content is calculated using the following formula:
[At~As] X [Ws/Wt] X F~ X F~
[15]
HIGH-PRESSURE LIQUID CHROMATOGRAPHY
where A t = peak area of (TC + 0.27 ETC + 20.43 ATC + 0.18 E A T C
317
of
sample A s = peak area of (TC + 0.27 ETC)
of reference standard
W s = weight of reference standard (rag) W t = weight of sample (mg)
F1 = assigned value of TC reference standard (t~g/mg) F~ = dilution factor C o m m e n t s on the A s s a y M e t h o d
The chromatography of a TC sample takes approximately 12 ram; however, it usually takes 4 rain to stabilize the baseline at the end of each analysis. Therefore, each TC sample can be assayed at 16-rain intervals. A chromatogram indicating separation of TC's is shown in Fig. 8. The relative standard deviation of the TC determination is 0.66% with quant:.tative iniection, without an internal standard, using a septumless valve injector (Micromeritics, Inc., Noreross, Georgia).
5 6
L
i
I
I
0
4
8
12
MINUTES
FIG. 8. Chromatogram of tetracyclines. (1) 4-Epitetracycline, (2) tetracycline, (3) chlortetracycline, (4) 4-epianhydrotetracycline, and (5) anhydrotetracycline.
318
METHODS FOR THE STUDY OF ANTIBIOTICS
[15]
The method is capable of detecting the presence of 0.3% EATC in TC and the sensitivity of the method to TC is approximately 10 ng per sample injected. Alternative Method for Tetracycline 26
Chromatoqraphic Conditions Detector: 254 nm UV Column: stainless steel, 1.8 mm i.d. X 2250 ram, packed with Pellionex, CP-128 (Reeve Angel, Clifton, New Jersey) Column temperature: 50 ° Mobile phase: 30.0% (v/v)ethanol, 0.10 M Na ÷, 2 mM EDTA at pH 4.6 Flow rate: 60 ml/hr Sample size: 5 ~l containing about 10 ~g of TC ]3. Oxytetracycline 27
Chromatographic Conditions Detector: 254 nm UV Column: Stainless steel, 2 mm i.d. X 1500 mm packed with SCXZIPAX (DuPont) Column temperature: ambient Mobile phase: 10 mM EDTA and 10 mM KH~PO at pH 7.0 Flow rate: 1 ml/min C, D a u n o m y c i n 28
Chromatographic Conditions Detector: 254 nm Column: stainless steel 2 mm i.d. X 150 mm packed with Micropak Si-10 (Varian Aerograph, Palo Alto, California) Column temperature: ambient Mobile phase: isopropanol: 0.5 M acetate buffer (pH 4.5) (90:10) Flow rate: 0.42 ml/min Column pressure: 34 arm (500 psi) Sample size: 5 ~l containing about 0.5 mg/ml A. G. Butterfield, D. W. Hughes, N. J. Plund, and W. L. Wilson, Antimicrob. Ag. Chemother. 4, 11 (1973). ~7K. Loettler, Varian Instrument, "Liquid Chromatography at Work," 17. R. E. Majors, Varian Instrument, "Liquid Chromatography at Work," 7.
[15]
HIGH-PRESSURE
LIQUID
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C'~lc'x~
320
METHODS FOR THE STUDY OF ANTIBIOTICS
[15]
Acknowledgment The author is indebted to his many colleagues who provided unpublished data for this article.
[15] I s o l a t i o n o f A n t i b i o t i c s b y C o u n t e r c u r r e n t
Distribution
B y LYMAN C. CRAIG and JOHN SOGN I. Introduction . . . . . . . . . . . . . . . . . II. Uses of CCD in the Antibiotic Field . . . . . . . . . . A. Isolation from Growth Medium . . . . . . . . . . . B. Final Purification . . . . . . . . . . . . . . . C. Testing for Purity . . . . . . . . . . . . . . . D. Molecular Weight Determination by the Method of Partial Substitution . . . . . . . . . . . . . . . . E. Isolation of Fragments in Structural Studies . . . . . . . III. Nonideality in CCD . . . . . . . . . . . . . . . IV. Systems . . . . . . . . . . . . . . . . . . V. Apparatus Used in CCD . . . . . . . . . . . . . .
. . . . .
320 321 321 324 327
.
328 330 331 331 341
. . . .
I. I n t r o d u c t i o n Countercurrent distribution (CCD) is a relatively simple extraction process specifically designed for the purpose of separating relatively small amounts of mixtures of solutes under mild conditions in the laboratory. I t is an extension of one of the oldest known separation methods--simple partition between two immiscible solvents. In its simplest form it involves only a very small number of separatory funnels, but modern, fully automatic, commercially available distribution trains of up to 1000 cells ~ allow one to separate cleanly a pair of solutes differing in their partition ratios in a suitable solvent system by no more than 10%. The available apparatus and the m a n y possible modes of operation will be discussed in a later section. The discontinuous, stepwise nature of the process permits a useful mathematical interpretation of the progress of the separation and the probable purity of the separated components, an interpretation more exact than that possible for any other known countercurrent separation process. The theory behind the operation and the mathematical interpretation of the data are discussed fully elsewhere ~,3 and will not be treated here. L. C. Craig and T. P. King, Fed. Proc., Fed. Amer. Soc. Exp. Biol. 17, 1126 (1958). 2 L. C. Craig and D. Craig, in "Technique of Organic Chemistry" (A. Weissberger, ed.), 2nd ed., Vol. III, part 1, p. 149. Wiley (Interscience), New York, 1956. s C. J. O. R. Morris and P. Morris, "Separation Methods in Biochemistry," p. 559. Wiley (Interscience), New York, 1963.
320
METHODS FOR THE STUDY OF ANTIBIOTICS
[15]
Acknowledgment The author is indebted to his many colleagues who provided unpublished data for this article.
[15] I s o l a t i o n o f A n t i b i o t i c s b y C o u n t e r c u r r e n t
Distribution
B y LYMAN C. CRAIG and JOHN SOGN I. Introduction . . . . . . . . . . . . . . . . . II. Uses of CCD in the Antibiotic Field . . . . . . . . . . A. Isolation from Growth Medium . . . . . . . . . . . B. Final Purification . . . . . . . . . . . . . . . C. Testing for Purity . . . . . . . . . . . . . . . D. Molecular Weight Determination by the Method of Partial Substitution . . . . . . . . . . . . . . . . E. Isolation of Fragments in Structural Studies . . . . . . . III. Nonideality in CCD . . . . . . . . . . . . . . . IV. Systems . . . . . . . . . . . . . . . . . . V. Apparatus Used in CCD . . . . . . . . . . . . . .
. . . . .
320 321 321 324 327
.
328 330 331 331 341
. . . .
I. I n t r o d u c t i o n Countercurrent distribution (CCD) is a relatively simple extraction process specifically designed for the purpose of separating relatively small amounts of mixtures of solutes under mild conditions in the laboratory. I t is an extension of one of the oldest known separation methods--simple partition between two immiscible solvents. In its simplest form it involves only a very small number of separatory funnels, but modern, fully automatic, commercially available distribution trains of up to 1000 cells ~ allow one to separate cleanly a pair of solutes differing in their partition ratios in a suitable solvent system by no more than 10%. The available apparatus and the m a n y possible modes of operation will be discussed in a later section. The discontinuous, stepwise nature of the process permits a useful mathematical interpretation of the progress of the separation and the probable purity of the separated components, an interpretation more exact than that possible for any other known countercurrent separation process. The theory behind the operation and the mathematical interpretation of the data are discussed fully elsewhere ~,3 and will not be treated here. L. C. Craig and T. P. King, Fed. Proc., Fed. Amer. Soc. Exp. Biol. 17, 1126 (1958). 2 L. C. Craig and D. Craig, in "Technique of Organic Chemistry" (A. Weissberger, ed.), 2nd ed., Vol. III, part 1, p. 149. Wiley (Interscience), New York, 1956. s C. J. O. R. Morris and P. Morris, "Separation Methods in Biochemistry," p. 559. Wiley (Interscience), New York, 1963.
[15]
ISOLATION OF ANTIBIOTICS BY CCD
321
The problem of the isolation in pure form of an active principle which has been shown by some bioassay to be present in the tissues of either plants or animals presents to the natural products chemist a definite challenge and fascination. The principle is usually present in a very small amount per unit weight of tissue, often has poor stability, and may be bound by secondary forces to components of the tissue. Tissues contain many other compounds that can interfere with isolation of a single component, and the task may be easy or very difficult depending on the physical properties of the active principle. II. Uses of C C D in the Antibiotic Field CCD has been applied to the study of antibiotics in a number of different ways. It has been used to isolate the antibiotic principles directly from the growth medium. Most often it is used to perform the final purification after some preliminary purification has been achieved by simple extraction or other bulk techniques. When material thought to be pure has been isolated without resort to CCD, as by chromatography or crystallization, CCD can be used as an easily interpreted and demanding analytical test of purity. In addition, after isolation of the pure antibiotic, CCD can be of substantial use in characterization studies. It is ideally suited to determination of molecular weight by the method of partial substitution ~ and has been used in a number of structural studies to isolate fragments and degradation products. Each of these uses will be discussed below. A. Isolation from the G r o w t h M e d i u m
It has long been realized that a basic requirement for the isolation is a convenient quantitative bioassay to monitor the extraction and subsequent fractionation procedures. However, it was not until highly selective liquid-liquid or liquid-solid systematic procedures were developed in the 1940's that rapid advances were made and isolation of many active principles became almost routine. Countercurrent distribution was demonstrated to be an almost ideal tool for the purpose and was subsequently shown to be made even more so by integration with paper chromatography, thin-layer chromatography and zone electrophoresis. The improvement and standardization of antibiotic assays in combination with extraction and fractionation procedures made the isolation of many pure antibiotics a relatively simple procedure and placed the important question of purity on a very high level. The first step in a quite general procedure is the demonstration that 4 A. R. Battersby and L. C. Craig, J. Amer. Chem. Soc. 74, 4023 (1952).
322
METHODS FOR THE STUDY OF ANTIBIOTICS
[15]
biological activity (antibiotic activity in the case of a suspected antibiotic) can be extracted from the tissue or culture broth by a suitable organic solvent (chloroform, ether, butanol, etc.). A known volume of this extract is then shaken with a volume of water or buffered solution chosen so that two approximately equal volumes of two immiscible phases are formed. Each phase is then assayed. If all or nearly all the biological activity is found to be in one of the phases either phase must be modified or replaced by other pairs of immiscible solvents so that the activity will be partitioned between the two phases to give a ratio between the limits of 0.1 and 10. At this point it will not be known whether one or several substances of biological activity are present or what the nature of the active principle might be. Nonetheless, the basic information is now at hand for a preliminary countercurrent distribution which can be made in a series of test tubes or more conveniently in a small hand-operated train of 20-30 tubes. 5 The probability of success in isolation of the active principle is already quite high. After a few transfers have been made, both phases in each tube are assayed and partition ratios in terms of biological activity are determined. From this information it is possible to derive a hint as to whether or not more than one active substance is present and to modify the system or volumes so that a more efficient separation will be achieved. For instance, if the partition ratio in each tube seems to be approximately 0.2 or less, the upper phases can be doubled in volume and thereby achieve the equivalent of an operating ratio equal to 0.4. Conversely if it is too high, the lower phase can be doubled. Continuation of the distribution to higher numbers of transfers will permit a distribution pattern based on biological activity to be constructed from which an estimate of complexity can be made on the basis of the contour of the curve. At this point, however, it will be more useful to analyze by other techniques a sufficient number of the tubes to permit a curve to be superimposed on the activity curve. The analytical techniques most useful here include ultraviolet absorbance at various wavelengths for the case where absorbance is found, ninhydrin, residue weight5 (if the system itself is devoid of a nonvolatile component), carbohydrate color tests, etc. If, for instance, the activity curve exactly superimposes an absorbance curve this is a strong indication that the unknown active principle has absorption at the wavelength chosen. If the system does not contain a nonvolatile component, analysis by residue weight is the most informative since this permits activity:weight or absorbance:weight 5L. C. Craig, in "Analytical Methods of Protein Chemistry" (P. Alexander and R. J. Block, eds.), Vol. I, p. 122. Pergamon Press, Oxford, 1960.
[16]
ISOLATION OF ANTIBIOTICS BY CCD
323
ratios to be applied. If a single symmetrical superimposing band is obtained by activity, weight, and absorbance it is very probable that achievement of a pure antibiotic has been reached. This can be checked easily by determination of partition ratios. If a pure solute is present the ratios should not v a r y across the band. ~ The first application of this general procedure for isolation of a new antibiotic by CCD was published in 1946 ~ and will be described briefly here. In this work the initial steps were slightly different from those described above. The initial ethyl ether extract of the Aspergillus ustus culture broth was evaporated to dryness and weighed to see how much nonvolatile material had been extracted. This residue was then found to be soluble in aqueous buffer at pH 11 but to be precipitated on acidification. The antibiotic activity was found to be in the precipitate, which indicated it to be an acid. A sample of the precipitate dissolved in ethanol showed absorption in the ultraviolet with a plateau in the range of 250-270 ran. It was then found that the precipitate as judged by absorbance at 260 mn was about equally distributed between the two phases of a system made from cyclohexane and a pyrophosphate buffer adjusted to pH 8.31 with sulfuric acid. A preliminary distribution indicated more than one component and proceeding to 24 transfers gave the pattern shown in Fig. 1. In order to obtain this pattern, sulfuric acid was added to each tube, whereby after shaking, all the antibiotic activity was transferred to the upper phases. A theoretical distribution curve "~was superimposed over the two active bands in the pattern. This indicated the center band to be nearly pure, but the one on the right to be a mixture. Evaporation of the solvent from tubes in the central band, in contrast to those in the right band, gave crystals that were pure by a single recrystallization. The combined material from tubes 16-24 was then subjected to a second distribution in which the buffer had a higher pH. This gave the pattern shown in Fig. 2. Again the main band yielded a crystalline pure antibiotic with a different melting point and composition from that of the first crystalline antibiotic. Further active material was found to be present in tubes 20-24, but the fractionation was not pursued further. Both pure compounds were found to be chlorine containing, one with three atoms of chlorine, the other with two. This isolation was a very easy one in terms of present day methodology, but it illustrates the simple general approach. Another early illustration was the first isolation of a radioactive penicillin produced by a culG. T. Barry, Y. Sato, and L. C. Craig, J. Biol. Chem. 174, 221 (1948). G. H. Hogeboom and L. C. Craig, J. Biol. Chem. 162, 363 (1946),
324
METHODS
FOR THE
S T U D Y OF A N T I B I O T I C S
[15]
24.6
12 II 0 0 c~
"6
8
I0 9 8
7 B
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(4÷**)
5
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0
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I J J I I i
4
6
8
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~ I I /~'.~,..~t
~
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12 14 16 18 20 22 24
Tube number
Fzo. 1. First countercurrent distribution pattern of a new antibiotic. ture containing s o d i u m sulfate, in which the sulfur w a s radioactive s as s h o w n in Fig. 3.
B. F i n a l P u r i f i c a t i o n
CCD has proved to be an invaluable tool in the ultimate purification of a wide range of complex natural products, including a large number of antibiotics. The variety of materials separated can be seen in the table in Section IV. In many eases CCD has been the first method found to be capable of resolving closely related members of an antibiotic family. In some cases other methods have subsequently been worked out to perform the same separation, but in some groups, such as the polymyxins and linear gramicidins, CCD remains the only acceptable purification method. Actually the early experience with countercurrent distribution in the polypeptide antibiotic field did much to dispel belief at that time in the commonly held reliance for evidence of purity on sharp crystallinity and failure to change the properties of a preparation by fraction crystallization. In the polypeptide field the tyrocidine and gramieidin antibiotics s y. Sago, G. T. Barry, and L. C. Craig, J. Biol. Chem. 174, 217 (1948).
[161
ISOLATION
OF
ANTIBIOTICS
BY
CCD
325
(*÷**)
5196 I
15 14 iZl
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Tube number
FI~. 2. Second countercurrent distribution pattern of a new antibiotic. were sharply crystalline and were regarded as pure because repeated fractional crystallization did not change their properties. Even chromatography in its state of development up to 1950 failed to show t h a t these preparations were mixtures. The gramicidins were one of the first peptide antibiotics to be looked at by CCD. 9 T h e y were distributed in a 54-tube distribution train using the method of alternate withdrawal with 100 transfers in a solvent system of b e n z e n e - c h l o r o f o r m - m e t h a n o l - w a t e r (15: 15:23:7). This modest distribution was sufficient to indicate the presence of at least three distinct components. When a 100-tube machine was used 1° and 340 transfers were applied, the three components, gramicidins A, B, and C, were largely separated. These peptides were found to v a r y in the aromatic amino acid located at position eleven in the peptide chain. Gramicidin A contains t r y p t o p h a n , B contains phenylalanine, and C contains tyrosine. When the C C D was continued to 2000 transfers in a 500J. D. Gregory and L. C. Craig, J. Biol. Chem. 172, 839 (1947). ~+L. C. Craig, J. D. Gregory, and G. T. Barry, Cold Spring Harbor Syrup. Quant. Biol. 14, 24 (1949).
326
METHODS FOR THE STUDY OF ANTIBIOTICS
[16]
1400 , A Biological l
~iactivity ,.
1200
Weight
i
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800
i
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e
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Fro. 3. Countercurrent distribution pattern of a crude mixture containing radioactively labeled penicillin G. Ordinate: weight of a 1-ml aliquot in 0.01 mg (O - 0 ) ; activity in 100 units vs Staphylococcus aureus ( C ) - - - O ) .
tube machine, 11 the gramicidin A peak was further separated into two components known as valine-gramicidin A and isoleucine-gramicidin A, the difference again being a single amino acid replacement. In this case, and more strongly in the case of the tyrocidines, which will be discussed later, nonideal distribution behavior was encountered, resulting in skewed distribution patterns. The series of papers dealing with the structure of the bacitracin antibiotics 12-~ served to show that reliable and clear-cut structural work could be done without achieving a single crystalline preparation along the way. Acceptance of this view was greatly supported by the development of quantitative amino acid analysis and the various techniques developed for the determination of the amino acid sequence of proteins. Several years after our CCD studies showing that crystalline preparations of tyrocidine and gramicidin could be resolved into individual molecular species differing only in their amino acid content, Brockman and 11L. K. Ramachandran, Biochemislry 2, 1138 (1963). W. Hausmann, J. R. Weisiger, and L. C. Craig, J. Amer. Chem. Sac. 77, 723 (1955). 1, L. C. Craig and W. Konigsberg, ]. Org. Chem. 22, 1345 (1957). 14L. C. Craig, W. Hausmann, and J. R. Weisiger, J. Amer. Chem. Sac. 76, 2839 (1954).
[16]
mOLATION OF ANTIBIOTICS BY CCD
327
his collaborators duplicated our procedures with the actinomycin antibiotics. The method of countercurrent distribution had been introduced into Germany in a series of lectures delivered at various universities in 1948.15 Brockman and PfenningTM first demonstrated that the crystalline preparation of aetinomycin C was impure with the system ethylether/5.6% HC1, but it was not a satisfactory system because of a slow transformation of the antibiotic at the low pH. Moreover, low solubility of the antibiotic in the aqueous phase limited the choice at a higher pH. They overcame this difficulty by using 30% urea for the lower phase and a mixture of methyl-n-butyl ether and dibutyl ether for the upper phase. TM This system resolved the preparation of actinomycin into four bands. The urea in the aqueous phase provided much greater solubility and probably had a beneficial effect in promoting dissociation of aggregates, thus providing a linear partition isotherm. Another additive to water for the aqueous phase was sodium naphthalene fl-sulfonate. Each of the actinomyein families A, B, C, and X was resolved into several individual peptide antibiotics. Countercurrent distribution thus made possible an extensive study of a large class of interesting natural products. Brockman and his collaborators later developed systems for separation and identification of individual actinomycins by chromatography, as has often been done with other antibiotics and natural.products after the initial breakthrough has been made with CCD.
C. Testing for Purity The general rules for establishing purity by CCD were developed during World War II in the antimalarial field. 1. The method even in its clumsy stage of development at that time revealed substantial amounts of impurities in the standard reference samples of such antimalarials as Atabrine, Chloroquine, and Plasmoquine, which had been carefully prepared to serve as controls and which had been documented as to purity with every other technique available at the time. The impact of these results was decisive and CCD became the final test of purity for every preparation of a new antimalarial before it. was put into clinical trial. Countercurrent distribution came into use in the antibiotic field toward the close of the war with penicillin. Earlier preparations of amorphous penicillin had been so successful in the treatment of syphilis that a single large injection was sufficient to eliminate the infection and this procedure had become widely used in the Army. Unexpectedly, however '° L. C. Craig, Fortschr. Chem. Forsch. 1, 292 (1949). '" H. Broekmann and N. Pfenning, Naturwissenscha]ten 39, 429 (1952). 1, L. C. Craig, H. Mighton, E. Titus, and C. Golumbie, Anal. Chem. 20, 134 (1948)
328
METHODS FOR THE STUDY OF ANTIBIOTICS
[16]
large numbers of cases began to reappear with the disease, and it was feared that the antibiotic was losing its effectiveness owing to mutation of the parasite, as was known to occur with streptomycin for the treatment of tuberculosis. At about this time Craig and Hogeboom had secured samples of amorphous penicillin from different producers and had developed systems for fractionating them? 8 Systems made from an organic solvent such as ethyl acetate or isopropyl ether equilibrated with 2 M P04 buffer adjusted to a suitable pH proved ideal for the separation of the four different penicillins known at that time, G, K, F, and X. The proportions of the four penicillins were found to be different in the preparations from the different producers. The separation was superior to that obtainable by chromatographic techniques in the state of the art at that time even though only 25 tubes were available in the rather clumsy steel distribution apparatus. It so happened that yield of antibiotic activity in terms of assay units was the control used for choosing the optimum strain of mold for producing the amorphous antibiotic. The CCD analysis, however, showed that this led to preparations consisting almost exclusively of penicillin K. This type of penicillin partitioned most in favor of the organic phase and was found to be eliminated most rapidly from the body through the kidneys, presumably before it could be effective in curing the infection. This seemed to explain the loss of effectiveness of the preparations then being used. In any case the producers converted their process to the preparation of crystalline penicillin G in a remarkably short time, and the effectiveness of the commercial antibiotic was thus restored. A large reference sample of crystalline penicillin G was prepared and carefully standardized for purity. CCD in the phosphate systems played a major role in this work. D. Molecular Weight Determination by the Method of Partial Substitution The determination of the true molecular weight of polypeptide antibiotics can often present a problem when they show a strong tendency to associate, as is the case with the tyrocidine antibiotics. A general and reliable way of overcoming this difficulty was devised for gramicidin S which involved partial substitution of a functional group and subsequent fractionation of the mixture by countercurrent distribution. 19 Amino acid ~gL. C. Craig, G. H. Hogeboom, F. H. Carpenter, and V. du Vigneaud, J. Biol. Chem. 168, 665 (1947). 1, A. R. Battersby and L. C. Craig, J. Amer. Chem. Soe. 73, 1887 (1951).
[16]
ISOLATION
OF ANTIBIOTICS
329
B Y CCD
analysis had shown the presence of five different amino acids in equal amount. Since no free carboxyl group or alpha-amino group could be detected, it was thought to be most likely a cyclic pentapeptide with the single delta amino group of the ornithine free to account for the basic properties. There remained the possibility that it could be a cyclic decapeptide, a pentadecapeptide or even larger. The method devised for unequivocally settling this point was based on the theory that a reagent such as fluorodinitrobenzene, reacting with the amino group, if added in insufficient amount to cover all the amino groups would result in a mixture of all possible forms--partially substituted, completely substituted, and unsubstituted peptide. This theory was substantiated for gramicidin S 19 as shown in Fig. 4. Here the partially substituted reaction mixture was distributed to 120 transfers in a 100tube train. Analysis was made by weight and by absorbance at 350 nm. The right portion of the pattern containing the unsubstituted peptide is the effluent or withdrawal series. The fundamental series contained two well-separated bands both of which showed absorbance at 350 nm. There-
0.9 I " 0.8 0.7
¢o
0 cw
"C3 0.6 8 -~ o.5 E 0.4
~
F-~
~-m ,~
/
0.3 0.2
-I
0.1
t,. 0
20
, 40
J1 60
Tube number
80
I00
120
IO0
Tronsfer number
FIG. 4. Countercurrent distribution pattern of partially substituted DNP gramicidin S.
330
METHODS FOR THE STUDY OF ANTIBIOTICS
[15]
fore, two substitutable amino groups were shown to be in the gramicidin-S molecule. This demonstrated that it was a decapeptide. Moreover, from the known extinction coefficient of a DNP-amino acid and the weight extinction ratios of each band at 350 nm the molecular weight of the solute in each band could be calculated. The correct molecular weight of a number of antibiotic polypeptides has been established by this general method.
E . I s o l a t i o n o f F r a g m e n t s in Structural S t u d i e s
Antibiotic polypeptides nearly always contain amino acid residues different from the known amino acids derived from proteins. They often contain carbohydrate moieties, cyclic structures, etc. Partial hydrolysis by acid is required since the known proteolytie enzymes usually do not bring about selective hydrolysis. CCD is excellent for fraetionating the complex mixtures resulting from acid hydrolysis. It not only offers adequate selectivity, but has sufficient capacity so that further structural studies can be carried out on a fragment when it is needed. The covalent structures of the tyrocidins,2° the polymyxins,21 the bacitracins, 1~ subtilin, 22 nisin, 23 and many others were determined this way.
With polymyxin B1 21 difficulty was encountered in separating the partial hydrolysis mixture because of the polarity of the products. This was overcome by converting the antibiotic to the DNP derivative and subjecting that to partial hydrolysis. It was found that attachment of the DNP group greatly reduced the polarity and thus provided a wider range of systems and in addition greatly reduced the dialyzability of the peptides. A reasonably clean group separation of the derivatized from free peptides was easily accomplished by dialysis before applying CCD. Derivatized fragments were also found to be more suited to CCD separation in the partial hydrolysis studies leading to the sequence of bacitracin n. 12 Recently Gross and co-workers 22 have used CCD extensively not only in purifying subtilin starting material, 24 but also in isolation of the hydrolysis fragments leading to the determination of its covalent structure. It contains 32 amino acid residues and could be fragmented enzymically 20T. P. King and L. C. Craig, J. Amer. Chem. Soe. 77, 6671 (1955). ~1W'. Hausmann,J. Amer. Chem. Soe. 78, 3663 (1956). ~2H. H. Kiltz and E. Gross, Hoppe 8eyer's Z. Physiol, Chem. 354, 802 (1973). 2aE. Gross and J. L. Morell, J. Amer. Chem. Soe. 93, 4634 (1973). ~4A. Stracher and L. C. Craig, J. Amer. Chem. Soe. 81, 696 (1959).
[16]
ISOLATION OF ANTIBIOTICS BY CCD
331
and with B r C N as is customary with proteins. It contains five rings in which each contains a single S atom. The same workers in this elegant study have also determined the covalent structure of nisin, '-'3 a closely related antibiotic polypeptide with 34 amino acid residues and five rings. Both antibiotics contain dehydro peptide linkages and thus have poor stability. I I I . N o n i d e a l i t y in C C D
Polypeptide antibiotics often show a strong tendency to associate in aqueous solution, particularly if the solution contains inorganic salt. Salt tends to increase hydrophobic interactions which seems to be the main parameter promoting association. ~ The tyrocidine polypeptide antibiotics are outstanding examples of association phenomena. T h e y were first resolved into individual peptides by countercurrent distribution and their monomeric molecular weights established by the partial substitution method. 4 Their association properties have been studied by sedimentation, ~'~ nuclear magnetic resonance, 26 thin fihn dialysis, ~7 and by countercurrent distribution. '-'~ In the latter, the complex intermolecular interaction is reflected by the obvious deviation from ideality shown in Fig. 5. Here at the start where the solute concentration is highest, there is little resolution up to 530 transfers apparently due to the heterointeraction of the A, B, and C species. As the distribution proceeds to higher numbers of transfers and the concentration drops, bands appear by 1030 transfers. The separation improves up to 2050 transfers but by 3000 transfers has deteriorated. Apparently homo-association is favored after 530 transfers, but after 2050 transfers the partition isotherm is such that dilution favors nonideality and the separation suffers. The isotherms of the individual tyrocidines are shown also in Fig. 5. It was of interest that a strong dissociating system which gave a linear partition gave a very poor resolution of A, B, and C. 29
IV. S y s t e m s
The most important consideration for the application of countercurrent distribution to the isolation of polypeptide antibiotics is the choice :~ S. L. Laiken, M. P. Printz. and L. C. Craig, Biochem. Biophys. Res. Comrm~n. 43, 595 (1971). :" A. Stern, W. A. Gibbons, and L. C. Craig, J. Amer. Chem. Soc. 91, 2794 ('1969). 27M. Burachik, L. C. Craig, and J. Chang, Biochemistry 9, 3293 (1970). :s R. C. Williams and L. C. Craig, Separ. Sci. 2, 487 (1967). ,-9M. A. Ruttenberg, T. P. King, and L. C. Craig, Biochemistry 5, 2857 (1966).
332
METHODS
FOR
THE
STUDY
[16]
OF ANTIBIOTICS
K=2.03 75
550
2050
fransfers
transfers
- Recycle begun at
!100
transfers
K=1.87
50
yrocidine
C
~1~ ~ 2"57 25 0OJ ~E~L o
~ B
~ ~emoved i
o= oc
200 |030
L......
400 K= 1.90
r
600
3000
T
200
,
,
,
400
-i
~
600
/
transfers
o
~ 5o
Tyrocidine A Partition coefficients as function of concentration in system 0.01NHCI: Chloroform:Methanol I : 2 : 2
transfers
K =2.81 K= 1.91
0
i
B
5
I0
Conch in upper phase
25
0~ 40O
(mg/ml)
K=2.51
600
800
600
SO0
0
200
400
Tube number
Fro. 5. Countercurrent distribution patterns of the separation of individual tyrocidines and partition isotherms of tyrocidines A, B, and C.
of a system with good capacity and high selectivity. There appears to be no substitute for experience in the choice of solvents for this problem. Theoretical predictions have been of little aid. The organic chemist in trying to choose a solvent for fractional crystallization gains experience in solubility properties and similarly can learn to manipulate solvents to achieve favorable partition ratios with given solutes. Partition ratios are often referred to as a measure of relative solubilities in the two phases of the system. This view is much too simple and does not give the correct relationship. In a given system the limit of solubility can be reached in one phase but not in the other although the two are in equilibrium. At this point a rather sharp break in the partition isotherm will occur. A partition ratio seems to be a balance of molecular interactions which in general are the same forces which determine solubility but whose relative importance are different in the two solvent environments and are unpredictable. In choosing a system it is helpful to have a list available of the systems which have been of practical use in the isolation of polypeptide
[161
ISOLATION OF ANTIBIOTICS BY CCD
333
antibiotics. Such a list is given in the table. No attempt has been made to make this a complete survey of the literature. In this list the number of transfers used is also given. If a system has a high selectivity or if the separation is relatively easy, relatively few transfers are required. The majority of separations listed in the table have not been made with high numbers of transfers. This may be a reflection of the lack of suitable equipment as well as good selectivity. In any case the full separating potential of the method has either not been needed or has not been utilized. One of the major advantages of CCD as compared to other separation techniques offering high separating power is the way it can be scaled up to give gram quantities of pure materials or even larger amounts. For this purpose the first consideration is the choice of a system with high capacity as well as good selectivity. For example, in the separation of the streptomycins there was the problem of compounding a system with sufficient solubility in the organic phase. The streptomycins are highly polar solutes. This was solved by adding so-called "carriers ''3" to the system. These were long-chain fatty acids which had the effect of bringing a higher proportion of the antibiotic into tile organic phase by associating strongly with the polar solute, and thus reducing its polarity. Titus and Fried 31 had earlier shown that p-toluenesulfonic acid had this property. The sulfonic acids, other detergents and trichloroacetic acid have this effect, probably by ion pairing. O'Keefe e t a l 2 2 worked out a steady-state scheme for large-scale separation of the streptomycins in these systems using separatory funnels. The more recently developed CDCD train can be set to follow this scheme automatically, and thus save the labor involved in the use of separatory funnels. When it is desired to influence the partition ratio in favor of the more polar phase which almost always is mostly water, additives which change the water structure can be effective. Urea or formamide or salts which have a so-called "salting in" effect can be used. Phenol is a particularly interesting solvent for highly polar solutes. It forms a suitable system with water in which the phenol phase contains about 12% water at room temperature and has broad solubility properties. It is the heavier phase. The surface tension properties are such that the phases separate rather slowly, and not always cleanly. Nonetheless, the capacity and selectivity of the system are usually quite good. Addi3OG. W. E. Plaut and R. B. MeCormack, J. Amer. Chem. Soc. 71, 2264 (1949). 31E. Titus and J. Fried, J. Biol. Chem. 168, 393 (1947). ~ A. E. O'Keefe, M. A. Dolliver, and E. T. Stiller, J. Amer. Chem. Soc. 71, 2452 (1949).
334
METHODS FOR THE STUDY OF ANTIBIOTICS
[16]
SOLVENT SYSTEMS FOR THE COUNTERCURRENT DISTRIBUTION OF ANTIBIOTICS: PART A
Antibiotic (s)
Number of transfers
Actinomyein C, C~, C~, Ca
System
180
Methyl butyl ether + dibutyl ether (71:29)/30% urea a Actinomycin X, X0, X~, X.o 243 Methyl butyl ether/1.5 % Na naphthalene fl-sulfonateb Actinomycins 40 Ethyl ether/5.6 % HC1c 4O Dibutyl ether + methyl butyl ether (71:29)/5 % Na xylol sulfonate c 254 Methyl-n-butyl ether/1.75% Na naphthalene fl-sulfonate d Actithiazic acid 24 Benzene/petroleum ether/80% aqueous ethanol (3:2:5) ~ 24 n-Butanol/1 M phosphate, pH 6.94/ Aglycones of olivomycin 49 Ethyl acetate/hexane/ethanol/H20 (8: 4: 6: 7)a and chromomycin Aa CH2CI~h Amicetin Angustmycin A 40 n-Butanol/pyridine/H20 (100: 1: 100) i Antifungal polypeptide 25 n-Butanol/0.1 M phosphate pH 8.7i produced by B. subtilis Antimycins 800 CC14/Skellysolve B/methanol/H20 (80:20:87: 13) k 40 CC14/methanol/H20 (10: 7:1) z Aureomycin and terra100 n-Butanol/0.01 N HC1~ mycin BA-90, 912 150 n-Butanol/1% acetic acid n BA-181, 314 100 CHC13/ligroin/methanol/H20 (3 : 1 : 3 : 1) ° BA-6903 100 CHC13/ligroin/methanol/H:O (3: 1:3:1) p Bacitracins 221 Amyl alcohol~n-butanol~50 mM phosphate, pH 7.0 (4:1:5)q 150 Amyl alcohol/n-butanol/50 mM phosphate, pH 7.0 (1:1:2) r 2-Butanol/1.7 % aqueous acetic acid' 90O 2-Butanol/3 % aqueous acetic acid t 241 CHC13/methanol/H20 (2: 2 : 1)" Bostrycoidin 24 CCl4/aqueous NH4OH, pH 10~ Cephalosporins 15 Hexane/i-propyl ether/acetone/0.5 M phosplmte, pH 6.0 (25:8:25:25) w 8O CC14/phenol/2,4,6 trimethylpyridine/H20/10 N H2SO4 (40:40:7.1:350:1) x 8O 2-Butanol/ethanol/0.1 M Na~HPO~/0.1 M citric acid/(NH4)2SO4 (5 ml : 30 ml: 50.5 ml : 14.7 ml : 30 g)~ .a H. Brockmann and N. Pfennig, Naturwissenschaften 39, 429 (1952).
[15]
ISOLATION OF ANTIBIOTICS BY CCD
335
b H. Brockmann, H. Longe, and H. GrSne, Naturwissenschaften 40, 224 (1953). H. Brockmann and N. Pfennig, Hoppe-Seyler's Z.Physiol. Chem. 292, 77 (1953). H. Brockmann and H. GrSne, Chem. Ber. 87, 1036 (1954). H. G. Schneider, G. M. Toner, and F. M. Strong, Arch. Biochem. Biophys. $7, 147 (1952). s R. Schenk and A. F. DeRose, Arch. Biochem. Biophys. 40, 263 (1952). M. G. Brazhnikova, E. V. Krugliak, and A. S. Mesentsev, Antimierob. Ag. Chcmother. 1965, 858. h C. DeBoer, E. L. Caron, and J. Hinman, J. Amcr. Chem. Soc. 75, 499 (1953). i H. Yunsten, K. Ohkuma, Y. Ishii, and H. Yonehara, J. Antibiot. 9A, 195 (1956). i J. Babad, A. Pinsky, R. Turner-Graft, and N. Sharon, Nature (London) 170, 618 (1952). k W. Lin and F. M. Strong, J. Amer. Chem. Soc. 81, 4387 (1959). t y. Harada, K. Uzu, and M. Asai, J. Antibiot. l l A , 32 (1958). m R. J. Hickey and W. F. Phillips, Anal. Chem. 9.6, 1640 (1954). " K. V. Rao and l). W. Renn, Antimicrob. Ag. Chemother. 1965, 77. " l). W. Renn, M. Kugelman, and K. V. Rao, Antimicrob. Ag. Chemothcr. 1963, 80. ~' K. V. Rao and S. C. Brooks, Antimicrob. Ag. Chemother. 1961, 491. q G. G. F. Newton and E. P. Abraham, Biochem. J., 47, 257 (1950). G. C,. F. Newton, E. P. Abraham, H. W. Florey, N. Smith, and J. Ross, Brit. J. Pharmacol. 6, 417 (1951). G. T. Barry, J. ]). Gregory, and L. C. Craig, J. Biol. Chem. 175, 485 (1948). t L. C. Craig, J. R. Weisiger, W. Hausmann, and E. J. ttarfenist, J. Biol. Chem. 199, 259 (1952). " W. Konigsberg, and L. C. Craig, J. Org. Chem. 27, 934 (1962). F. J. Cajori, T. T. Otani, and M. A. Hafflilton, J. Biol. Chem. 208, 107 (1954). ~' tI. S. Burton and E. P. Abraham, Biochem. J. 50, 168 (1951). E. P. Abraham, G. G. F. Newton, and C. W. Hale, Bioehem. J. 58, 94 (1955). tion of a l i t t l e n - b u t a n o l or acetic a c i d i m p r o v e s t h e s e p a r a t i o n of t h e phases. P h e n o l v a p o r is q u i t e toxic which r e s t r i c t s its use to C C D t r a i n s in a good hood or c a r r i e d out in t h e a u t o m a t i c t r a i n s enclosed in a hood. I n general c o u n t e r c u r r e n t d i s t r i b u t i o n has been used for s e p a r a t i o n s of a c t i v e p r i n c i p l e s o n l y a f t e r a t t e m p t s w i t h o t h e r p r o c e d u r e s h a v e failed. T h i s has been t r u e e s p e c i a l l y w i t h t h e p o l y p e p t i d e hormones. O b v i o u s l y the k e y to success lies in finding a s y s t e m w i t h o p t i m u m s e l e c t i v i t y . P h e n o l - w a t e r s y s t e m s h a v e s e l e c t i v i t i e s different f r o m a l m o s t a n y o t h e r s y s t e m s a n d in a d d i t i o n h a v e a high d i s s o c i a t i n g p o w e r for those solutes which h a v e a s t r o n g t e n d e n c y to self-associate. A good e x a m p l e of t h e a d v a n t a g e of these s y s t e m s is f o u n d in t h e i s o l a t i o n of t h e edeine antii)iotics f r o m a s t r a i n of Bacillus brevis. T h e successful e l u c i d a t i o n of t h e s t r u c t u r e s 33 shown in Fig. 6 could n o t h a v e been done w i t h o u t C C D in t h e phenol s y s t e m s . T h e edeines are v e r y b a s i c a n d e x t r e m e l y polar. A f t e r e x t e n s i v e t r i a l s w i t h all o t h e r s e p a r a t i o n t e c h n i q u e s i n c l u d i n g c h r o m a t o g r a p h y , a n d C C D in alcohol s y s t e m s t h e C C D p a t t e r n shown in Fig. 7 3.~T. P. Hettinger and L. C. Craig, Biochemistry 9, 1224 (1970).
336
METHODS FOR THE STUDY OF ANTIBIOTICS
[16]
SOLVENT SYSTEMS FOR THE COUNTERCURRENT DISTRIBUTION OF ANTIBIOTICS: PART B
Antibiotic (s)
Number of transfers
Celesticetin Chloramphenicol
200 24
Cirramyein Clupeine and salmine Comirin Coumeromycins
50 150 20 200
Cycloheximide
12 48 200
Demetric acid Diazomycins DNP-bacitracins
100 183 209 203
DNP-streptothricin
60
Drosopholins A and B
8 8 54 60
Duramycin Endomycins Etamycin Fervenulin Flaveolin Gramicidin S Gramicidins (linear) Grisein Griseoluteins H-277 (streptothricinolike)
24 -
-
9 36 36 100 2000 15 l0 29
System n-Butanol/acetic acid/H20 (4: 1:5) ~ Heptane/i-amyl alcohol/0.2 M phosphate, pH 6.7 (79:21:100) b Benzene/SSrensen's buffer pH 8.0 c n-Propanol/3 M sodium acetate (3:2) d n-Butanol/10% aqueous pyridine (1:5) e CHCla/i-propanol/O.067 M phosphate, pH 7.0 and 8.2 (1:1:2)s Benzene/H2Oo CCl4/CHC13/methanol/H20 h Benzene/Skellysolve B/methanol/H20 (3:2:4:1) ~ Phenol/H~Oi 2-Butanol/3% aqueous acetic acid k CHC13/acetic acid/H20 (2:2:1) k Benzene/CHCl~/aeetic acid/0.1 N HCI (1:1"2:1) 1 n-Butanol~methanol~acetic acid/H20 (18:2: 1:19) ~ Hexane/ether/50% aqueous methanol (2:3:5)" CHCI3/0.5 M phosphate, pH 6.9 ° n-Butanol~0.2 M NH4OAc pH 5.35p Ethyl acetate/n-propanol/O.1 M NH4OAc (3:1:3)q Benzene/heptane/methanol/O.125% Na~SO~r Benzene/methanol/H~O (10: 10:2) * n-Butanol/0.067 M phosphate pH 5.5 t CHCl~/methanol/0.01 N HCI (10:7:3)" CHC13/85% aqueous methanol ~ Benzene/CHCls/methanol/H.~O (15: 15:23 : 7) ~ Benzene/CHCls/methanol/H:O (15: 15:23: 7) ~ 10% (w/v) phenol in CHCIJ2 M phosphate pH 6.7 • Methyl-isobutyl ketone/0.5 M phosphate pH 6.0u n-Butanol/5% aqueous p-toluenesulfonic acid"
a H. Hoeksema, G. F. Crum, and W.H. DeVries, Antibiot. Annu. 1964]1966, 837. b A. J. Glazko, W. A. Dill, and M. C. Rebstock, J. Biol. Chem. 183, 679 (1950). oH. Koshiyama, M. Okanishi, T. Ohmori, T. Miyaki, M. Matsuzaki, and H. Kawaguchi, J. Antibiot. 16A, 59 (1963). d F. S. Scanes and B. T. Tozer, Biochem. J. 63, 565 (1956). e W. G. C. Forsyth, Biochem. J. 69, 500 (1955). s j. Berger, A. J. Schocher, and H. Spiegelberg, Antimicrob. Ag. Chromother. 1966, 778.
[16]
ISOLATION OF ANTIBIOTICS BY CCD
337
0 J. H. Ford and B. E. Leach, J. Amer. Chem. Soc. 70, 1223 (1948). h R. Corbaz, V. Prelog, and H. Zahner, Helv. Chim. Acta 38, 1445 (1953). i R. L. DeVault, H. Schmitz, and I. R. Hooper, Antimicrob. Ag. Chemother. 1965, 776. i K. V. Rao, S. C. Brooks, Jr., M. Kugelman, and A. A. Romano, Antibiot. Annu. 19§9/1960, 943. k L. C. Craig, W. Hausmann, and J. R. Weisiger, J. Biol. Chem. 200, 765 (1953). l j. R. Weisiger, W. Hausmann, and L. C. Craig, J. Amer. Chem. Soc. 77, 3123 (1955). m T. Goto, Y. Hirata, S. Hosoya, and N. Komatsu, Bull. Chem. Soc. Jap. 30, 729 (1957). '~ F. Kavanagh, A. Hervey, and W. J. Robbins, Proe. Nat. Acad. Sci. U.S. 37, 570 (1051). o F. Kavanagh, A. Hervey, and W. J. Robbins, Proc. Nat. Acad. Sci. U.S. 38, 555 (1952). , O. L. Shotwell, F. H. Stodola, W. R. Michael, L. A. Lindenfelser, R. G. I)worschack, and T. G. Pridham, J. Amer. Chem. Soc. 80, 3912 (1958). q L. C. Vining and W. A. Taber, Can. J. Chem. 35, 1461 (1967). r Q. R. Bartz, J. Standiford, and T. H. Haskell, Antibiot. Annu. (1954/1955), 777. 8 T. E. Eble, E. C. Olson, C. M. Large, and J. W. Shell, Antibiot. Annu. 1959/1960, 227. t B. Takahashi, J. Antibiot. 6A, 11 (1953). u R. Schwyzer and P. Sieber, Helv. Chim. Acta 40, 624 (1957). v j. l). Gregory and L. C. Craig, J. Biol. Chem. 172, 839 (1948). w L. K. Ramachandran, Biochemistry 2, 1138 (1963). F. A. Kuehl, Jr., M. N. Bishop, L. Chaiet, and K. Folkers, J. Amer. Chem. Soe. 73, 1770 (1951). F. Tausig, F. J. Wolf, and A. K. Miller, Antimicrob. Ag. Chemother. 1964, 59. N. Komatsu and Y. Saburi, J. Antibiot. 5, 522 (1952).
was obtained at 600 transfers 34 with the system made by equilibrating 80% phenol with 0.15 M ammonium acetate-0.30 M acetic acid. For analysis ether was added to the tube to be analyzed. This transferred
RNH~NH~NH~I1~NH OH 0 O~ NH2~/\OH o
OH
0 O~0 H
FXG. 6. Structure of edeines. R = H or - C ( N H : ) = N H ~4T. P. Hettinger and L. C. Craig, Biochemislry 7, 4153 (1968).
338
METHODS FOR THE STUDY OF ANTIBIOTICS
[15]
SOLVENT SYSTEMS FOR THE COUNTERCURRENT DISTRIBUTION OF ANTIBIOTICS: PART C
Antibiotic (s) Hamycin Homomycin Hygromycin Leucomycins (erythromycin-like) Levomycin Levorins Licheniformins Lincomycins
Luteomycin Magnamycin Micrococcin Monamycins
Number of transfers 300 40 40 100 30 54 60 24 1000 930 500 60 60 8 ---
Monazomycin Myeobacillin Mycomycin Neamine and neomycin Neomycin
40 24 8 90 -
-
-
-
49 Nisin fragments Nisins
PA-114 A and B PA-312
150 20 336 49 49 100
System
CHCla/methanol/0.1 M sodium acetate (2:2:1) a n-Butanol/0.25 M phosphate pH 4.6 b n-Butanol/ethyl acetate/H~O (1.2: 0.5: 1.9) b n-Butanol/H~O adjusted to pH 3 with acetic acid c Benzene/petroleum ether/phosphate buffer, pH 7.25 (35:65:100) d Benzene/methanol/H20 (5:5:1) e CHC18/methanol/borate buffer pH 8.2s Phenol/0.003 N HClg n-Butanol/H20 adjusted to pH 4.2 with HC1h n-Butanol/H~O ~ 2-Butanol/H2Oi Ethyl acetate/McIlvaine's buffer pH 5.8 k Benzene/sodium acetate pH 4.5 l CC14/CHC13/acetic acid/H~O/ethanol" Ethyl acetate/cyclohexane/methanol/H.~O (12: 10: 10: 7) n 60-80 ° bp petroleum ether/methanol/H~O (10:10:1) ~ n-Butanol/acetic acid/H20 ° n-Butanol/0.1 M phosphate pH 8.7 p CHC13/2% aqueous phosphate pH 7.0 q n-Butanol/5 % aqueous 2-ethyl-butyric acid pH 6.9 ~ n-Butanol/aqueous p-toluenesulfonic acid" Pentasol/borate buffer pH 7.3 or 7.6 t n-Butanol/acetic acid/H~O (3 : 1 : 4) ~ Methanol~n-butanol/acetate buffer ~ n-Butanol~methanol~2 N acetic acid, 0.25 M NaCl, adjusted to pH 3 with NaOH w n-Butanol/aqueous ammonium acetate pH 2.8-3.0 • Benzene/methanol/H20 (2:1: 1)Y Toluene/methanol/H20 (4:3: 1)~ Toluene/60% aqueous methanol"
a B. N. Ganguli and V. M. Doctor, Appl. Microbiol. 16, 43 (1967). b K. Isono, S. Yamashita, Y. Tomiyama, S. Suzuki, and H. Sakai, J. Antibiot. 10A, 21 (1957). c R. L. Mann, R. M. Gale, and F. R. Van Abeele, Antibiot. Annu. 196311964, 167. a y. Sano, J. Antibiot. 7A, 93 (1954). e H. E. Carter, C. P. Schaffner, and D. Gottlieb, Arch. Biochem. Biophys. 53, 282 (1954).
[15]
ISOLATION OF ANTIBIOTICS
BY
CCD
339
I V. A. Tsiganov and E. P. Yakovleva, Antibiotiki (Moscow) 14, 387 (1969). a R. K. Callow and T. S. Work, Biochem. J., 558 (1952). h A. D. Argoudelis, J. A. Fox, and T. E. Eble, Biochemistry 4, 698 (1965). A. D. Argoudelis, J. A. Fox, and T. E. Eble, Biochemistry 4, 710 (1965). J R. R. Herr and M. E. Bergy, Antimicrob. Ag. Chemother. 1962, 560. k S. Hosoya, N. Komatsu, M. Soeda, Y. Saburi, K. Okada, and S. Wutanabe, J. Antibiot. 6B, 1 (1953). R. L. Wagner, F. A. Hochstein, K. Murai, N. Messina, and P. P. Regna, J. Amer. Chem. Soc. 76, 4684 (1953). m N. G. Heatley and H. M. Doery, Biochem. J. 50, 247 (1951). " C. H. Hassall and K. E. Magnus, Nature (London) 184, 1223 (1959). " K. Akasaki, K. Karasawa, M. Watanabe, H. Yonehara, and H. Umezawa, J. Antibiot. 16A, 127 (1963). p J. Babad, A. Pinsky, R. Turner-Graft, and N. Sharon, Nature (London) 170, 618 (1952). q W. 1). Celmer and I. A. Solomons, J. Amer. Chem. Soc. 74, 2245 (1952). r B. E. Leach and C. M. Teeters, J. Amer. Chem. Soc. 73, 2794 (1951). R. L. Peck, C. E. Hoffhine, Jr., P. Gale, and K. Folkers, J. Amer. Chem. Soc. 71, 2590 (1949). rE. A. Swart, D. Hutchison, and S. Waksman, Arch. Biochem. Biophys. 24, 92 (1949). " E. Gross and J. L. Morell, J. Amer. Chem. Soc. 9 3 , 4634 (1971). N. J. Berridge, G. G. F. Newton, and E. P. Abraham, Biochem. J. 52, 529 (1952). N. J. Berridge, Nature (London) 169, 707 (1952). G. C. Cheeseman and N. J. Berridge, Biochem. J. 71, 185 (1959). W. D. Celmer and B. A. Sobin, Antibiot. Annu. 1956/19[i6, 437. B. K. Koe, B. A. Sobin, and W. D. Celmer, Antibiot. Annu. 1966/1957, 672.
12 4,8 44 I 1.o 4.0 36 0.8"~ 32 ? I,& 28 vie
0.6 ~
A
.
?
,
2.4
v
II --OJ
0
0.4 ~
E 02
I
f,
E E
t
2.0
16 12
25
i/,/
j~23 8
08 0.4
0
100
~ . ' / 2oo
~ ' r - - - _ - . - ~ 3oo 40o Tube No.
5O0
Fro. 7. Countercurrent distribution pattern showing the isolation and separation of the edeines.
340
METHODS FOR THE STUDY OF ANTIBIOTICS
[16]
SOLVENT SYSTEMS FOR THE COUNTERCURRENT DISTRIBUTION OF ANTIBIOTICS: PART D
Antibiotic(s)
Number of transfers
Peliomycin Penicillins
Polymyxins
Polypeptin Protactinomycin-like antibiotic Protactinomycins Protomycin Quadrifidins
100 100 24 24 36 11 1789 1332 329 624 24
System CC14/CHC13/methanol/H.,O (2:2:3:1) a Benzene/Skellysolve B/80% ethanol (2:3:5) ~ Ethyl ether/2 M phosphate pH 4.8b Ether, furan, CHC18, isopropyl ether or ethyl acetate vs 1-3 M phosphate, pH 4.60-5.12 ~ CttC18/1 M phosphate pH 5.3 a Methyl amyl acetate/1 M phosphate pH 5.2 ~ 2-Butanol/0.1 h r HCll n-Butanol/2-butanol/0.1 N HC1 (6:30:40)a n-Butanol/2 % dichloroacetic acid (6: 7)a 2-Butanol/isopropyl ether/0.1 N HC1 (2: 1:3) ^ Ether/0.5 M phosphate pH 6.8 i
24 30 8 8 8
Ether/67 mM phosphate, various pH valuesi Benzene/methanol/0.001 N HC1 (10:2:8) k Benzene/hexane/H20 (1 : 25 : 26) l Benzene/H20 ~ Quinocyclines CC14/CH2CI~/H20 (1:1:2) adjusted to pH 4.1 with acetic acid m Rhodomycins 24-550 n-Butanol/O.1 M phosphate, pH 7.2, other pH's 6.0, 7.5~,o,~ Rifamycins 100 Benzene/petroleum ether/methanol/0.01 N HC1 (15:5: 10:5)q Rotaventin 11 Benzene/CHC13/methanol/H20 (15: 30:23 : 7 ) ~ Rotaventin-like anti11 Benzene/CHC13/methanol/H20 (15:30:23:7)' fungal antibiotic Rubidin 24 Ether/H~O adjusted to pH 4 with HCI t Rubiflavin 40 Ethyl acetate/0.1 M phosphate pH 6.5 ~ Rubradirin 200 Hexane/acetone/H~O (5 : 5 : 1)" Rubromycin -Benzene/dioxane/H20 (3: 12:2) w Staphylomycin 100 Toluene/methanol/H~O (4 : 3 : 1)• 35 Methyl isobutyl ketone/50 mM phosphate pH 6.0~ Stendomycin a H. Schmitz, S. B. Deak, K. E. Crook, Jr., and I. R. Hooper, Antimicrob. Ag. Chemother. 1963, 89. b L. C. Craig, G. H. Hogeboom, F. H. Carpenter, and V. du Vigneaud, J. Biol. Chem. 168, 665 (1947). G. T. Barry, Y. Sato, and L. C. Craig, J. Biol. Chem. 174, 221 (1948). d E. Martin, J. Berky, C. Godzesky, P. Miller, J. Tome, and R. W. Stone, J. Biol. Chem. 203, 239 (1953). C. R. Barrels and M. A. Dolliver, J. Amer. Chem. Soc. 72, 11 (1950). ] W. Hausmann and L. C. Craig, J. Amer. Chem. Soc. 76, 4892 (1954). a T. Suzuki, H. Inouye, K. Fujikawa, and Y. Suketa, J. Biochem. 54, 25 (1963).
[16]
ISOLATION OF ANTIBIOTICS BY CCD
341
h W. Hausmann and L. C. Craig, J. Biol. Chem. 198, 405 (1952). ~M. N. Donin, J. Pagano, J. D. Dutcher, and C. M. McKee, Antibiot. Annu. 1955/1954, 179. J R. Q. Marston, Brit. J. Exp. Pathol. 30, 398 (1949). k K. Sugamara, J. Antibiot. 16A, 115 (1963). i l l . M. Doery, J. F. Gardner, H. S. Burton, and E. P. Abraham, Antibiot. Chemother. 1, 409 (1951). m W. D. Celmer, K. Mural, K. V. P~ao, F. W. Tanner, and W. S. Marsh, Antibiot. Annu. 1957/1958, 484. n H. Brockmann, K. Bauer, and I. Borchers, Chem. Ber. 84, 701 (1951). ° H. Brockmann and I. Borchers, Chem. Bet. 86, 261 (1953). p H. Brockmann and P. Patt, Chem. Ber. 88, 1455 (1955). P. Sensi, A. M. Greco, and R. BaUotta, Antibiot. Annu. 1989/1960, 262. S. Hosoya, N. Komatsu, and M. Soeda, J. Antibiot. 5, 451 (1952). S. Hosoya, N. Komatsu, and M. Soeda, J. Anlibiot. 5, 525 (1952). t A. K. Banerjee, G. P. Sen, and P. Nandi, Antibiot. Annu. 1955/1956, 640. A. Aszalos, M. Jelinek, and B. Berk, Antimicrob. Ag. Chemother. 1964, 68. C. E. Meyer, Antimicrob. Ag. Chemother. 1964, 97. w H. Brockmann and K.-H. Renneberg, Naturwissenschaften 40, 59 (1953). F. W. Eastwood, B. K. Snell, and A. Todd, J. Chem. Soc. (London) 1960, 2286. R. Q. Thompson and M. S. Hughes, J. Antibiot. 16A, 187 (1963).
all the antibiotic to the aqueous phase. Two more extractions with ether removed all the phenol. For recovery the aqueous phase was lyophylized after extraction of the phenol with ether. The residue left after lyophylization was the acetate of the edeine.
V. Apparatus Used in CCD A major objection to the choice of countercurrent distribution as the separation method for a problem requiring highly selective separation is the relatively larger expenditure of time and effort required as compared to other fractionation techniques, such as chromatography. This difference can be largely overcome with the proper organization and availability of CCD equipment. Judging from the questions and statements made in the literature, it would appear that the majority of potential users of CCD do not know what is available as far as distribution trains and supporting techniques are concerned. It therefore seems desirable to include a short section that deals with the subject. There are several reasons why countercurrent distribution equipment has never received much publicity. One is that the manufacturers have been few and have been only small independent shops. In fact, the one who made the first commercially available trains and who has probably supplied the largest number in use today has never advertised his product
342
METHODS FOR THE STUDY OF ANTIBIOTICS
[16]
SOLVENT SYSTEMS FOR THE COUNTERCURRENT DISTRIBUTION OF ANTIBIOTICS: PART E
Antibiotic(s)
Number of transfers
Streptomycin
24 24 24 24 24
Streptonigrin Streptovitacin A
100 2500
Streptozotocin Subtilin Subtilin fragments Succinimycin 5-OH, 7-C1 Tetracycline
775 23 720 100 500 500 30 600 1600 2140 1205 800
Thermoviridin Tyrocidines
U12, 241 (rhodomycin-like) U-12, 898 U-13, 714 Xanthomycins Zizanin
-
-
-
-
24 50
System n-Butanol/5 % p-toluenesulfonic acid ~ n-Butanol/3 % p-toluenesulfonic acidb 5 % Stearic acid in pentasol/.5 % NaHCOa, 1% NaC1c 13% Laurie acid in pentasol/0.375 M borate, 0.12 M phosphate, final pH to 7.15 d 5 % Stearic acid in pentasol/0.5 M borate final pH to 7.3, 7.6, or 7.8~ Ethyl acetate/3% phosphate pH 7.5s n-Amyl alcohol/isoamyl alcohol/H20 (12:17:29)0 Methyl ethyl ketone/H~Oh n-Butanol (or 2-butanol)/H20i n-Butanol/acetic acid/H20 (3:1 : 4)i Benzyl alcohol/1.75 M sodium acetate pH 5.7k n-Butanol/0.01 N HC1 ~ n-Butanol/ethyl acetate/0.01 N HC1 (7:3:10) ~ n-Butanol/0.4 M phosphate pH 6.05 m CHC18/methanol/0.1 h r HC1 (2:2:1) ~ CHC13/methanol/0.1 N HC1 (2:2:1) ° Benzeue/CHC13/methanol/H~O (10: 20:23 : 7) ° CHC13/methanol/0.1 N HC1 (2: 2:1)p Pyridine/ethyl acetate/H2Oq n-Butanol/H20r 2-Butanol/H~O* CHCla/0.1 M phosphate pH 4.4t CC14/methanol/H20 (5:4:1) ~
E. Titus and J. Fried, J. Biol. Chem. 168, 393 (1947). b E. Titus and J. Fried, J. Biol. Chem. 174, 57 (1948). G. W. E. Plaut and R. B. McCormack, J. Amer. Chem. Soc. 71, 2264 (1949). d A. E. O'Keefe, M. A. Dolliver, and E. T. StilleL J. Amer. Chem. Soc. 71, 2452 (1949). E. A. Swart, J. Amer. Chem. Soc. 71, 2942 (1949). I K. V. Rao and W. P. Cullen, Antibiot. Annu. 1959/1960, 950. a T. E. Eble, M. E. Bergy, C. M. Large, R. R. Herr, and W. G. Jackson, Antibiot. Annu. 1968/1959, 555. h R. R. Herr, T. E. Eble, M. E. Bergy, and H. Jahnlee, Antibiot. Annu. 1969/1960, 236. i N. G. Brink, J. Mayfield, and K. Folkers, J. Amer. Chem. Soc. 73, 330 (1951). i H. H. Kiltz and E. Gross, Hoppe-Seyler's Z. Phylsiol. Chem. 364, 802 (1973). k T. H. Haskell, R. H. Bunge, J. C. French, and Q. R. Bartz, J. Antibiot. 16A, 67 (1963). zj. H. Martin, L. A. Mitscher, P. A. Miller, P. Shu, and N. Bohonos, Antimicrob. Ag. Chemother. 1967, 563. m D. M. Schuurmans, B. H. Olson, and C. L. San Clemente, Appl. Microbiol. 4, 61 (1956). L. C. Craig, J. D. Gregory, and G. T. Barry, Cold Spring Harbor Symp. Quant. Biol. 14, 24 (1949). ° A. R. Battersby and L. C. Craig, J. Amer. Chem. Soc. 74, 4019 (1952). T. P. King and L. C. Craig, J. Amer. Chem. Soc. 77, 6624 (1955). q C. E. Meyer, Antimicrob. Ag. Chemother. 1968, 432. " M. E. Bergy, T. E. Eble, R. R. Herr, C. M. Large, and B. Bannister, Antimicrob. Ag. Chemother. 1962, 614. • M. E. Bergy and R. R. Herr, Antimicrob. Ag. Chemother. 1964, 80. t C. B. Thorne and W. H. Peterson, J. Biol. Chem. 176, 413 (1948). K. Ishibashi, J. Antibiot. 16A, 88 (1962).
[15]
ISOLATION OF ANTIBIOTICS BY CCD
343
at a scientific meeting or in a scientific journal. This is in contrast to the advertising propaganda of the makers of chromatography equipment which constantly bombard the research worker at scientific meetings and in journals. Another reason is the fact that a train, if reasonably well taken care of, will last indefinitely and does not need to be replaced every few years. In the United States two makers of distribution equipment will be mentioned although a few other individually designed trains have been in use. They are the Post and the Raymond trains, which are mechanically different. In other countries there is the Quickfit design made in England, the yon Metsch design made in Germany and one made in Switzerland by F. Schmidiger, CH-4000 Basel 9. The Raymond design and manufacturing has now been absorbed by the Milton Roy Company, 5000 Park Street, N., St. Petersburg, Florida. Its description is in the literature ~,35 and will not be described here. The earlier Post designs are also well described in the literature ~,~ and will not be further described here. There are a number of improvements in his equipment, however, which are available and which have not been described in the literature. The modern Post design of equipment is a modification of that originally developed at Rockefeller and subsequently improved by Post and his associates. Mr. Post has retired, but the equipment is still available from Spectrum, Inc., 430 Middle Village Station, New York City, which have taken over the shop. For the initial exploratory work on a naturally occurring mixture thought to contain an antibiotic the semiautomatic train shown in Fig. 8 can be highly recommended. It is a 30-tube train with each tube providing for l0 ml of lower phase and a maximum of 15 ml of upper phase. An automatic filling device is provided. An inexpensive supporting arrangement contains a small motor and timer which does the equilibration, times the settling interval and then moves the train to the decant position at which position it rings a bell to let the operator know that a single cycle has been completed. The operator then tilts the train forward by hand to make the transfer and resets the motor for the next cycle. The advantage of this over the completely hand-operated train is that the operator is free to do other things at the bench if the settling period is longer than a minute. The flexibility of the small hand-operated train is not sacrificed. In fact if desired the train, supported in its frame. can be lifted off the unit containing the motor and timer and operated by hand directly on the bench. This is advisable for washing the train after removal of the sample. The 30-tube train shown in Fig. 8 call be a5 S. Raymond, Anal. Chem. 30, 1214 (1958).
344
METHODS FOR THE STUDY OF ANTIBIOTICS
[16]
FIO. 8. Semiautomatic countercurrent distribution assembly.
replaced by sixty tubes arranged in two rows. In this case, however, the tubes are smaller, providing for 5/3 ml volumes instead of 15/10. All the Post trains are equipped with butt joints pressed together by a spring in order to provide ready and convenient access to each tube. With the loager trains containing 100-1000 tubes the time and labor required to remove or position each butt joint is appreciable. A new design of the spring clamp (A) shown in the photograph of Fig. 9 reduces the time required to place or remove the butt joints. The most versatile distribution train available is a modification of the CDCD train first described in 1963 by Post and Craig ~6 and available from Spectrum. The model described in 1963 permits steady-state operation of CCD and therefore extended CCD to the possibility of preparing much larger quantities of material. Its potential and high separating power was originally demonstrated by the fractionation of a commercial sample of bacitracin27 A modification of the tube design shown in Fig. 9 now permits the ~ O. Post and L. C. Craig, Anal. Chem. 35, 641 (1963). s~ L. C. Craig, W. F. Phillips, and M. Burachik, Biochemistry 8, 2348 (1969).
[16]
ISOLATION OF ANTIBIOTICS BY CCD
345
FIG. 9. New design of a single tube of a counter double-current distribution train. (A) Spring clamp, (B) interchangeable lower phase extension, (C) Teflon CCDCDCD converter, (D) swiveling butt joint.
train to be adjusted so that it can be used either as a CCD train or as a C D C D (steady state) train. Moreover, a modification of the part of the tube where the heavier phase settles permits variation of the heavier phase volumes as well as the lighter phase. This versatility is accomplished by the use of butt joints and suitable clamps, part B of Fig. 9, as shown in Fig. 10. Interchangeable extensions of various lengths can be attached. The switch from the C D C D mode to the CCD mode of operation is accomplished by virtue of a part, C (Fig. 9), made from Teflon which permits the tube D, with its butt joint to be moved from one tube to the next in series. It, therefore, on the transfer step can allow the heavier phase to flow to the adjoining tube in the series (the C D C D mode) or return to the equilibration chamber of the same tube (the CCD mode). The original rotatory evaporator apparatus 3s which was designed for recovery of antibiotic solutes following their separation by countercurrent distribution has now been modified by different apparatus manufacturers ~ L. C. Craig, J. D. Gregory, and W. Hausmann, Anal. Chem. 22, 1462 (1950).
346
METHODS FOR THE STUDY OF ANTIBIOTICS
[15]
FIG. 10. Photograph of extensions and clamps for CCD or CDCD tube extensions. (Buchler, Buchi, Rinco, and others) and is widely used in almost every laboratory. Buchler Instruments also market a device called the Rotary Evapo-mix which will evaporate the contents of 10 tubes from the CCD train simultaneously under vacuum and at a controlled temperature.
[17]
SPECTROPOLARIMETRY OF ANTIBIOTICS
[17] Spectropolarimetry By
347
of Antibiotics
LESTER A. MITSCHER, MOHINDER S. BATHALA, a n d THEODORE S. SOKOLOSKI
I. Introduction and Background . . . . . . . . . . . . . II. hnportant Equations for Data Reduction and Curve Comparison . III. Some Specific Examples Involving Antibiotics . . . . . . . . . A. Oleandomycin . . . . . . . . . . . . . . . . . B. Spectinomycin . . . . . . . . . . . . . . . . . C. Potassium Benzylpenicillin . . . . . . . . . . . . . D. Hetacillin and 6-Epihetacillin . . . . . . . . . . . . E. Tetracycline . . . . . . . . . . . . . . . . . F. Chloramphenicol . . . . . . . . . . . . . . . . G. An Enantiomeric p-Phenyl Analog of Chloramphenicol . . . . . H. Chelocardin and Its Interaction with Aluminum Ions . . . . I. Interaction of an Antibiotic with a Biopolymer . . . . . . . IV. Choice and Optimization of Experimental Parameters . . . . . . A. Choice of Solvent . . . . . . . . . . . . . . . . B. Temperature . . . . . . . . . . . . . . . . . C. Atmosphere . . . . . . . . . . . . . . . . . D. Cells . . . . . . . . . . . . . . . . . . . . . E. Instrument Calibration . . . . . . . . . . . . . . F. Slit Widths . . . . . . . . . . . . . . . . . . G. Scanning Speed . . . . . . . . . . . . . . . . H. Gain Settings . . . . . . . . . . . . . . . . . I. Sample Concentration . . . . . . . . . . . . . . J. Survey Measurements . . . . . . . . . . . . . . K. Blank . . . . . . . . . . . . . . . . . . . L. Reproducibility . . . . . . . . . . . . . . . .
347 355 355 356 359 360 362 363 364 366 367 367 369 369 369 370 370 ;170 ;571 371 371 371 372 372 373
I. Introduction and Background S p e c t r o p o l a r i m e t r y e n c o m p a s s e s t h e c o m p l e m e n t a r y c h i r o p t i c a l techn i q u e s of o p t i c a l r o t a t o r y dispersion ( O R D ) and c i r c u l a r d i c h r o i s m ( C D ) a nd a m o r e r e c e n t m e t h o d , m a g n e t i c c i r c u l a r d i c h r o i s m ( M C D ) . T h e basic d o g m a of t h e c h i r o p t i c a l m e t h o d s can be s u c c i n c t l y s t a t e d a nd m u s t n o t be v i o l a t e d . I f the s o l u t i o n c o n f o r m a t i o n of a m o l e c u l e is known, t h e n its a b s o l u t e c o n f i g u r a t i o n can, in principle, be d e t e r m i n e d by O R D - C D m e a s u r e m e n t s . C o n v e r s e l y , if a m o l e c u l e ' s a b s o l u t e configur a t i o n is k n o w n , t h e n its solution c o n f o r m a t i o n can be d e t e r m i n e d by O R D - C D - - e s p e c i a l l y if i n f o r m a t i o n f r o m o t h e r t e c h n i q u e s , such as n u clear m a g n e t i c r e s o n a n c e ( N M R ) a n d X - r a y , is included in the analysis. W i t h some well e x p l o r e d c h r o m o p h o r e s , such as k e t o n e s in rigid molecules, s e l n i e m p i r i c a l p r e d i c t i v e rules h a v e been w o r k e d o u t w h i ch e n a b l e
348
METHODS FOR THE STUDY OF ANTIBIOTICS
[17]
one to extract the absolute configuration or solution conformation directly from the spectral findings. More commonly with antibiotics, the spectra are interpreted by comparison with the spectra of model molecules chosen with great care so as to contain the chromophore in question in a molecule where closely analogous electronic and conformational effects are at play. When an antibiotic is absorbed to an optically active biopolymer, such as lipoprotein or D N A - R N A , the electrons in the absorbed drug find themselves in a newly asymmetric environment. This frequently leads to the generation of new optically active transitions, characteristic of the binding, allowing much useful information to be deduced about the nature of the binding. When conformational changes follow the binding, the spectral transformations may be dramatic. It is in this latter area of "extrinsic" spectral bands t h a t the greatest future potential of O R D - C D lies. Readily available commercial instrumentation capable of high precision measurement over an acceptably wide spectral range has been available only since approximately 1950 for ORD and roughly since 1960 for CD, although the methods themselves are much older. An enormous literature has developed, particularly following the early work of Carl Djerassi and his school. A number of significant books have since appeared outlining the theoretical basis of the chiroptical methods and reviewing applications to specific chromophoric groups and classes of compounds. 1-12 The reader is directed to an examination of these for a definition of terms and general background. In the authors' opinion, the book 1p. Crabb6, "ORD and CD in Chemistry and Biochemistry." Academic Press, New York, 1972. 2p. Crabb6, "Optical Rotatory Dispersion and Circular Dichroism in Organic Chemistry." Holden-Day, San Francisco, 1965. "Spectroscopic Approaches to Biomolecular Conformation" (D. W. Urry, ed.). The American Medical Association, Chicago, 1970. ~B. Jirgensons, "Optical Rotatory Dispersion of Proteins and Other Macromolecules," Springer-Verlag, Berlin and New York, 1969. 5"Methods in Pharmacology," Vol. 2, "Physical Methods" (C. F. Chignell, ed.), Chapter 11. Appleton, New York, 1972. s L. Velluz, M. Legrand, and M. Grosjean, "Optical Circular Dichroism." Academic Press, New York, 1965. 7"Optical Rotatory Dispersion and Circular Dichroism in Organic Chemistry," (G. Snatzke, ed.). Sadtler, Philadelphia, 1967. 8C. Djerassi, "Optical Rotatory Dispersion." McGraw-Hill, New York, 1960. ' P. Crabb~, "An Introduction to the Chiroptical Methods in Chemistry." Impressors Offsali-G, Colonia del Valle, Mexico 12, D. F., 1971. ~op. Crabb~, "Applications de la Dispersion Rotatoire Optique et du Dichroisme Circulaire Optique en Chimie Organique." Gauthier-Villars, Paris, 1968. 11C. A. Bush, Optical rotatory dispersion and circular dichroism, in "Physical Techni-
[l~l
SPECTROPOLARIMETRY OF ANTIBIOTICS
349
edited by Snatzke ~ is the single most useful reference, the chapters by Chignell 5 and Urry 3 deal best with extrinsic Cotton effects and Crabb6's recent book 1 contains the most lucid exposition of the many semiempirical rules elaborated for predicting the chiroptical effects of the various chromophores which have been studied. Because of the relative molecular complexity of antibiotics, the chiroptical methods were only occasionally applied to them until fairly recently. Because molecular conformation is felt to be as significant as optical asymmetry in dictating the kind and intensity of chiroptical spectra as well as the kind and intensity of bioactivity of most drugs, it is not surprising that the chiroptical methods are being widely utilized in attempts to uncover and codify the laws of the molecular biology of drug action. The antibiotics clearly fit into this context, but surprisingly, considering their importance and the volume of work involved, this sub ieet has not yet been reviewed. As a very brief precis of the phenomenology underlying the chiroptical methods, the reader is reminded that light passing through a solution containing a molecule possessing a chromophoric grouping is absorbed at discrete wavelengths. The study of this constitutes conventional UV-VIS photometry and the extent of absorption is roughly equivalent to the probability of a given electronic transition taking place. When the solute is optically active, and circularly polarized light is used, the right and left circularly polarized components are unequally absorbed (eL # eR). This effect is often as much as 10,000 times weaker than the bulk absorption (e), so that a very sensitive instrument utilizing a very intense light source is required to generate alternately left and right circularly polarized light and then to measure continuously their relative intensities as a function of wavelength. Figure 1 shows that e and A~ occur at closely similar wavelengths, but their relative intensities are not closely related, for A, is a measure of molecular asymmetry in the vicinity of the chromophore and not, therefore, dependent on the same molecular characteristics that generate e. At the same time that e and A~ signals are being generated by passage of light through a chromophore, the plane polarized beam is also being differentially diffracted because ne does not equal ~m anymore than eL equaled eR. The differential diffraction results in a rotation of the plane of polarized light as compared with the incident ques in Biological Research," 2nd ed., Vol. 1, Part A, "Optical Techniques" (G. Oster, ed.). Academic Press, New York, 1971. ,2D. J. Caldwell and H. E. Eyring, "The Theory of Optical Activity." Wiley (Interscience), New York, 1971.
350
[17]
METHODS FOR THE STUDY OF ANTIBIOTICS
+
I/...
So
//,,''\), WAVELENGTH
WAVELENGTH
FIG. 1. A comparison of e and be for a hypothetical chromophore. Curve b has been normalized. plane. This effect can be measured b y use of an i n s t r u m e n t t h a t accurately measures the angle of a plane of polarized light. If only the sodium D line is reported, one obtains the familiar [a]D. When, however, the results are plotted as a function of X, the result is an optical r o t a t o r y dispersion curve. Thus, three different phenomena result from the interaction of a chromophore with incident polarized light: UV, ORD, and CD. Each encodes significant structural information and requires a different instrument for measurement. The underlying molecular phenomenon is generally assumed to be excitation of molecular electrons to higher energy level orbitals. The electrons are presumed to follow helical paths in the course of this excitation process. If the molecule is symmetrical in the vicinity of the chromophore (I), no preference for a right- or left-handed helical
n-.>
Tr*
(i)
(2}
(I) path will be experienced, so *L -- cR and nL ---- na and no optical activity accompanies this transition. However, unsymmetrical conformation or substitution (II) will lead to a definite preference, and optical activity
5 ~ 1
R23 2
(3)(CHAIR)
S,
2
hY
nL f/TR; ~L# ER
6 (5)
(II)
R (3)(TWIST)
[17]
SPECTROPOLARIMETRY OF ANTIBIOTICS
351
results. The carbon bearing the R group (R =/= H) in formulas (3) and (4) (II) has four different substituents and is a classical asymmetric center. The dependence of chiroptical phenomena on molecular conformation and molecular asymmetry can be readily assimilated from such considerations. This grossly oversimplified exposition documents another important rule. The ORD and CD curve of a given substance encodes the same information regarding molecular asymmetry and the methods are, therefore , complimentary. Specific examples to be given shortly will help the reader comprehend the practical and strategic considerations which may lead the investigator to choose one or the other of these methods as most appropriate in a given case. Table I lists the four classes of chromophores recognized in chiroptical studies and some examples of each type encountered in the study of antibiotics. Crabb5 (pp. 185-191) ~ has an excellent tabular summary of the Cotton effects generated by a wide variety of chromophores and the rules for curve rationalization. Table II lists the families of antibiotics which have been studied spectropolarimetrically and the purposes of the particular study. The literaTABLE I TYPES OF CHROMOPHORES O1~" INTEREST IN CHIROPTICAL STUDIES OF ANTIBIOTICS
Type Symmetrical chromophore in an unsymmetrical molecular environment Inherently disymmetric chromophore due to lack of symmetry of the chromophore, or molecular twisting Homoconjugated. Chromophores interact through space rather than through the molecular framework. Exciton coupled
Extrinsic. Optical activity induced in a symmetrical molecule by binding to an optically active biopolymer (I)NA, RNA, protein). A special subcase exists when the bound molecule also has optical activity in its own right
Examples Ketone n -~ ~r* (erythromycin, fusidic acid, Actidione) benzene ring (chloramphenicol) a,B-Unsaturated ketones (methymycin) conjugated dienes (Leucomycin A3) conjugated ene-one system (tetracyclines) Peptides in helical arrays or otherwise conformationally fixed at angles ~180 ° (tyrocidin S, bacitracin, stendomycin). Other chromophores of similar energy content fixed at angles ~ 180 ° relative to one another (chromomycin, anhydrotetracyclincs, chelocardin) (Actinomycin -I- I)NA, bleomycin + I)NA, etc.)
352
METHODS FOR THE STUDY OF ANTIBIOTICS
[17]
TABLE II CttIROPTICAL STUDIES OF ANTIBIOTICS
Antibiotic a n d / o r family Actinomycin Chlorampheaicol Fusidic acid Glutarimide family ~3-Lactam antibiotics
Macrolide family Peptide family
Tetracycline family Miscellaneous types Azoxyketones Streptothricins Rhodomycins Antimycins Chromomycins Bluensomycin Griseofulvin X-537A I n t e r a c t i o n of antibiotics with biopolymers Bleomycin types Actinomycins Kanchanomycin Netropsins Sulfonamides
Purpose of s t u d y Conformational analysis and solvent interactions a-d Conformational analysis, °-g assignment of absolute configuration,/' g a n d interaction with chelating ionsg Absolute configuration h-i Absolute configuration k-° Investigation of the electronics of the chromophore ~-t a n d q u a n t i t a t i v e analysis ~ Conformational analysis r Conformational analysis, " - ' ' ~' structure d e t e r m i n a t i o n , " ~'' b, solvent effects . . . . . ' Conformational analysis, d'-~' solvent effects,X, ,'. a'-k'. ~,-o,, ~, interaction with chelating ions, k', g" "-~' solvent effects, d'' ,,,g,-k,, ,,,-o,, ~, a n d absolute configuration-structure analysis °''*'' ~' Conformational analysis, ~ ' - ' ' ' ~"' ~ structure studies, ~ ion binding, ~'-'',¢'. ~a q u a n t i t a t i v e analysis ~* Absolute configuration, dd' *o solvent effects, dd' °° conformational analysis d~' ** N a t u r e of the chromophore H Assignment of absolute stereochemistryga Assignment of absolute stereochemistry hh Assignment of absolute stereocheinistry,"' Ji n a t u r e of thc chromophore~, ii Assignment of absolute stereochemistry, kk ion binding kk Structural analysis kk I o n binding u
W i t h D N A =m W i t h D N A . . . . . . pp DNAqq DNA~r, u Plasma albumin ~u
H. Ziffer, K. Yamaoka, a n d A. B. Mauger, Biochemistry 7, 996 (1968). b F, Ascoli, P. DeSantis, M. Lener, and M. Savino, Biopolymers 11, 1173 (1972). D. M. Crothers, S. L. Sabol, D. I. Ratner, and W. Mueller, Biochemistry 7, 1817 (1968). d F. Ascoli, P. DeSantis, a n d M. Savino, Nature (London) 227, 1237 (1970). • L. A. Mitscher, F. Kautz, a n d J. Lapidus, Can. J. Chem. 47, 1957 (1969). I L. A. Mitscher, P. W. Howison, J. B. Lapidus, and T. D. Sokoloski, J. Med. Chem. 16, 93 (1972). o L. A. Mitscher, P. W. Howison, a n d T. D. Sokoloski, J. Med. Chem. 16, 98 (1972). h R. Bucort, M. Legrand, M. Vignau, J. Tessier, a n d V. Delaroff, C. R. Acad. Sci. 257, 2679 (1963).
[17]
SPECTROPOLARIMETRY OF ANTIBIOTICS
353
R. Bucort and M. Legrand, C. R. Acad. Sci. 258, 3491 (1964). i W. O. Gotfredsen, W. V. Daehne, S. Vangedal, A. Marquet, I). Arigoni, and A. Malera, Tetrahedron 9.1, 3505 (1965). k F. Johnson, L. G. Duquette, and H. E. Hennis, J. Org. Chem. 33, 904 (1968). l T. Okuda, M. Suzuki, and Y. Egawa, Chem. Pharm. Bull. 8, 335 (1960). '~ T. Okuda and M. Suzuki, Chem. Pharm. Bull. 9, 1014 (1961). n M. Suzuki, Y. Egawa, and T. Okuda, Chem. Pharm. Bull. 11, 582 (1963). ° T. Okuda, Chem. Pharm. Bull. 7, 259 (1959). 7, [. Z. Siemion, J. Lisowski, B. Tyran, and J. Morawiec, Bull. Acad. Po/. Sci., Ser. Sci. Chim. 20, 549 (1972). q R. Nagarajan and D. 0. Spry, J. Amcr. Chem. Soc. 93, 2310 (1971). S. Kukolja, P. V. Demarco, N. D. Jones, M. O. Chaney, and J. W. Paschal, J. Amer. Chem. Soc. 94, 7592 (1972). L. Neelakanthan and D. W. Urry, Abstr. 158th Meet. Amer. Chem. Soc. 1969, B10L176. t F. Snatzke, Bull. Acad. Pol. Sci. Set. Sci. Chim. 21, 167 (1973). " C. E. Rasmussen and T. Higuchi, J. Pharm. Sci. 60, 1608 (1971). •' T. J. Perun, R. S. Egan, P. H. Jones, J. R. Martin, L. A. Mitscher, and B. J. Slater, Antimicrob. Ag. Chemother. 116 (1969). w S. Omura, A. Nakagawa, N. Yagisawa, Y. Suzuki, and T. Hata, Tetrahedron, 28, 2839 (1972). R. S. Egan, T. J. Perun, J. R. Martin, and L. A. Mitscher, Tetrahedron 29, 2525 (1973). L. A. Mitscher, B. J. Slater, T. J. Perun, P. H. Jones, and J. R. Martin, Tetrahedron Lett. 4505 (1969). J. R. Martin, T. J. Perun, and R. S. Egan, Tetrahedron 28, 2937 (1972). "' C. Djerassi and O. Halpern, Tetrahedron 3, 255 (1958). b, p. Kurath, J. R. Martin, J. Tadanier, A. W. Goldstein, R. S. Egan, and ]). A. Dunnigan, Helv. Chim. Acta 56, 1557 (1973). ~' C. Djerassi and J. A. Zderic, J. Amer. Chem. Soc. 78, 6390 (1957). d' l). W. Urry and A. Ruiter, Biochem. Biophys. Res. Commun. 38, 800 (1970) ~" S. Laiken, M. Printz, and L. C. Craig, J. Biol. Chem. 244, 4454 (1969). /' P. M. Bayley, Biochem. J. 125, 90 (1971). ~' B. E. Isbell, C. Rice-Evans, and G. It. Beaven, F E B S Lclt., 192 (1972). h' D. W. Urry, Biochemistry 11, 487 (1972). i, D. W. Urry, A. L. Ruiter, B. C. Starcher, and T. A. tiinners, Antimicrob. Ag. Chemother., p. 87 (1968). i' F. Quadrifoglio and D. W. Urry, Biochem. Biophys. Rcs. Commun. 29, 785 (1967). k, T. Funk, F. Eggers, and E. Grell, Proc. Ear. Biophys. Congr., 1st, Vol. 3, 37 (1971). i, M. A. Ruttenberg, T. P. King, and L. C. Craig, Biochemistry 5, 2857 (1966). '~' M. A. Ruttenberg, T. P. King, and L. C. Craig, J. Amer. Chem. Soc. 87, 4196 (1965). "' M. Bodarlszky and A. Bodaaszky, Nature (London) 220, 73 (1968). o' Yu. A. Orchinuikov, V. T. Ivanov, B. F. Bystrov, A. I. Miroshnikov, E. N Shepel, N. D. Abdullaev, E. F. Efremov, and L. B. Senyavina, Biochem. Biophys. Res. Commun. 39, 217 (1970). ~" D. Balasubramaniau, J. Amer. Chem. Soc. 89, 5445 (1967).
354
METHODS FOR THE STUDY OF ANTIBIOTICS
[17]
q' V. T. Ivanov, I. A. Laine, N. D. Abdullaev, V. Z. Pletnev, G. M. Lipkind, S. F. Arkhipova, L. B. Senyavina, E. N. Meshcheryakova, and E. M. Popov, Khim. Prir. Soedin. 7, 221 (1971). "' K. A. Zykalova, G. N. Tishcheako, G. A. Kogan, aad V. T. Ivanov, Isv. Akad. Nauk. SSSR, Ser. Khim., p. 1547 (1970). ~' K. Vogler, R. O. Struder, P. Lanz, W. Lergier, E. Boehri, and B. Fust, Helv. Chim. Acta 46, 2823 (1963). t, N. W. Cornell and D. G. Guiney, Jr., Biochem. Biophys. Res. Commun. 40, 530 (1970). -' N. A. Podduknaya and N. Ya. Krasnobrizhii, Zh. Obshch. Khim. 41, 46 (1971). •' A. Bodanzsky, M. Bodanzsky, K. L. Perlman, and D. Perlman, J. Antibiot. 25, 325 (1972). ~" U. Ludescher and R. Schwyzer, Helv. Chim. Acta. 54, 1637 (1971). ~' L. A. Mitscher, B. Slater-Eng, and T. Sokoloski, Antimicrob. Ag. Chemother. 2, 66 (1972). • ' L. A. Mitscher, A. C. Bonacci, B. J. Slater, A. K. Hacker, and T. D. Sokoloski, Antimicrob. Ag. Chemother., p. 111 (1969). y' A. H. Caswell and J. D. Hutchison, Biochem. Biophys. Res. Commun. 43, 625 (1971). •' L. A. Mitscher, A. C. Bonacci, and T. D. Sokoloski, Antimicrob. Ag. Chemother., p. 78 (1968). ~a L. A. Mitscher, A. C. Bonacci, and T. D. Sokoloski, Tetrahedron Lett. 1968, 5361 (1968). b~L. A. Mitscher, W. Rosenbrook, Jr., W. W. Andres, R. S. Egan, J. Schenk, and J. V. Juvarkar, Antimicrob. Ag. Chemother., p. 38 (1970); J. Amer. Chem. Soc. 92, 6070 (1970). cc R. F. Miller, T. D. Sokoloski, L. A. Mitscher, A. C. Bonacci, and B.-A. Hoener, J. Pharm. Sci. 62, 1143 (1973). ~4 W. J. McGahren and M. P. Kunstman, J. Amer. Chem. Soc. 92, 1587 (1970). " W. J. McGahren and M. P. Kunstman, J. Org. Chem. 37, 902 (1972). ss H. Taniyama and Y. Sawada, Chem. Pharm. Bull. 20, 596 (1972). ga H. Brockmann, Jr. and M. Legrand, Tetrahedron 19, 395 (1963). hh M. Kinoshita, S. Aburaki and S. Umezawa, J. Antibiot. 25, 373 (1972). ii N. Harada and K. Nakanishi, Accounts Chem. Res. 5, 257 (1972). iJ N. Harada, K. Nakanishi, and S. Tatsuoka, J. Amer. Chem. Soc. 91, 5896 (1969). k~ C. B. Barlow and L. Anderson, J. Antibiot. 25, 281 (1972). kk, A. Brossi, M. Brumann, and F. Burdhardt, Helv. Chim. Acta. 45, 1292 (1962). zl H. Degani, H. L. Friedman, G. Navon, and E. M. Kosower, Chem. Commun., p. 431 (1973). m,~ W. C. Krueger, L. M. Pschigoda, and F. Reusser, J. Antibiot. 26, 424 (1973). ~ R. B. Homer, Arch. Biochem. Biophys. 129, 405 (1969). oo y. Courtois, W. Guschlbauer, and P. Fromageot, Eur. J. Biochem. 6, 106 (1968). ~" K. Yamaoka and H. Ziffer, Biochemistry 7, 1001 (1968). qq P. A. Friedman, T.-K. Li, and I. H. Goldberg, Biochemistry 8, 1545 (1969). ~ C. Zimmer, G. Luck, H. Thrum, and C. Pitra, Eur. J. Biochem. 26, 81 (1972). ~ C. Zimmer, K. E. Rinert, M. Thrum, U. Waehnert, and G. Loeber, J. Mol. Biol. 56, 329 (1971). u C. Zimmer and G. Luck, F E B S Lett. 10, 339 (1970). ~ G. C. Wood and S. Stewart, J. Pharm. Pharmacol. 2S, Suppl., 248s (1971).
[17]
SPECTROPOLARIMETRY OF ANTIBIOTICS
355
ture is widely scattered, and the authors apologize for inevitable accidental errors of omission.
I I . I m p o r t a n t E q u a t i o n s for D a t a R e d u c t i o n a n d Curve Comparison
ORD M o l e c u l a r r o t a t i o n = [~]~ = [a]x X M / 1 0 0 = (a X 100 X M)/l X ~:' A m p l i t u d e = a --- ([~]1 - [~b]2)/100
CD Specific ellipticity = [~I,]x = (~I, X 104)/l × c' Molecular ellipticity = [O]x = ([xI,] X M ) / 1 0 0 = 3300 [A~] = (3300 X O D X M)/c × 1 A n i s o t r o p y f a c t o r = g = Ae/e
Comparison o] ORD-CD Data a = 40.28Ae = 0.0122 [~]
Meaning o] Terms ~I, = ellipticity in degrees = scale setting x degrees ellipticity for full scale M = g r a m molecular w e i g h t of sample c = c o n c e n t r a t i o n in g / m l c' = c o n c e n t r a t i o n in g / l i t e r l = light p a t h in c m = degrees r o t a t i o n [a]× = specific r o t a t i o n at w a v e l e n g t h ~ in n m [O]t - [~]~ = the difference b e t w e e n the a m p l i t u d e s of the molecular r o t a t i o n s of the p e a k a n d t r o u g h of a g i v e n C o t t o n effect [~]x = eL - ~R O D = optical d e n s i t y difference III. Some Specific Examples
Involving
Antibiotics
T h e spectra described in this section were obtained using a J a s c o ins t r u m e n t and the r e m a r k s a p p l y specifically to d a t a obtained in this way. T h e principles a p p l y also to the C a r y instrument, but one anticipates
356
METHODS FOR THE STUDY OF ANTIBIOTICS
[17]
that there would be some small differences in operating procedure and strategy.
A. Oleandomycin (5)
Favorable Ae/~; expected response o] instrument; representation o] data; ORD/CD ]actorable. The ORD and CD spectra are illustrated CH
Ou /O'~'CH2
CH31~.I."CH3
N(CH3)2
L c.,. CH'30 ~ . , 0 " ~ CH3
0 "I
~""Z--"CH3
~--~OCH 3 u " ""~'-0 CH3 H (6)
HO CH3HN..-.~,,,,,'~ ~
-v VHo.~OH
CH5
CH3 (7)
for this substance in methanol as solvent (Fig. 2). There are two chromophoric groups in the molecule, the a-epoxyketone function absorbing at 302 nm (n--> ~*) and the lactone n--) ~r* transition at 215 nm. The Ac/c ratio of both chromophores is favorable for measurement because of their relatively high local asymmetry and relatively low total absorptivity. At both high (700 nm) and low (200 nm) wavelengths the Xe lamp output is weak and the readings are noisy. In the 700 nm region, the molecule does not absorb light, so the low lamp output simply results in greater uncertainty in absolute intensity than would otherwise be tolerated. This can be overcome substantially by increasing instrument response, by increasing the cell pathlength or the concentration of the solution. These
SPECTROPOLARIMETRY OF ANTIBIOTICS
[17]
357
H IN
/S~cH3
CH5 HN ~
/ ~
CH3
C"
/S~
.CH3
0 CH5 (10)
(9)
H~. .O...H N----.// I
H/. '~.(O
HO "" O-,,J ^ ~CH~, -3 ~ L + ".H
H
O .O O ~H"" ~H"'"
devices cannot be used to improve the readings in the 200-nm region because the sample is also absorbing light fairly strongly (±,/c decreases). It is customary to record the wavelength of the last reading which the investigator believes to be reproducible and reliable, and this point is often indicated by an asterisk after the intensity in the experimental section of the paper. For purposes of data presentation, the following form is convenient: CD spectrum of oleandomycin (c = 0.11, MeOH): [0]3440, [0]3025685, [0]~50- 190. (c = 0.86, MeOH): [0]~40- 500, [01212- 10860, [0121o - 8000*. It is clear from this that it was necessary to employ two different concentrations to get the data.
358
METHODS FOR THE STUDY OF ANTIBIOTICS
HOCH2 H+NHCOCHCI2 HO~H
CH20H 0,2CHCOHHN~~I NO?_ (12)
c
H
[17]
Q (13)
~
C
H
O,~,H..O~,H..O
3
0
0
(14) O R D spectrum of oleandomycin (c = 0.11, M e O H ) : [ ¢ ] 7 0 0 - 470, [¢]589 - 470, [¢]~00 -- 700, [¢]400 -- 2185, [¢]850 - 2655, [¢]3~o - 4690, [¢]29a 0, [¢]27s q- 1875, [¢]264 0, [@]2~5 -- 3590, [¢]220 - 3120".
It is interesting to note t h a t at the scale used in measuring this spectrum, the instrument pen, which makes a 1 mm-wide line, introduces a potential error as large as ± 1 5 6 [@] units, when the blank is also considered, for this is the magnitude indicated for each millimeter of pen deflection even when no noise is present. For a peak of [¢] 350 - - 4690, this error is about 3%, and hence not too serious, but at the sodium D line uncertainty represents about 30% of the reading itself. Thus, readings of [aid using such a spectropolarimeter should be done with much more
70 --
0
x
[]
/
-2
z~-4 '<
V<,- KETONE n ~¢r*
-6
'~'-e -I0
LACTONE n--).lr * 200
I
300
I
I
I
400 600 WAVELENGTH, nm
FZG. 2. The optical rotatory dispersion spectra of oleandomycin in methanol.
(
I 700
) and circular dichroism (---)
[17]
SPECTROPOLARIMETRY OF ANTIBIOTICS
359
concentrated solutions than this--indeed, concentrations that would be too great for successful measurement of the whole ORD curve in the UV region, where absorption by the sample would not allow sufficient light to pass through the sample for accurate readings. It can be seen immediately from the CD spectrum that both Cotton effects are negative in sign. Note that, like UV, tile peak intensities fall away to vanishing values when the peak is traversed. This allows convenient factoring of the spectra of molecules containing more than one chromophore if they are far enough apart in the molecule so that their electrons do not overlap to form a new ehromophoric system, and if their energy levels are far enough apart so that the Cotton effects occur at fairly widely separated wavelengths (here a nm = 87 nm, and the valley between the two troughs nearly touches the baseline at 250 nm). The ORD curve can also be factored fairly successfully in this favorable case. Both lobes of the a-epoxyketone Cotton effect are displaced negatively by the first (negative) lobe of the lactone transition at 225 nm. The average [4] value, ([~k]~- [4]~)/2 =--2815, occurs almost exactly at 302 nm (the point of maximal CD and UV absorption), while the [4~] = 0 point lies at 293 nm. Because of sample absorption it was not possible to trace the second (positive) lobe of the ORD lactone Cotton effect, but the first (negative) lobe is clearly visible (at 225 nm). With both transitions clearly negative in sign, it is easily understandable that methanol solutions of oleandomycin should be lcvorotatory at the sodimn D line. Because the CD curve can almost always be factored more easily than the ORD curve, the CD spectrum is generally the one measured. Negative peaks for each transition are seen in both spectra, and, since the absolute configuration of oleandomyein is known, the sign and intensity of these two bands can, in principle, be used to determine the solution conformation of the antibiotic by processes analogous to those already used for erythromyein.1'~
B. Spectinomycin (7) CD analysis easier than ORD : verification o) result via octant projection; detection o) ketal formation. Spectinomycin presents an interesting ease in which the ketone group is ahnost completely masked as a ketal in hydroxylic solvents. The ORD curve (Fig. 3) of a commercial sample shows only a slight break in smoothness in the region where an a-hydroxyketone would be expected to absorb. The inflection is so weak, in la t/. S. Egan, T. J. Perun, J. R. Martin, and L. A. Mitscher, Tetrahedron 29, 2525 (1973).
360
METHODS FOR THE STUDY OF ANTIBIOTICS
[17]
:f o
2
x
Z
-2
b
-4
X
-6
N
-8 -I0
200
I
t
I
i
I
Z,O0
400
500
600
700
WAVELENGTH, nm
FIO. 3. T h e optical r o t a t o r y dispersion ( - - - ) and circular dichroism (
) spectra
of spectinomycindihydrochloridein water. fact, that assignment of a sign to the Cotton effect would have to be done with great circumspection. The intensity would be impossible to decide. The hump at about 250-260 nm is likely the second lobe of this Cotton effect and, since it is at lower X and is plus, one infers that a weak negative lobe should have been present somewhere around 300 nm. The CD spectrum takes the guesswork out of this sort of analysis because a weak, but definite, negative Cotton effect is in fact present at 300 nm. This peak is somewhat more intense in MeOH. The relative lack of C ~ 0 character is easily confirmed by measurement of the IR spectrum of spectinomycin dihydrochloride pentahydrate in KBr. It is interesting to note that this negative Cotton effect is the most proximate to the visible region but is insufficient to overcome the strongly dextrorotatory background so the substance is dextrorotatory at the sodium D line. The origin of the second Cotton effect at about 210 nm in this sample is not known at present--it may be due to an impurity. Figure 4 shows an octant projection of the X-ray crystal conformation of spectinomycin. One sees from this that the observed negative Cotton effect is consistent with theoretical prediction as all nonmodal and unbalanced atoms are in the lower left back octant.
C. Potassium BenzylpeniciUin (8) ORD more use]ul than CD ; ef]ect o] bu]yer. The penicillins represent a type II chromophore in that the amide (B-lactam) function lacks the ground-state symmetry of the ketone functions and the chromophore is further complicated by interaction with the n-electrons of the sulfur atom and lack of planarity of the bicyclo ring system. For reasons of maximal
[17]
SPECTROPOLARIMETRY
OF ANTIBIOTICS
361
I I I
l
'~II ~.c.3 -
', (
c.3
~
• o
I
~=N
!
()
~ !
H (~)CH 3
I! |
FIa. 4. T h e o c t a n t p r o j e c t i o n for spectinomycin.
solution stability, the O R D - C D spectra in Fig. 5 were measured in water at pH 6.5. The spectra (Fig. 5) show two maxima, one centered about 230 nm, which was measurable in its entirety, and the approach slopes of the second occurring at wavelengths below about 200 nm, where sample absorption and failing lamp intensity make accurate measurements impossible. When citrate buffers were used to get better pH control, end absorption prevented accurate measurement below 225 nm, and consider6 ,q. 5
b- -
X ~3
4
~2
/, -
/
rO I - \ \ /
b_
x o -I 200
J
I
]
250 300 350 WAVELENGTH, nm
I
400
Fro. 5. T h e optical r o t a t o r y dispersion ( ) and circular dichroism ( - - - ) spectra of p o t a s s i u m benzylpenicillin in water at p H 6.5.
362
METHODS
FOR
THE
S T U D Y OF A N T I B I O T I C S
[17]
5
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FIG. 6. The optical rotatory dispersion (ORD) and circular dichroism (CD) spectra of hetacillin ( ) and epihetacillin (---) in 0.1 M citrate buffer, pH 6.5. able spectral information was lost. When this happens, ORD will be more useful as the first lobe of the 230 nm Cotton effect can be measured entirely, while the 230 nm C D Cotton effect is barely measurable past its peak. The 230 nm positive Cotton effect is directly attributable to the fl-lactam because 6-aminopenicillanic acid gives a closely similar spec. trum and because hydrolysis of the fl-lactam bond leads to disappearance. Higuchi has used this fact to develop a rapid, sensitive and precise assay for many penicillins by measuring the intensity of the first lobe of the 230 nm ORD Cotton effect before and after the addition of penicillinase. TM Chiroptical studies have yet to achieve their full promise in quantitative analysis, but a few papers have now appeared suggesting that interest in this area has been awakened. The expense of the equipment has had a depressing influence on this effort. D. Hetacillin (9) and 6-Epihetacillin (10)
O R D more useful than CD; potential use in assay of epimerization; kinetics. From the standpoint of potential for analysis, the curves of hetacillin and 6-epihetacillin in Fig. 6 are instructive. T h e y also illustrate another case where the ORD spectrum is more useful than the CD. Because of extensive end absorption in the UV region caused by the citrate 14C. E. Rasmussen and T. Higuchi, J. Pharm. Sci. 60, 1608 (1971).
[17]
SPECTROPOLARIMETRY OF ANTIBIOTICS
363
buffers used, it was not possible to trace reliably even the first Cotton effect in its entirety. Because the first lobe of an ORD Cotton effect occurs at longer wavelengths than the CD maximum, it was possible to trace the position and intensity of this lobe even though the peak CI) absorption was not obtained with satisfactory precision. The background rotation in the penicillin family is relatively constant, so that [~] is a relatively significant and comparable indicator of molecular structure and concentration in the pencillin family. The difference in peak intensity in the ORD is of sufficient magnitude that a sensitive assay for the extent of epimerization in a given family could easily be developed. This would be far more precise and rapid than current microbiological methods, and much easier than NM1R differences, which amount to only a few Hertz and require significantly greater concentrations. Furthermore, preliminary work in this laboratory has shown that the kinetics of epimerization can be easily followed by monitoring the peak intensity vs time under a variety of conditions. E. Tetracycline (11)
Interpretation o] spectra by the use of analogs; solution con]ormation; ion binding; assay o] epimerization. In the tetracycline molecule, there are two ehromophorie areas. The twisted fl-triearbonyl system of ring A absorbs at about 263 nm in 0.03 N HC1 solution. The remaining bands in the spectrmn at 360, 320, 290, and 230 nm are generated by the conjugated fl-diketoaryl system of rings B, C, and D. Accurate factoring of these multiple bands in the ORD would be extremely difficult, with the necessary approximations rendering each successive peak assignment toward the UV region more and more subiect to doubt. On the other hand, the CD spectrum allows a much more secure analysis of changes affecting the A ring separately from those affecting the BCD rings. These factors are clear on examination of Fig. 7, where the relevant spectra are recorded. The finding that, under identical conditions of measurement, a wide variety of tetracyelines give closely similar CD spectra allows the conclusion that they possess not only the same absolute configurations, but also nearly the same solution conformation) ~ This conclusion does not, however, hold up at other pH's. ''; There ,ire no available simple model systems from which to treat these spectra from first principles. so empirical and careful comparative curve analysis must be done. ~'~L. p. ~"L. 66
A. Mitschcr, A. C. Bonacei, and T. D. Sokoloski, Antirnicrob. Ag. Chemotl~er., 78 (1968). A. Mitscher, B, Slater-Eng, and T. Sokoloski, A~limicrob. Ag. Cl~ernother. 2, (1972).
364
METHODS FOR THE STUDY OF ANTIBIOTICS 6
12
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FIG. 7. The optical rotatory dispersion (---) and circular dichrolsm ( of tetracycline hydrochloride in 0.03 M hydrochloric acid.
) spectra
A p p a r e n t l y because the A ring chromophore of 4-epitetracycline is significantly less twisted t h a n t h a t of (11), the C D band at about 263 nm is d r a m a t i c a l l y less intense in epitetracycline. This has led to a rapid, sensitive C D assay for a mixture of these m a t e r i a l s . " Finally, because of the ease with which one can "see" both spectroscopic regions of the tetracyclines, extensive ion binding studies have given much useful information about the influence of p H and structural features on this phenomenon. 1~,1~,1s-2°
F. Chloramphenicol (12) UnJavorable A~/~ ratio. T h e C D spectrum in Fig. 8 represents t h a t of a most difficult spectroscopic case. Chloramphenicol possesses an inherently symmetrical chromophore (substituted benzene ring) a s y m m e trically perturbed by the flexible side chain (Class I ) . Three bands can
"R. F. Miller, T. D. Sokoloski, L. A. Mitscher, A. C. Bonacci, and B.-A. Hoener, J. Pharm. Sci. 6o., 1143 (1973). lSL. A. Mitscher, A. C. Bonacci, B. J. Slater, A. K. Hacker, and T. D. Sokoloski, Antimicrob. Ag. Chemother., p. 111 (1969). 19A. H. Caswell and J. D. Hutchison, Biochem. Biophys. Res. Commun. 43, 625 (1971). 2oL. A. Mitscher, A. C. Bonacci, and T. D. Sokoloski, Tetrahedron Lett. 1968, 5361 (1968).
[17]
SPECTROPOLARIMETRY
OF ANTIBIOTICS
365
IOr-
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550 400 200 250 ~00 WAVELENGTH, nm FIo. 8. The relationship between degrees ellipticity (proportional to pen deflection) and photomultiplier voltage (PM voltage) and wavelength for chloramphenicol (0.8%) in methanol. Scale = 2 millidegrees per centimeter. Curve a: using a l-ram cell. Curve b: using a 0.1-mm cell.
be measured in the spectroscopically accessible region, the benzenoid 1Lb band (ca. 340 nm), 1L~ band (ca. 260 gm), and the ~B band (ca. 220 nm). These increase in intensity in approximately 10-fold increments on going to the UV from the visible, necessitating at least two successive dilutions in order to measure the spectrum. Even then, the ~c/e ratio is very small, so little light passes through the solutions; this makes the spectra noisy, and the small signals must be measured repeatedly before satisfactory values are obtained. An instrument with signal averaging capabilities would have made the determination of this spectrum much easier. The normalized spectrum has been published. 21 Figure 8 is a tracing of the actual spectra and the photomultiplier voltage readings. As the Xe lamp intensity decreases or sample absorption of light increases, the photomultiplier (PM) voltage across the lamp is increased to compensate for diminished light arriving in the detector system. The less light received, the more noisy is the spectrmn and the more difficult it is to locate the precise ellipticity induced by the sample. In our hands, if the P M voltage exceeds approximately 0.8, the instrument records artifacts (on one famous occasion when an inappropriate concentration was used, ..1L. A. Mitscher, P. W. Howison, J. B. Lapidus, and T. D. Sokoloski, J. Med. Chem. 16, 93 (1972).
366
METHODS FOR THE STUDY OF ANTIBIOTICS 2
[17]
21.-
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FIG. 9. The circular dichroism spectrum of L-threo-l-p-phenylphenyl-2-dichloroacetamido-l,3-propanediol in Cupra A solution. we were able to obtain a positive Cotton effect for benzldehyde), and the data cannot be used. This places limits on the amount of sample that can be used to obtain a weak Cotton effect, and renders the data considerably less precise than those one gets with chromophores having a better Ac/c ratio. Molecules with chromophores in more rigid molecular frameworks normally give more intense signals. G. An Enantiomeric p-Phenyl Analog of Chloramphenicol (13)
Use of chelation to fix conformation and simplify chromophore. One interesting device for solving the problem introduced in flexible molecules by their possession of numerous conformations which contribute different increments to the overall molecular rotation, is to run the spectrum in a solution containing a chelating ion which not only "freezes" the molecule into a single rotomeric state, but also possesses a different, more favorable, chromophore. Cupra A is an alkaline aqueous solution of Cu (II) ions which does this admirably (Fig. 9). The methanol CD spectrum of L-threo-(1S,2S)-l-p-phenylphenyl-2-dichloroacetamido-l,3-propanediol (13) is difficult to obtain with any degree of accuracy and is hard to compare with that of (12) because of the different electronic nature of the chromophore itself (p-¢ instead of p-NO2). In Cupra A solvent, however, the chromophore is the d--> d orbital transition of the Cu atom and the stereochemistry of the side chain determines the positions, signs, and intensities of the bands, greatly facilitating the assignment of absolute stereochemistry in this otherwise difficult spectroscopic antibiotic c l a s s . 22
= L. A. Mitscher, P. W. Howison, and T. D. Sololoski,J. Med. Chem. 16, 98 (1972).
[17]
367
SPECTROPOLARIMETRY OF ANTIBIOTICS
8
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FIG. 10. The circular dichroism (CD) and ultraviolet (UV) spectra of chelocardin in the presence ( - - ) and absence (---) of aluminum ion at pH 7.5.
H. Chelocardin (14) and Its Interaction with Aluminum Ions C D detects ion binding better than UV. Chelation with metal ions affects the solubility, the stability, and the clinical performance of the tetracycline antibiotics, so studies of this phenomenon are of some significance. Often this can be done by less complex procedures, but in some key respects and cases, CD offers striking advantages. Figure 10 illustrates the UV and CD spectra of chelocardin at pH 7.25 in the presence and in the absence of an excess of AI(III) ions. In the UV spectrum, there are intensity differences accompanying the chelation, but these are not dramatic. In the CD, however, it appears that a conformational change may be occurring which results in dramatic intensity differences upon chelation. It is obvious in this case that CD is a more sensitive spectroscopic tool for the study of chelation than is UV. Several other examples with different antibiotics are listed in Table II.
I. Interaction of an Antibiotic with a Biopolymer The reader is encouraged to examine the paper of Krueger et al. 2a for an excellent recent study showing the spectroscopic changes resulting from the interaction of antitumor antibiotics of the bleomycin type with DNA. The study of molecular interactions increasingly "~bsorbs the energies of spectroscopists and is producing important information in careful hands. It is important, however, that the inexperienced consider certain warnings. It is not difficult to demonstrate binding of antibiotics to significant biopolymers. Several studies demonstrate, in fact, that CD measurements frequently give more dramatic evidence of binding than UV 23 W. C. Krueger, L. M. Pschigoda, and F. Reusser, J, Antibiot. 26, 424 (1973).
368
METHODS FOR THE STUDY OF ANTIBIOTICS
[17]
studies carried out under the same conditions. The studies require considerable experimental skill and great care in the interpretation of the results. Postulated conformational perturbations of either the substrate or the biopolymer or both should be assigned cautiously. When an optically inactive antibiotic, like netropsin, binds to a biopolymer, it now finds itself in an asymmetric environment, with the result that cL-~ ~ and induced optical activity ("extrinsic" Cotton effect) results. In such studies, the CD spectrum of the biopolymer itself is often altered. It is common practice to interpret these changes in terms of a new conformation for the biopolymer induced as a consequence of the binding. This may well be justified. It is also possible, however, that the binding generates new pathways for electron exaltation in the biopolymer as well as it does for the drug. Partly for this reason, the interpretation of such studies is difficult and the results are accepted with a degree of justified caution. Another factor compelling caution is that we are not yet able to determine more than the overall shape of biopolymers from their CD spectra alone. Binding of antibiotics frequently shows great specificity, and, if only a few residues in a large molecule are affected by the binding, one may not see any perceptible change in the CD spectrum even though strong binding may be occurring. In those cases where a spectral change is apparent, we are often unable to define precisely the nature of that change. When the antibiotic is also optically active--which is the usual case--the situation becomes accordingly even more difficult to interpret. Nevertheless, much useful data and interesting specular:on has come out of these studies and when the conclusions are supported by data from another independent spectroscopic probe, confidence in the result is increased. Furthermore, the quantitative nature of the binding is not dependent upon the interpretation of the results in conformation terms, and so it is possible to determine the number of moles of antibiotic bound to a biopolymer and often to discriminate between different sites. Because current spectropolarimeters are monobeam instruments, it is necessary to perform repeated measurements in which one or more component is progressively added and their spectral contributions subtracted in order to arrive at a final result. This requires great stability and precision in measurement and repeated determinations in order to arrive at reliable results. Because of the relative insensitivity of the chiroptical methods, it often requires substantial concentrations of biopolymer. In some cases, this may lead to molecular aggregations which not only may be unphysiological, but also have altered spectroscopic properties as compared to the monomers. Despite these warnings, the ORD-CD methods are increasingly being used for such studies, and to a considerable extent these studies represent
[17]
S P E C T R O P O L A R I M E T R Y OF ANTIBIOTICS
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some of the most exciting work underway at present. References cited in footnotes 3 and 5 are excellent sources of information on this subject.
IV. Choice and Optimization of Experimental Parameters The vast majority of the chiroptical studies in the United States in general, and on antibiotics in particular, have been carried out with the Cary-Varian model 6024 or the Jasco O R D / U V / C D - 5 instrument, '-'~ the latter with or without the various modifications of SproulJ GEach of these vendors will supply detailed information about the design and use of their instruments. The following parameters are directly under the spectroscopist's control, and a proper choice requires considerable experience and has a definite bearing on the quality of the experimental results.
A. Choice of Solvent The majority of the O R D - C D spectra of antibiotics have been run in water or alcohols. M a n y antibiotics are good chelating agents and chelation alters their spectra so water should be double distilled and redistilled from EDTA. Spectrograde methanol, free as far as possible from acid, should be used to avoid ketal formation with ketones. Dioxane, purified as described by Fieser (method A),27 is an excellent solvent for less polar solutes. When collateral conformational information is available from a complimentary technique, such as N M R , the CD spectra should, if possible, be run in the same solvent.
B. Temperature Normally measurements are run at room temperature. The thermometer should be placed in the cell compartment, and this figure should be reported. Conformational instability can be detected by making measurements at a variety of temperatures. Very low temperatures (ca. --190 °) are used to freeze substances into their preferred conformations. Use of methyl cyclohexane-isopentane (1:3) and ether-isopentane-ethanol (5:5:2) mixtures as solvents is recommended. A special jacketed cell has been constructed for use with these conditionsJ s Both /-camphor and :' Applied Physics Corporation, 2724 South Peck Road, Monrovia, California 91016. :~Japan Spectroscopic Co., Ltd., 2967-5, Ishikawacho, Hachioji City, Tokyo 192, Japan. USproul Scientific Instruments, P. 0. Box 110, Boulder Creek, California 95006. 2TL. F. Fieser and M. Fieser, "Reagents for Organic Synthesis," Vol. I. Wiley, New York, 1967. 2sG. Barth, W. Voelter, H. S. Mosher, E. Bunnenberg, and C. Djerassi, J. Amer. Chem. Soc. 92, 875 (1970).
370
METHODS FOR THE STUDY OF ANTIBIOTICS
[17]
/-fenchone are readily available in pure form and are essentially devoid of either asymmetric solvation and conformational flexure; their use as a control for the magnitude of nonconformational effects due to changes in refractive index, volume contraction or expansion, and the like, is of considerable value. C. Atmosphere Ozone is generated in the instrument and for health as well as spectroscopic reasons should be swept out from the optical train by use of a constant flow of dry nitrogen gas while the instrument is on. This will prevent corrosion of the mirrors and also increases instrumental penetration in the 185-220 nm range. That region is of particular significance with peptides. The Cary instructions call for a considerably greater rate of N2 flow than the Jasco. D. Cells
Ordinary quartz UV cells can be used if they are sufficiently strainfree. Strain will cause light scattering and also interfere with proper polarization of the light. One test for this is to run a blank using sensitive settings and the suspect cell. Then reverse the cell in the holder and repeat the measurements. The two blank scans will track one another closely in strain-free cells--otherwise the cell should not be used. Pretested cells can be obtained from the instrument manufacturer at a price premium. If a cell becomes chipped, it usually becomes useless. A warm acidic methanol solution is excellent for removing traces of most antibiotics, and a dilute pepsin solution is valuable for removing residual protein. No trace of acid or base must be left in the cells. The cells should be placed as nearly as possible in the identical position in the holder and the light train for optimal reproducibility in precise work. Each investigator is encouraged to develop his own protocol for this. Of course, the cell should always face in the same direction. E. Instrument Calibration
d-10-Camphor sulfonic acid can be obtained commercially from a variety of sources, and 0.1% aqueous solution in a 1-cm cuvette is used to verify the proper calibration of the instrument as its O R D / C D maxima in the 290 nm region is conveniently easy to obtain. The solution can be sealed in a cuvette with a water-insoluble adhesive to guard against contamination and evaporation and then used at intervals as a
[17]
S P E C T R O P O L A R I M E T R Y OF ANTIBIOTICS
371
primary standard. This should be done daily for precise work and should be done at least weekly. The wavelength settings can be checked against samples of known X..... ; use of a neodymium glass available from the manufacturer (x ..... 586.0 nm----- 10 A), or, more cumbersomely, use of the line spectrum produced by a mercury vapor lamp. F. Slit Widths The slits are programmed to open automatically in regions where the Xe arc lamp has a lower output. It is advisable to open the slits manually or to displace the entire program toward more open slits in regions of significant sample absorption to allow sufficient light to pass through the sample and, thus, allow more accurate readings to be obtained. This will not only improve the signal to noise ratio, but at the same time decrease ~he ability to resolve maxima which are close to one another. These conflicting demands often require a compromise based on operator iudgment.
G. Scanning Speed When a spectrum contains sharp narrow peaks, or when it is necessary to use low gain settings to reduce instrument noise, slower scan speeds are necessary to allow for proper pen response and prevent shape distortions. Broad peaks and ORD approach slopes can be scanned more rapidly. A wide variety of scan speeds are available through use of the appropriate instrument settings, and operator judgment plays a significant role here also in obtaining optimal spectra.
H. Gain Settings At lower settings, lower pen responsiveness and sensitivities are obtained. This is usually done in order to filter random noise in regions of low net light transmission. One needs to choose a setting which allows for the most rapid pen response consistent with acceptable pen chatter and curve reproducibility and detail. In our opinion, it is better to have a slightly noisy spectrum and to take the average line width for calculation than to have a smooth curve with a sluggish pen response. The judgment that comes with experience is especially needed in this area.
I. Sample Concentration The optimum sample concentration is almost always determined by empirical experimentation. A balance must be found between the desire
372
METHODS FOR THE STUDY OF ANTIBIOTICS
[17]
to achieve the maximum possible ORD-CD pen deflection and the need to avoid excessive concentrations, which would result in insufficient light penetrating through the sample for accurate measurement. As a rule of thumb, a concentration roughly 10 times greater than would be used for normal UV measurements is a satisfactory place to begin. This concentration should not give photomultiplier voltage readings above approximately 0.8, or the ORD-CD readings will be noisy at best and inaccurate at worst. It is possible with some instruments to obtain apparent Cotton effects using, for example, benzaldehyde if the solution is too concentrated. It is advisable to check each instrument for this effect and for the concentrations, if any, that will give such spurious peaks. When quite accurate measurements are needed at longer wavelengths, especially ORD measurements at the sodium D line, it is seldom possible to choose a single satisfactory concentration to use for measurement of a spectrum through the entire spectral range. A high initial concentration is used, and, when unsatisfactorily high extinctions are obtained, the sample is either diluted or placed in a cell with a narrow path and the measurement continued. In almost every case, greater absorptions are encountered at lower wavelengths. The proper concentration is, then, another factor calling for initiative and judgment by the operator.
J. Survey Measurements Survey measurements are of great value in determining the necessary instrumental settings and concentrations. A prior determination of the UV spectrum is of great value for maximal ORD-CD readings are obtained at wavelengths close to those of the UV absorption maxima. One places the sample in the instrument and scans by hand near the UV maxima to see whether sufficient light will pass and to select the appropriate scale. Be careful to allow sufficient time for the pen to respond. After a few measurements have been made in a given antibiotic family, one can usually guess in advance what concentrations will be appropriate.
K. Blank Currently available instruments operate using a single beam so that a baseline solvent spectrum must be obtained after the sample has been measured. Care must be taken that the same settings are used for both blank and sample in order that the measurements be meaningful. Despite the occasional temptation to speed up the blank determination, it must be remembered that one is doing a difference spectrum and both scans are of the same intrinsic importance. With determinations of interactions
[18]
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between antibiotics and biopolymers, successive scans are needed and a high premium is placed on instrument stability to avoid the accumulation of errors. With CD measurements, zero sample ellipticities are encountered rather near the Cotton effect, so the blank scan can cover a narrow spectral range (from contact point with the sample spectrum to contact point or loss of sensitivity). For ORD, the whole spectral range must be rescanned. Direct difference measurements can be obtained conveniently with the Cary instrument by purchase of suitable accessories.
L. Reproducibility This varies somewhat with wavelength as some regions (notably at the longest and shortest wavelengths) are inherently more noisy than others. In student hands and with the exercise of average care, amplitude reproducibility of approximately 5-10% is expected. With special care and experience, precisions of 1-3% can be obtained. In absolute terms this is really not as bad as it may seem, for A~ is often only about l0 -~ X c. Thus specific ellipticity is very small compared with bulk absorption. Modern spectropolarimeters are remarkable in that they can measure these rather small differences with high precision. The use of computer summation of successive scans will improve the reproducibility even more.
[18] D i f f e r e n t i a l P u l s e P o l a r o g r a p h y o f A n t i b i o t i c s By HOWARD SIEGERMAN I. II. III. IV.
Introduction . . . . . . . . Limitations and Advantages . . . . Equipment and Analysis Procedure . . Specific A n a l y s i s C o n d i t i o n s . . . . A. C h l o r a m p h e n i c o l . . . . . . B. S t r e p t o m y c i n S u l f a t e . . . . . C. T e t r a c y c l i n e H y d r o c h l o r i d e . . . D. P e n i c i l l i n . . . . . . . . E. N e o m y c i n S u l f a t e . . . . . . F. C e p h a l o g l y c i n . . . . . . .
.
.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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373 375 377 381 382 383 384 385 387 387
I. Introduction Polarography and its more sensitive successor, differential pulse polarography, have been used infrequently to complement the accepted microbiological techniques for antibiotic analysis. Clinical chemists, microbi-
[18]
DIFFERENTIAL PULSE POLAROGRAPItY OF ANTIBIOTICS
373
between antibiotics and biopolymers, successive scans are needed and a high premium is placed on instrument stability to avoid the accumulation of errors. With CD measurements, zero sample ellipticities are encountered rather near the Cotton effect, so the blank scan can cover a narrow spectral range (from contact point with the sample spectrum to contact point or loss of sensitivity). For ORD, the whole spectral range must be rescanned. Direct difference measurements can be obtained conveniently with the Cary instrument by purchase of suitable accessories.
L. Reproducibility This varies somewhat with wavelength as some regions (notably at the longest and shortest wavelengths) are inherently more noisy than others. In student hands and with the exercise of average care, amplitude reproducibility of approximately 5-10% is expected. With special care and experience, precisions of 1-3% can be obtained. In absolute terms this is really not as bad as it may seem, for A~ is often only about l0 -~ X c. Thus specific ellipticity is very small compared with bulk absorption. Modern spectropolarimeters are remarkable in that they can measure these rather small differences with high precision. The use of computer summation of successive scans will improve the reproducibility even more.
[18] D i f f e r e n t i a l P u l s e P o l a r o g r a p h y o f A n t i b i o t i c s By HOWARD SIEGERMAN I. II. III. IV.
Introduction . . . . . . . . Limitations and Advantages . . . . Equipment and Analysis Procedure . . Specific A n a l y s i s C o n d i t i o n s . . . . A. C h l o r a m p h e n i c o l . . . . . . B. S t r e p t o m y c i n S u l f a t e . . . . . C. T e t r a c y c l i n e H y d r o c h l o r i d e . . . D. P e n i c i l l i n . . . . . . . . E. N e o m y c i n S u l f a t e . . . . . . F. C e p h a l o g l y c i n . . . . . . .
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373 375 377 381 382 383 384 385 387 387
I. Introduction Polarography and its more sensitive successor, differential pulse polarography, have been used infrequently to complement the accepted microbiological techniques for antibiotic analysis. Clinical chemists, microbi-
374
METHODS FOR THE STUDY OF ANTIBIOTICS
[1@]
ologists, and others involved in antibiotic analysis have been unaware of the potentialities and scope of the polarographic method. This is unfortunate, for differential pulse polarography has several attractive features as far as antibiotic analysis is concerned. These advantages include: Qualitative and quantitative analysis Analysis of mixtures Good sensitivity Functional group analysis Starting material and degradation product identification Response to antibiotics of widely varying structure Speed of analysis Analysis of enzymes (e.g., fl-lactamases) In this article emphasis will be focused on the differential pulse polarographic (DPP) technique as applied to the analysis of such common antibiotics as chloramphenicol, tetracycline, streptomycin, penicillin, cephaloglycin, and neomycin. These are by no means the only antibiotics that can be analyzed, but they serve to illustrate how the technique can be applied to analysis of a variety of different structures comprising different functional groups. Viewing antibiotics in the light of the organic functional groups that they contain m a y seem awkward at first, but it is the key to success in a proposed D P P analysis. Some antibiotics, e.g., ehloramphenicol, tetracyline, and streptomycin, contain electroactive functional groups, such as nitro functions, activated earbonyls, activated double bonds, and aldehyde groups, that respond well to this method of analysis. Other antibiotics, such as penicillin and neomycin, contain no eleetroactive functional groups yet they are easily converted into electroactive structures through simple functionalization reactions, e.g., hydrolysis, nitrosation. Differential pulse polarography involves measurement of solution constituents by microelectrolysis at a dropping mercury electrode (DME). The microelectrolysis current 1 is plotted on a recorder as a function of the electrode potential of the DME. The microeleetrolysis of an antibiotic at the D M E yields a peak-shaped current-voltage curve, which contains both qualitative and quantitative information. Not only can the peak position aid in identifying the antibiotic, but the peak height responds linearly to solution antibiotic concentration. Differential pulse polarography differs from its less sensitive predecessor, dc polarography, both in the manner in which the potential of the 1More properly, a current difference which results from electronic subtraction of two current samples. The potential-time waveform which gives rise to the current difference operation is employed primarily to eliminate background effects which interfere at high sensitivities.
[18]
375
DIFFERENTIAL PULSE POLAROGRAPHY OF ANTIBIOTICS
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-14
-t
Fie. 1. Differential pulse polarography of chloramphenieol. DME, dropping mercury electrode ; SCE, saturated calomel electrode. D M E is varied with time, and in the manner in which current is displayed on the recorder Y axis. For the present discussion it is sufficient to note t h a t differential pulse polarography is preferred to classical dc polarography because it is more sensitive (by a factor of 100-1000) through signal-to-noise enhancement, the readout is easier to use and quantitate (peak-shaped vs S-shaped curves), and there is no added inconvenience to the user (commercial instrumentation for D P P is readily available). Figure 1 shows the differential pulse polarographic response for a 1.3 X 10-~ M chloramphenicol solution in 0.1 M acetate buffer, p H 4, while Fig. 2 shows the dc response for the same solution. The signal-tonoise enhancement of D P P over dc is obvious. For a more complete discussion of the inner beauty and workings of D P P , the interested reader is referred to the literature3 -~
II. Limitations and Advantages M a n y industrial and federal laboratories are concerned strictly with antibiotic activity. For them, only common microbiological assays such -"E. P. Parry and R. A. OsteD'oung, Anal. Chem. 37, 1634 (1965). 3 H. D. Siegerman and G. W. O'Dom, Amer. Lab. 4(6), 59 (1972). 4j. Flato, Anal. Chem. 44, 75A (1972).
376
[18]
METHODS FOR THE STUDY OF ANTIBIOTICS
1.5 xlO-s M CHLORAMPHENICOL O.1 M Acetate Buffer DC-DME
IItlItltttlIltItlt
SIGNAl_
T
0
I
I
!
I
!.
I
I
I
I
-0.1 - 0 2 - 0 . 3 - 0 . 4 - 0 5 - 0 6 - 0 7 - 0 8 - 0 9 POTENTIAL r E vs s c E]
I
-I0
I
-11
I
-12
I
-I.3
I
-14
-1.5
FIo. 2. Direct current polarography of chlorampheni~ol. DME, dropping mercury electrode; SCE, saturated calomel electrode. as the cylinder plate assay or the turbidimetric assay are suitable or even acceptable. D P P can function here only as a possible check on a microbiological assay--it can never replace that assay since the primary information sought is the microbiological activity of the antibiotic. Also if many assays are required, the microbiological technique has the advantage that simultaneous assays can be run on many plates with only a small increase in operator time and little, if any, increase in capital equipment expenditure. What the microbiological assay lacks is the capability to function as a rapid screening technique. Most often assays require overnight treatment. This may be totally unsatisfactory if a quick answer is sought concerning the presence or absence of an antibiotic in a given matrix (e.g., feed additives). D P P can be most valuable in cases such as these since the technique is rapid and straightforward. Often, more information than just antibiotic activity is required. In the development of new antibiotic species--either natural or synthetic-sensitive, rapid analytical methods, usually involving functional group determinations, are necessary. Frequently it is necessary to ensure that the antibiotic is not contaminated with unacceptable amounts of starting materials, degradation products, reaction by-products, etc. Also, an analyst occasionally may be required to identify an unknown antibiotic, or
[18]
DIFFERENTIAL
PULSE
POLAROGRAPHY
OF A N T I B I O T I C S
377
25 mV MODULATION I SEC/DROP 2 mV/SEC
l
SIGNAL
3_ 0 5,uA
T
-0 2
-Q4 -06 POTENTIAL [E vs SCE]
- 08
FIG. 3. Differential pulse polarography of ehloramphenieol and tetracycline. SCE, saturated calomel electrode. a mixture of two dissimilar antibiotics. For cases such as these, DPP can be used to advantage, since structural information is sought rather than gross microbiological activity. Figures 3 and 4 illustrate some interesting applications of the DPP technique to two of the cases described above. Figure 3 shows the DPP response to a mixture of two antibiotics--chloramphenicol and tetracycline--at the parts per million level. Both antibiotics can be identified and quantitated in the presence of the other. Analysis times are of the order of minutes rather than hours. Without prior separation of the antibiotics, such a n analysis would be impossible by the microbiological technique. In Fig. 4 the presence of 6-aminopenicillanic acid, a penicillin d~gradation substance, is easily recognized in a proprietary penicillin product, since it exhibits a peak at --0.64 V, well removed from the penicillin peak at --0.4 V.
III. Equipment and Analysis Procedure To perform differential pulse polarographic analyses of antibiotics, one requires an instrument, a recorder (preferably X-Y, but strip chart will do), a suitable cell system, and a nitrogen supply. The actual analysis is quite simple. The sample is dissolved in the proper supporting electrolyte (see Section IV), and a measured volmne (usually 10 ml) of the sample solution is pipetted into the polarographic cell (Fig. 5), fitted with a reference electrode and a platinum wire counter
378
[18]
METHODS FOR THE STUDY OF ANTIBIOTICS
O.IM ACETATEDUFFER,pH4 DIFFERENTIAL PULSEMODE
0 2 ~A ~
l ^
0
,
,
4.20ppm TETRACYCLINE •H
2.40ppm
,
,
,
,
,
,
,
,
,.
--o.i -o2 -o.3 -o4 -0.5 -o.s -0.7 -O.S - .9 -I.o -II POTENTIAL [E
vs
!
,
,
-12 -13 -~.4
SC E]
FI~. 4. Differential pulse polarography of 5 p p m proprietary penicillin Jr-17 ppm of 6-aminopenicillanic acid in 1 M NaOH. SCE, saturated calomel electrode.
electrode2 The solution is purged of dissolved oxygen for 10 min using high quality nitrogen purified with an oxygen scrubbing system. 6 After the purging period the nitrogen stream is directed over the solution, and a nitrogen blanket is maintained over the sample during the actual electrochemical experiment. The dropping mercury electrode is immersed into the solution and the differential pulse polarogram is run. Typical instrument settings are as follows: scan rate, 2 mV/sec; scan direction, "--"; scan range, 1.5 V; initial potential, 0 V; modulation amplitude, 25 mV; operating mode, differential pulse; current range, 0.5 /~A full scale; output offset, off; display direction, " ~ " ; drop time, 1 sec; low pass filter, off. These instrument settings refer to the Princeton Applied Research Model 174 Polarographic Analyzer. Actual switch positions will be governed by the nature and concentration of the antibiotic in question. 5 One version of a commercially available polarographic cell is shown in Fig. 5. This cell has the advantage t h a t the electrodes and purge tube remain fixed to the cell top, while the sample solution is contained in a beaker-style cell b o t t o m which can be quickly removed from the cell top. Thus, changing samples and rinsing electrodes between runs can be accomplished quickly and conveniently. "Why Outgassing--and How," Application Note 108, Princeton Applied Research Corporation, P. O. Box 2565, Princeton, New Jersey 08540.
[18]
DIFFERENTIAL PULSE POLAROGRAPHY OF ANTIBIOTICS
379
-!
6
7
8 I~i ¸
-9
Fro. 5. Polarographic cell and associated glassware. 1, D M E contact tube; 2, purge tube with two-way stopcock; 3, mechanical drop timer; 4, saturated calomel reference electrode; 5, cell top; 6, reference electrode salt bridge tube; 7, platinum wire counter electrode; 8, dropping mercury electrode capillary; 9, demmmtable cell bottom.
380
METHODS FOR THE STUDY OF ANTIBIOTICS
[18]
The antibiotics in solution often can be identified by the position and pattern of the peaks obtained. For antibiotics that do not require prior functionalization, concentrations are best determined by the method of standard additions since matrix effects are, to a large degree, eliminated; alternatively, a calibration curve can be used. The standard additions method involves spiking the solution in the cell with a small, known volume of a standard solution of the antibiotic followed by rerunning the DPP scan. A convenient one-line equation yields the original solution concentration, Cu = (ilvCs)/[i2v + (is -
il)V]
where Cu = original concentration, C. -- concentration of standard solution, il = original peak height, i2 = "spiked" peak height, v = volume
1.4-
1.2-
T
1.0-
z~ 0 . 8 hi D: Q:
0.6laJ a.
0.4-
0.2-
ppm
FIG. 6. Differential pulse polarography peak current vs concentration for ehloramphenicol in 0.1 M acetate buffer, p H 4; differential pulse mode; peak current measured at --0.27 V vs saturated calomel electrode.
[18]
DIFFERENTIAL
PULSE
POLAROGRAPHY
OF ANTIBIOTICS
381
of s t a n d a r d solution " s p i k e , " V = original simple volume in polarographic cell. This m e t h o d of calculation assumes that, other p a r a m e t e r s being the same, p e a k height is a linear function of the antibiotic concentration. Such linearity is shown in Figs. 6 and 7 for D P P analysis of chloramphenicol and tetracycline. If m a n y analyses are to be run, m u c h calculation time can be saved b y incorporating the equation into a p r o g r a m m a b l e calculator.
IV. Specific Analysis Conditions Suitable D P P analysis conditions for a variety of antibiotics are given in the table. The information presented here is by no means restrictive. For example, other aqueous buffers are suitable for polarography of 0.5
04-
03-
Z w
~ 0.2-
w
0.1
ppm
Fro. 7. DPP peak current vs concentration for tetracycline hydrochloride in 0.1 M acetate buffer, pH 4; differential pulse mode; peak current measured at --1.01 V vs saturated calomel electrode.
382
[18]
METHODS FOR T H E STUDY OF ANTIBIOTICS SPECIFIC ANALYSIS CONDITIONS FOR DIFFERENTIAL PULSE POLAROGRAPHY OF ANTIBIOTICS
Concentration b
Supporting
Antibiotic Chloramphenicol Streptomycin Tetracycline hydrochloride Penicillin G potassium Ampicillin • 3H~O Nitrosated penicillin G potassium
electrolyte 0.1 M Acetate buffer, pH 4 1 M NaOH, 1 mM EDTA 0.1 M Acetate buffer, pH 4 1 M NaOH 1 M NaOH 2.4% NaOH
E, (V vs SCE) a
(ppm)
-0.27
0.1
-1.52, - 1.64
1
- 1 . 0 1 , -1.18,
0.2
--1.31 -0.37 -0.36 -0.35, -0.76, -0.93, - 1.14,
0.2 0.2 1
--1.33
Penicillin, enzyme treated 6-Aminopenicillanic acid Neomycin sulfate Cephaloglycin
0.1 M Acetate buffer, pH 4 1 M NaOH 0.1 M Acetate buffer, pH 4 1 M H~SO4
-0.05
18.6
-0.64 -0.19, - 0 . 3 6
1 50
0.935
1
Where multiple peaks occur, the italicized Ep indicates the peak that was quantitated. b Lowest concentration investigated in this study. chloramphenicol, and a large number of tetracycline, penicillin, and cephalosporin species can be analyzed using the appropriate supporting electrolyte listed in the table. Also, by optimizing instrument settings and solution conditions, it is quite conceivable that higher sensitivities than those listed can be obtained. Additional pertinent information concerning polarographic analysis of these antibiotics is described below.
A. Chloramphenicol The polarographic behavior of chloramphenicol centers on the reduction of the nitro group, a well-characterized and well-behaved electrode process. B y dc polarography Fossdal and Jacobsen 7 determined chloramphen:.col over a concentration range 0.3 to 600 ppm in 0.5 M acetate buffer, pH 4.7. These workers also used other electroanalytical techniques such as cyclic voltammetry, chronopotentiometry, coulometry, and ac polarography to study the electrochemical behavior of chloramphenicol. 7K. Fossdal and E. Jacobsen, Anal. Chim. Acta 56, 105 (1971).
[18]
DIFFERENTIAL
PULSE
POLAROGRAPHY
OF ANTIBIOTICS
383
0 96 pp~ 0.72 pp~A 1
T
SIGNAL
O0
± 50nA T
'
-0~.2
-0~.4 ' -016 POTENTIAL[EvsSCE]
FIG 8. Differential pulse polarography of sub-ppm chloramphenicol in 0.1 M acetate buffer, pH 4, differential pulse mode. SCE, saturated calomel electrode. Ae polarography was found to be not as sensitive as the de counterpart for chloramphenicol determination. With the D P P technique, chloramphenicol can be detected easily at the 0.1 p p m level in 0.1 M acetate buffer, p H 4, giving a 3-fold improvement in detection capability when compared to the dc technique. Typical results are shown in Fig. 8.
B. Streptomycin Sulfate L e v y and co-workers s observed well-defined de waves for 100 ~g/ml streptomycin in 3% t e t r a m e t h y l a m m o n i u m hydroxide. D o a n and RiedeP were able to detect only a minimum of 100 p p m streptomycin sulfate in 1 N N a O H by dc polarography. Using the D P P technique, 1 p p m can be easily detected (Fig. 9). The high sensitivity of D P P necessitated the use of a small amount of E D T A to complex and render electroinactive trace quantities of zinc (Ep = --1.44 V vs SCE in 0.1 M N a O H without E D T A ) present as an impurity in the supporting electrolyte. G. B. Levy el ed., J. Amer. Chem. Soc. 68, 528 (1946). 0 L. Doan and B. E. Riedel, Car~. Pharm. J. Sci. 96, 109 (1963).
384
[18]
METHODS FOR THE STUDY OF ANTIBIOTICS
SIGNAL
2OnA
W
-I.2
I
-I.3
I
-I.4
I
-I .5
I
-I .6
I
- I .7
I
- I .8
l
l
POTENTIAL [Evs S C E]
Fro. 9. Differential pulse polarography of streptomycin; 1 ppm streptomycin sulfate, 0.1 M Na0H, 1 mM EDTA. SCE, saturated calomel electrode.
C. Tetracycline Hydrochloride Caplis TM reported de polarographic results for 1.04 X 10-~ M (= 50 ppm) tetracycline hydrochloride in Clark and Lubs buffer p H 4.1. Similarly Doan and RiedeP employed a minimum concentration of 50 p p m using a Sorensen buffer p H 6.2. The latter workers obtained an unusually high diffusion current (20 ~A) for such a low concentration of electroactive material. Typical differential pulse polarograms of tetracycline hydrochloride in 0.1 M acetate buffer p H 4 are shown in Fig. 10. Thus, an increase in sensitivity of 250 times can be realized if the D P P technique is adopted rather than the dc technique. Although the tetracycline functional groups undergoing reduction have ~0M. E. Caplis, Electroreduction of tetracycline antibiotics, Ph.D. Thesis, Purdue University, Lafayette, Indiana, 1970.
[18]
DIFFERENTIAL PULSE POLAROGRAPHY OF ANTIBIOTICS
_L
385
2.10 ppm
50 nA
T
3-
SIGNAL
ppm £..
-08
-- 110
ppm
--112
POTENTIAL [EvsS C E]
FIG. 10. Differential pulse polarography of tetracycline hydroehloride in 0.1 M acetate buffer, pH 4, SCE, saturated calomel electrode. not been positively identified, based on the de polarography of anhydrotetracycline, isoehlortetraeyeline, and aureomyeinic acid, Doskocilova 1~ suggested that the first wave corresponds to reduction of the double bond in ring A conjugated to the adjacent earbonyl group and the second wave corresponds to reduction of the conjugated carbonyl at the C-11 to C-11a and C-12 positions. Other eleetroanalytical techniques have been used for analysis of chlortetraeyeline, tetracycline, doxyeycline, methaeyeline hydrochloride, and rolitetraeyeline. 1'-'-1' D. Penicillin
Although penicillin compounds are not electroaetive they can be easily converted to species that are amenable to examination by DPP. Penicillin compounds are converted to the corresponding penieilloie acid derivatives either by basic hydrolysis or enzymic reaction. For analysis of penicillin either reaction is suitable. As an added benefit the penieilloic acid content of a penicillin product can also be determined. 11D. Dosko~ilov~, Pharmazie 13, 548 (1958). 12j, S. Hetman, Lab. Pract. 12(8), 727 (1963). ~ A. Regosz and R. Kaliszan, Farma. Pol. 26(12), 1039 (1970); Anal. Abstr. 20, 4363 (1971). 14S. Silvestri, Pharm. Acta. Helv. 47, 209 (1972).
386
METHODS FOR THE STUDY OF ANTIBIOTICS
[18]
SIGNAL
-o;t
-o~s
-019
-Iio
-Ir.,
,.2
POTENTIAL [E vs. SCE]
Fie. 11. Differential pulse polarography of cephaloglycin in 1 M H=SO,. SCE, saturated calomel electrode. Benner 1~ developed a 2-hr assay of penicillin G in serum ultrafiltrate using fast-scan polarography with 1 M N a O H as the supporting electrolyre. D P P , however, offers higher sensitivity over the fast-scan technique--0.2 p p m vs 1 p p m for both penicillin G and ampicillin (see the table). The polarographic response of penicilloic acid produced by the reaction of a penicillinase enzyme (e.g., Nutrapen) with the parent compound and the enzymic stability of penicillins has been studied by Dusinsky. 1~-18 B y reversing the order of addition, it should be possible to analyze for 15E. J. Benner, Antimicrob. Ag. Chemother. 201 (1970). ~*G. Dusinsky, in "Scientiae Pharmaceuticae-II" (Proc. 25th Congr. Pharm. Sci. Prague, 1965), p. 241. Butterworths, London, 1966. 17G. Dusinsky, Proc. Con]. Appl. Phys. Chem. Vol. II, p. 431 (1971). ~ G. Dusinsky and P. Antolik, Nature (London) 206, 196 (1965).
[18]
DIFFERENTIAL PULSE POLAROGRAPHY OF ANTIBIOTICS
3S7
fl-lactamase concentration; i.e., increasing amounts of penicillin added to a fixed quantity of the enzyme should exhibit a rising then flat peak current-concentration response as the concentration of penicilloic acid increases to its maximum value. The benzene ring of the penicillin G is also susceptible to functionalization by nitrosation. The nitroso derivative yields good DPP response down to the 1 ppm level and is prepared as follows: An aliquot (0.1-1 ml) of an aqueous penicillin G potassium solution is added to 4 ml of 1 M HC1 in a 25-ml volumetric flask and held in a 70 ° water bath for 40 min. Two milliliters of 1 M NaNO~ is added, and after 10 min 3 ml of 20% NaOH. The solution is allowed to equilibrate to room temperature, diluted to volume, and analyzed. The application of the technique to the detection of 6-aminopenicillanie acid has been discussed earlier and is shown in Fig. 4.
E. Neomycin Sulfate After acid hydrolysis, neomycin can be detected by DPP as the furfural derivative. The hydrolysis reaction is carried out under reflux conditions in 1 M HC1. The solution is cooled, neutralized with sodium acetate to yield an acetate buffer supporting electrolyte, pH 4. F. Cephaloglycin A number of cephalosporin antibiotics including eephaloridine, cephalothin, and cephalexin have been examined previously by polarogra-
600-
5OO= ~_.4oocr rr
:300-
zoo-
I00,
0 ppm OF CEPHALOGLYCIN
FIG. 12. Differential pulse polarography peak current vs concentration for cephaloglycin in 1 M H2SO4.
388
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
p h y 15,19,2° w i t h s e n s i t i v i t y a t t h e 1 p p m level. D P P y i e l d s a w e l l - s h a p e d curve for I p p m c e p h a l o g l y c i n ( p r e v i o u s l y u n r e p o r t e d ) in 1 M H2S04 (Fig. 11) w i t h a l i n e a r p e a k c u r r e n t c o n c e n t r a t i o n r e l a t i o n (Fig. 12).
Acknowledgments The assistance of Lonel Brown, Jose Chang, and Marie Lefevre in obtaining some of the experimental data is gratefully acknowledged. ~9I. F. Jones, J. E. Page, and C. T. Rhodes, J. Pharm. Pharmacol., 20, 455 (1968); Anal. Abstr., 18, 2729 (1970). 2oD. A. Hall, J. Pharm. Sci. 62, 980 (1973).
[19] Proton Magnetic Resonance Spectroscopy of Antibiotics By GEORGE SLOMP I. II. III. IV.
Introduction . . . . . . . . . . . . . . . . . . Applicability . . . . . . . . . . . . . . . . . . Instrumentation and Sample Requirements . . . . . . . . . Interpreting the Spectrum . . . . . . . . . . . . . . A. Area Measurement . . . . . . . . . . . . . . . ]3. Spectral Analysis . . . . . . . . . . . . . . . . C. Chemical Shift Measurement . . . . . . . . . . . . V. Proposing a Structure . . . . . . . . . . . . . . . . A. Interpreting the Shifts . . . . . . . . . . . . . . B. Interpreting the Coupling Constants . . . . . . . . . . C. Trial Structures . . . . . . . . . . . . . . . . VI. Further Studies . . . . . . . . . . . . . . . . . A. Changing the Spectrum . . . . . . . . . . . . . . B. Changing the Sample . . . . . . . . . . . . . . .
388 390 392 394 394 395 396 396 397 398 400 400 400 403
I. I n t r o d u c t i o n P r o t o n m a g n e t i c r e s o n a n c e s p e c t r o s c o p y ( P M R ) of a n t i b i o t i c s is a specific a p p l i c a t i o n of t h e general n u c l e a r m a g n e t i c r e s o n a n c e ( N M R ) technique. T h e g r e a t e s t u t i l i t y of t h i s r e l a t i v e l y new f o r m of s p e c t r o s c o p y is in t h e a r e a of m o l e c u l a r s t r u c t u r e d e t e r m i n a t i o n , where it c o m p l e m e n t s some of t h e m o r e t r a d i t i o n a l m e t h o d s b u t d i s p l a c e s some others. T h e p r i n ciple is simple, b u t t h e i n s t r u m e n t a t i o n is c o m p l i c a t e d a n d expensive. T h e t e c h n i q u e is still u n d e r g o i n g r a p i d d e v e l o p m e n t , m o s t l y to i n c r e a s e its s e n s i t i v i t y . I t s a p p l i c a b i l i t y v a r i e s w i t h t h e size a n d n a t u r e of t h e u n k n o w n a n t i b i o t i c . I f t h e r e s u l t i n g s p e c t r a a r e difficult to a n a l y z e t h e y
388
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
p h y 15,19,2° w i t h s e n s i t i v i t y a t t h e 1 p p m level. D P P y i e l d s a w e l l - s h a p e d curve for I p p m c e p h a l o g l y c i n ( p r e v i o u s l y u n r e p o r t e d ) in 1 M H2S04 (Fig. 11) w i t h a l i n e a r p e a k c u r r e n t c o n c e n t r a t i o n r e l a t i o n (Fig. 12).
Acknowledgments The assistance of Lonel Brown, Jose Chang, and Marie Lefevre in obtaining some of the experimental data is gratefully acknowledged. ~9I. F. Jones, J. E. Page, and C. T. Rhodes, J. Pharm. Pharmacol., 20, 455 (1968); Anal. Abstr., 18, 2729 (1970). 2oD. A. Hall, J. Pharm. Sci. 62, 980 (1973).
[19] Proton Magnetic Resonance Spectroscopy of Antibiotics By GEORGE SLOMP I. II. III. IV.
Introduction . . . . . . . . . . . . . . . . . . Applicability . . . . . . . . . . . . . . . . . . Instrumentation and Sample Requirements . . . . . . . . . Interpreting the Spectrum . . . . . . . . . . . . . . A. Area Measurement . . . . . . . . . . . . . . . ]3. Spectral Analysis . . . . . . . . . . . . . . . . C. Chemical Shift Measurement . . . . . . . . . . . . V. Proposing a Structure . . . . . . . . . . . . . . . . A. Interpreting the Shifts . . . . . . . . . . . . . . B. Interpreting the Coupling Constants . . . . . . . . . . C. Trial Structures . . . . . . . . . . . . . . . . VI. Further Studies . . . . . . . . . . . . . . . . . A. Changing the Spectrum . . . . . . . . . . . . . . B. Changing the Sample . . . . . . . . . . . . . . .
388 390 392 394 394 395 396 396 397 398 400 400 400 403
I. I n t r o d u c t i o n P r o t o n m a g n e t i c r e s o n a n c e s p e c t r o s c o p y ( P M R ) of a n t i b i o t i c s is a specific a p p l i c a t i o n of t h e general n u c l e a r m a g n e t i c r e s o n a n c e ( N M R ) technique. T h e g r e a t e s t u t i l i t y of t h i s r e l a t i v e l y new f o r m of s p e c t r o s c o p y is in t h e a r e a of m o l e c u l a r s t r u c t u r e d e t e r m i n a t i o n , where it c o m p l e m e n t s some of t h e m o r e t r a d i t i o n a l m e t h o d s b u t d i s p l a c e s some others. T h e p r i n ciple is simple, b u t t h e i n s t r u m e n t a t i o n is c o m p l i c a t e d a n d expensive. T h e t e c h n i q u e is still u n d e r g o i n g r a p i d d e v e l o p m e n t , m o s t l y to i n c r e a s e its s e n s i t i v i t y . I t s a p p l i c a b i l i t y v a r i e s w i t h t h e size a n d n a t u r e of t h e u n k n o w n a n t i b i o t i c . I f t h e r e s u l t i n g s p e c t r a a r e difficult to a n a l y z e t h e y
[19]
P M R SPECTROSCOPY OF ANTIBIOTICS
389
can usually be manipulated to make them simpler, and several additional procedures are available when the interpretation is stalled. P M R spectroscopy is frequently employed in the study of time-dependent phenomena, such as the motions and interactions of large molecules of biological importance. 1,2 It also has quantitative applications, but its most widespread use is in the determination of molecular structure. It gives such minute structural detail that it is best used with more coarse methods like infrared and ultraviolet spectroscopy for detecting functional groups and characterizing unsaturation, or with mass spectrometry for molecular formula and fragment analysis. Since the integration of these analytical tools into a structure team most of the old fashioned degradations and chemical tests have been eliminated from structure proofs. The P M R method uses hydrogens as a probe for the molecular structure. It gives information on the environment around the hydrogens (i.e., what functional groups they are in), it tells which hydrogens are nearby each other (how the groups are connected), and it gives angular information (stereochemistry) on hydrogens which are vicinal, vinylic, or allylic. From this the rest of the structure can often be filled in. P M R is frequently augmented with the N M R of other nuclei, notably 13C. The two complement each other. Because the energy differences that are measured in this form of spectroscopy are so tiny (only slightly larger than thermal energy) and the whole radiofrequency spectrum which results is so narrow (the resolution must be in parts per billion) the instruments are quite costly. Even so, most laboratories are equipped for N M R studies at either 60, 100, or 220 MHz. Some commercial laboratories measure N M R spectra as a service. Although inherently an insensitive technique, compared to others, its great utility has generated many improvements. Recent advances based on relaxation (nuclear Overhauser effect3), complexation (shift reagents*), and pulsing (Fourier transform technique .r) help where the method was inherently weak previously. I~This series, Vol. 26, Section VII. D. W. Urry and M. Ohnishi, in "Spectroscopic Approaches to Biomolecular Conformation" (D. W. Urry, ed.), Chap. VII. American Medical Association, Chicago, Illinois, 1970. 3j. H. Noggle and R. E. Schirmer, "The Nuclear Overhauser Effect." Academic Press, New York, 1971. 4R. von Ammon and R. D. Fischer, Angew. Chem., Int. Ed. Engl. 11, 675 (1972). T. C. Farrar and E. D. Becker, "Pulse and Fourier Transform NMR." Academic Press, New York, 1971.
390
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
The reader is referred to other books, 6-1° reviews, 1~-13 and an introductory article TM for the theory of N M R , chemical shift, spin-spin coupling, double resonance, relaxation, etc., and for directions on how to obtain the best spectra. I t is intended here to discuss when and how to use P M R for molecular structure determination of antibiotics (as well as other organic molecules), what determinations to undertake or ask for, what size sample to provide, and especially how to interpret the data. 0 n l y recent or key references are cited, leaving it to the investigator to trace b a c k to the original contributions if he so desires.
I I . Applicability Success of the method depends greatly on the size of the molecule and the nature of the sample. Both influence the ability to analyze and interpret the N M R spectrum. Even when the spectrum cannot be completely "factored" much useful structural information can still be obtained. The analysis gets more difficult with larger molecules, having m a n y hydrogens, because of crowding and overlapping of the N M R signals. The best results have been on molecules of moderate size, up to about twenty nonmethyl hydrogens. Figure 1 shows the P M R spectrum which, together with MS, IR, and UV data, allowed a structure to be assigned to geldanamycin (I) 15 having 19 nonmethyl hydrogens, only 4 of which could not be analyzed. Larger molecules should be cleaved and studied in parts, as was done with lincomycin, TM streptolydigin, ~7 spectinomycin, TM and streptovaricin, TM 6j. W. Emsley, J. Feeney, and L. H. Sutcliffe, "High Resolution Nuclear Magnetic Resonance." Vols. 1 and 2. Pergamon, Oxford, 1965. 7L. M. Jackman and S. Sternhell, "Applications of Nuclear Magnetic Resonance Spectroscopy in Organic Chemistry." 2rid ed. Pergamon, Oxford, 1969. 8 E. D. Becker, "High Resolution NMR. Theory and Chemical Applications." Academic Press, New York, 1969. ~F. A. Bovey, "Nuclear Magnetic Resonance Spectroscopy." Academic Press, New York, 1969. 10N. Muller, in "Techniques of Chemistry" (A. Weissberger, ed.,), Vol. 1, Part IIIA, Chap. VII. Wiley (Interscience), New York, 1972. 11R. C. Ferguson and W. D. Phillips, Science 157, 257 (1967). 12 p. L. Corio, S. L. Smith, and J. R. Wasson, Anal. Chem. 44, 407R (,1972). la W. Mc.Farlane, Chem. Brit. 5, 142 (1969). 1~F. A. Bovey, Chem. Eng. News 43, 98 (1965). 15K. Sasaki, K. L. Rinehart, Jr., G. Slomp, M. F. Grostic, and E. C. Olson, J. Amer. Chem. Soc. 92, 7591 (1970). 1~G. Slomp and F. A. MacKellar, J. Amer. Chem. Soc. 89, 2454 (1967). 1TK. L. Rinehart, Jr., J. R. Beck, D. B. Borders, W. W. Epstein, T. H. Kinstle, L. D. Spicer, D. Krauss, and A. C. Button, Antimicrob. Ag. Chemother. 1963, 346 (1963).
[19]
P M R SPECTROSCOPY OF ANTIBIOTICS
391 s,
I H~O
u II
1
I
'
i
'
I
'
1
~'
t
M ]
(~
'
,o,
0 [
NO v~
i
r
II
B
/
H3
i r
R3.D
,
G
C
oJ~ I:
a=C
s3
'~3
F~
/'
![
CHF
v~*3
I1
~ J
~i ~!i~ Ti!'
-f,,
•
i
I
!"
[i
I : .... I0
I 9
,
[
, 8
I
7
.
I
6
..... I ......... I . . . . . . . . I . . . . . . . . . . . . . 5
6 , ppm
4
3
I............. I
from T M S
FIG. 1. Proton magnetic resonance spectrum obtained from a solution of geldanamycin acetate in deuterochloroform at 100 MHz.
for example. Cleavage or derivatization may also be necessary when large numbers of similar hydrogens are present, e.g., polysaccharides like neomycin, s° polyamino acids, ~ polyenes, :1 etc., since overlapping in one area would obscure the individual signals. Very large molecules like crythromycin, gramicidin, pimaricin, and novobiocin do not tumble well iH solution and therefore display broad signals that are poorly resolved and of little use in structure determination. On the other hand, molecules with relatively few hydrogens are difficult to analyze by P M R if they leave large gaps in the molecule in which there are no hydrogen probes. These are better studied by ~'~C N M R Y v-':~ P M R is most useful on molecules with continuous strings of neighboring hydrogens where their couplings can be used to identify the structure, working down a chain or around a ring. It also helps to know some history of the sample or other characteristics of the sample which give clues to what type of antibiotic to expect. 1~G. Slomp and F. A. MacKellar, Tetrahedron Lett. 521 (1962). ~' K. L. Rinehart, Jr., M. L. Maheshwari, F. J. Antosz, H. H. Mathur, K. Sasaki, and R. J. Schacht, J. Amer. Chem. Soc. 93, 6273 (1971). 2o M. Hichens and K. L. Rinehart, Jr., J. Amer. Chem. Soc. 85, 1547 (1963). 51R. C. Pandey, V. F. German, Y. Nishikawa, and K. L. Rinehart, Jr., J. Amer. Chem. Soc. 93, 3738 (1971). := J. B. Stothers, "Carbon-13 N M R Spectroscopy." Academic Press, N e w York, 1972. "~G. C. Levy and G. L. Nelson, "Carbon-13 Nuclear Magnetic Resonance for Organic Chemists." Wiley (Interscience), N e w York, 1972.
392
METHODS FOR T H E STUDY OF ANTIBIOTICS
[19]
TABLE I CONVENIENT SAMPLE SIZE FOR
PMR SPECTROSCOPY
Instrument type
Amount
Amount/400 mol. wt.
56-60 MHz 90-100 MHz 100 MHz, CW 100 MHz, FT 220-300 MHz, FT
12 ~moles 525 ~moles 1.25 ~moles 125 nmoles 50 nmoles
50 mg 10 mg 500 ~g 50 ~g 20 ~g
Unknown impurities should not be present in amounts greater t h a n about 5% lest an unrecognized signal from an impurity cause an error in the structure assignment. Signals from known impurities can be subtracted out of the spectrum. Binary mixtures are about four times more difficult to analyze t h a n are either of the components separately. Enantiomers are not distinguished except under special conditions using chiral solvents 24 or chiral complexing agents. 25 Presence of radicals, paramagnetic metal atoms or iron filings in the sample ruins the resolution and aborts the analysis.
III. Instrumentation and Sample Requirements M a n y kinds of N M R spectrometers are available commercially. 26 Most laboratories use a 90-100 M H z instrument for research. Some use 220-300 M H z superconducting spectrometers because sensitivity and resolution both improve as the frequency and magnetic field strength increase. Computerized spectrometers which add either spectra (CW) or pulsed interference patterns 5 (FT) to overcome noise increase the sensitivity by about 10- to 100-fold. Some instruments 2~ use a larger cell for samples of limited solubility. Sample requirements depend somewhat on the sensitivity of the particular m a k e of instrument and a lot on the L a r m o r frequency. A listing of convenient sample sizes is shown in T a b l e I. Often smaller amounts can be studied with extra care. Micro cells, which restrict the sample to the most closely coupled space, can be used, but they cause some loss of resolution and therefore are not recommended. W. H. Pirkle, R. L. Muntz, and I. C. Paul, J. Amer. Chem. Soc. 93, 2817 (1971). 25R. R. Fraser, J. B. Stothers, and C. T. Tan, J. Mag. Res. 10, 95 (1973). 26Anal. Chem. (Laboratory Guide issue) 45, 214 LG (1973). 27A. Allerhand, R. F. Childers, R. A. Goodman, E. Oldfield, and X. Ysern, Amer. Lab. 4 (11), 19 (Nov. 1972).
[19]
P M R SPECTROSCOPY OF ANTIBIOTICS
393
For P M R spectroscopy the sample should be a neat liquid or a concentrated solution of the recommended amount in enough solvent to make about 0.3 to 0.4 ml of solution. This makes the solvent requirement a rather stringent one. If proton-containing solvents are used they are usually deuterated to remove interference from the abundant solvent protons. Solvents with less than 0.5% of residual protons can be purchased and are acceptable. Each coupled deuterium (spin = 1) splits the residual proton signal into three lines of equal intensity. Two deuteriums give five lines with an intensity ratio of 1 : 2 : 3 : 2 : 1 owing to three overlaps in this triplet of triplets. The spectrum may v a r y slightly with solvent--especially the polar hydrogens which tend to associate. Often several solvents are used taking advantage of these shifts to uncover obscured lines. The most common solvent--the one to which most tabulations of shifts refer--is deuterochloroform. To give a suitable concentration in this solvent the samples must be of moderate molecular weight and must not be highly polar (not more than two or three hydroxyl groups). Polar hydrogens such as SH, NH, and especially OH exchange locations and therefore their collected signals often appear in the spectrum as a single [)road line at an averaged chemical shift. Sometimes it is so broad that it is undetected. The d-chloroform is easily evaporated, and the sample may be rerun in d6-benzene or d~-pyridine (having cone-shaped anisotropy effects) to scramble the lines. 28 Often this will uncover signals that were superimposed. A different scramble can then be obtained with d3-acetonitrile (rod-shaped anisotropy effects). For this reason the shift data in these solvents may not be reliable. For polar samples (most antibiotics d~,-dimethyl sulfoxide (DMSO) or dT-dimethyl formamide ( D M F ) are used. The latter, being less viscous, often gives sharper lines but has more residual proton absorptions to deal with. DMSO has a nice advantage; it binds water so tightly that exchange with OH or N H protons in the sample is prevented, and these can therefore be detected in the spectrum, coupled to any protons that were attached to the alpha carbon. :9 M a n y antibiotics will dissolve in deuterium oxide. These spectra should be calibrated with sodium 4,4-dimethyl-4-silapentane-l-sulfonate (DSS) ~° or sodium 3-trimethylsilyltetradeuteriopropionate (TSP) 31 as internal reference since tetramethyl silane (TMS) is not soluble in water. ~J. Ronayne and D. H. Williams, Annu. Rev. N M R Spectrosc. 2, 83 (1969). -~O. L. Chapman and R. W. King, J. Amer. Chem. Soc. 86, 1256 (1964). 30B. R. Donaldson and J. C. P. Schwarz, J. Chem. Soc. B 395 (1968). 31L. Pohl and M. Eckle, Angew. Chem. Int. Ed. Engl. 8, 381 (1969).
394
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
Exchange now produces a signal from the resulting HOD which could be large enough to obscure other absorptions. The difficulty can be minimized by several cycles of evaporation followed by addition of fresh D=,O. The final residual HOD can then be moved aside (upfield relative to the other signals) by observing the spectrum at a higher temperature revealing any signals that were hidden underneath it. Some samples can be made more soluble in D20 by the inclusion of some DC1 or NaOD to convert them to their salts. Carbamine hydrogens are shifted downfield by this treatment? 2 For some samples which are only moderately polar, d6-acetone or d~-methanol can be used in place of DMSO or DMF. When all else fails the sample can usually be dissolved in trifluoroacetic acid but the shift data are unreliable. Measuring the spectrum at elevated temperature also improves the solubility, but it lowers the sensitivity somewhat.
IV. Interpreting the Spectrum Three major types of information must be extracted from the spectrum before a molecular structure can be proposed for the sample. First, the relative area of each multiplet should be noted on the spectrum. The area tells how many hydrogens are represented by the multiplet. Next, the multiplets should be analyzed. These are the "pitchforks" on the spectrum in Fig. 1. The types and sizes (as measured by the coupling constant, J) of the multiplets tell how the hydrogens are arranged in the molecule. Last, the original absorption frequency (the so-called chemical shift) which results from the factoring should be obtained from the spectrum. The shift identifies the hydrogen type.
A. Area M e a s u r e m e n t
Areas can be measured with a planimeter, by cutting and weighing, or more easily from the step integral curve produced by the spectrometer. Relative areas can be determined by setting the total area equal to the number of hydrogens in the molecular formula, from mass spectrometry (MS) or elemental analysis, or by assigning a recognizable signal the appropriate number of hydrogens for calibration. If this is not satisfactory, a simple proton-containing derivative (acetate, methyl ester, tetramethylammonium salt, etc.) can be prepared, and the calibration can be taken from the area of the added proton signal. If adiacent multiplets come out as fractions, it usually means that partial overlapping is occurring. 3: G. Slomp and J. G. Lindberg, Anal. Chem. 39, 60 (1967).
[191
P M R SPECTROSCOPY OF ANTIBIOTICS
395
B. Spectral Analysis Factoring the multiplets is probably the most difficult part of the interpretation. It requires an understanding of spin-spin coupling and a foreknowledge of what the common multiplets look like in their various perturbations. The investigator is referred elsewhere 33-36 for a more comprehensive discussion of spin-spin coupling. The multiplets change as the strength of the coupling increases. They can be divided into two groups. First-order multiplets result from weakly coupled nuclei that have small couplings compared to the difference in their chemical shifts, J/Av ~ 0.15. These first-order multiples can be factored by inspection because they have approximately symmetrical patterns of N ~ 1 lines (where N is the number of coupled nuclei), and they are insensitive to the signs of the coupling constants. With practice considerable deviation from first-order appearance can be tolerated (J/±v up to 0.4) and the multiplets can still be recognized and factored. Second-order multiplets result from more strongly coupled nuclei, those which have large couplings compared to the difference in their chemical shifts, J/Av ~ 0.15. They give patterns which have more than N ~ 1 lines. These second-order multiplets can usually be recognized by their complexity and asymmetry. As J/Av increases the outside lines of the partners split, separate and move outward, diminishing in intensity. They are usually identified by the number and arrangement of the lines with reference to systematic plots of calculated spectra2 ,9 Like first-order multiplets, some of the line separations match up in the partners but these line separations, S, are rarely a measure of the coupling constant, J. For first approximations the line separations can be used in place of the coupling constants if an accuracy of ± 2 - 3 Hz can be tolerated. To measure the actual J values the multiplets must be analyzed mathematically per instructions in the literature 6 or occasionally they can be determined conveniently by construction methods27 The analysis can be checked by computing the spectrum 3s and if desired the accuracy of the J and ±v parameters can be improved by iteration, as 33E. W. Garbisch, J. Chem. Educ. 45, 311, 402, 480 (1968). 34R. J. Abraham, "Analysis of High Resolution Spectra." Elsevier, Amsterdam, 1971. ~J. N. Murrell, The theory of nuclear spin-spin coupling in high resolution NMR spectroscopy, in "Progress in NMR Spectroscopy" (J. W. Emsley, J. Feeney, and L. H. Sutcliffe, eds.), Vol. 6. Pergamon, Oxford, 1971. 3~p. Diehl, R. K. Harris, and R. G. Jones, in "Progress in NMR Spectroscopy" (J. W. Emsley, J. Feeney, and L. H. Sutcliffe, eds.), Vol. 3, Chap. 1. Pergamon Press, New York, 1967. 3, G. Slomp, Appl. Spectrosc. Rev. 2, 263 (1969). 38p. DieM, H. Kellerhals, and E. Lustig, Computer assistance in the analysis of high resolution NMR spectra, in "NMR" (P. Diehl, E. Fluck, and R. Kosfield, eds.), Vol. 6. Springer-Verlag, Berlin and New York, 1972.
396
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
First determine which multiplets are coupled partners by finding those with matching line separations using dividers. Check the assignment by noting the severity and direction of the intensity slant. In first-order multiplets the number of splittings must also match the partners' relative area. If there is any uncertainty in pairing the multiplets, it can be solved by decoupling 1~ them to see which ones are interacting and what pattern they decouple to. An I N D O R sweep is a convenient way to do this since it also locates buried partners. If the multiplet does not decouple to a singlet, additional partners should be sought. Next, factor the first-order multiplets by drawing in and labeling the pitchforks. Record the coupling constants, J, measured from the line separations then analyze the secondorder multiplets where possible. In the analysis be on the alert for deceptively simple spectra, virtual coupling, long-range coupling, and asymmetric nonequivalence. If there is difficulty in analyzing second-order multiplets they can usually be simplified by observing the spectrum at a higher Larmor frequency, which increases ±v relative to J. Switching solvents or adding a shift reagent may accomplish the same result. C. Chemical Shift Measurement
The frequency scale along the bottom of the spectrum is usually calibrated in Hz relative to internal TMS, a9 or a suitable substitute. ~°'31 For first-order multiplets the resonance frequency of the center of the multiplet is read from this scale. For second-order multiplets the resonance frequency is obtained from appropriate line frequencies by a mathematical, construction or computer analysis in accordance with instructions in the literature. If such analysis is not done, the resonance frequency may be approximated from the center of gravity of the multiplet. Since spectrometers operate at various Larmor frequencies~ the chemical shift is usually expressed in dimensionless units 8, which are common to all: ~ppm. = (robs. -- Vref.) X 106/Larmor frequency
where a positive value indicates the line was on the downfield side of TMS. V. Proposing a Structure
The results of the spectrum analysis are conveniently summarized in a diagram representing each hydrogen by a letter of the alphabet. The diagram for geldanamycin is shown in Fig. 2. 3~p. Laszlo, A. Speert, R. Ottinger, and J. Reissi, J. Chem. Phys. 48, 1732 (1968).
[19]
PMR
2.02
Ps
7.36
I ~1 A
6.54
B
SPECTROSCOPY OF ANTIBIOTICS 0.94
W3
2.47
6.5 3.0 5.12
11.5
397
N~ , ~ 5 0 ~ 0 ~
2.25
L
D
1.90 ~ Q - - ~ //~ 6.0 I I 9.5 ~3.6 I /z 1.11 V 3 I / I 4.38 G 3.51 / / " t0.0 I ~1 1.50 U ~ I 5.07 E 17 ~ / T ~ 1o9 FIG. 2. Analysis of the nuclear magnetic resonance spectrum of geldanamycin. Signals are labeled with a chemical shift, $, beside their symbol. Coupling constants, J, are shown beside the attaching lines. Couplings shown by the dashed lines were inferred. 5.79
C
1.76
1.0 S3
4.87 F 7.0 I
A. I n t e r p r e t i n g t h e Shifts T h e chemical shifts i d e n t i f y the h y d r o g e n type. A s s i g n m e n t is m a d e from e m p i r i c a l correlations u s i n g charts, 4°-44 tables, 45-49 a d d i t i v i t y rules, 5°-53 a n d model compounds. M a k e a list of p a r t i a l s t r u c t u r e s to fit each h y d r o g e n type, T a b l e II. "° K. Nukada, 0. Yamamoto, T. Suzuki, M. Takeuchi, and M. Ohnishi, Anal. Chem. 35, 1892 (1963). 41N. F. Chamberlain, "The Practice of NMR Spectroscopy with Spectra Structure Correlations for Hydrogen-l." Plenum, New York, 1974. 4~R. M. Silverstein and G. C. Bassler, "Spectrometric Identification of Organic Compounds," 2nd ed., p. 110. Wiley, New York, 1967. 4~O. Yamamoto, T. Suzuki, M. Yanagisawa, and K. Hayamizu, Anal. Chem. 40, 568 (1968). 4, N. F. Chamberlain, "Nuclear Magnetic Resonance Chemical Shifts of Oxygenated Unsaturated Aliphatics," Anal. Chem. 40, 1317 (1968). 4~N. S. Bhacca, D. P. Hollis, L. F. Johnson, E. A. Pier, and J. N. Shoolery, "NMR Spectra Catalog" Vols. 1 and 2. Varian Associates, Palo Alto, California, 1963. 46F. A. Bovey, "NMR Data Tables for Organic Compounds." Wiley, New York, 1967. *' W. Briigel, "Nuclear Magnetic Resonance Spectra and Chemical Structure," Vol. 1. Academic Press, New York, 1967. ,8 H. A. Szymanski and R. E. Yelin, "NMR Band Handbook." Plenum, New York, 1967. *9"Sadtler NMR Reference Spectra." Sadtler Research Laboratories, Philadelphia. Pennsylvania. 5oj. S. Martin and B. P. Dailey, J. Chem. Phys. 39, 1723 (1963). 51R. F. Zurcher, Helv. Chim. Acta 46, 2054 (1963). ~2A. I. Cohen and S. Rock, Steroids 3, 243 (1964). 5~U. E. Matter, C. Pascual, E. Pretsch, A. Pross, W. Simon, and S. Sternhell, Tetrahedron 25, 691, 2023 (1969).
398
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
TABLE II ABSORPTIONS IN THE SPECTRUM OF GELDANAMYCIN ACETATE
Shift, ~ Label 8.67 7.36 7.24 7.17 6.54 5.79 5.12 5.07 5.00 4.87 4.38 4.04 ~3.6 3.36 3.33 -~3.0 2.47 2.25 2.02 ~-~1.9 ~1.9 1.82 1.76 1.50 1.11 0.94 0.59
X A
Multiplet
Area Suggested structure
Singlet Doublet, S = 11.5 Singlet Singlet Doublet doublet, S = 11.5, 11.5 Doublet doublet, S = 9.5, 11.5 Broad doublet, S = 9.0, -~1 Broad singlet, S = ~ 1 Singlet Doublet doublet, S = 7.0, 4.0 Broad doublet, S = 9.5, ~ 1 Singlet Complex multiplet Singlet Singlet Complex multiplet Doublet doublet, J = -12.5, 7.0 Doublet doublet, J = -12.5, 5.8 Broad singlet, S = <1
1 Amide 1 Conj. vinyl Chloroform M 1 Aromatic B 1 Conj. vinyl C 1 Conj. vinyl D 1 Vinyl E 1 Allylic carbinol Y~ 2 Carbamate F 1 Carbinol G 1 Allylic ether H3 3 Aromatic methoxy I 1 Ether J3 3 Methoxy K3 3 Methoxy L 1 Allylic N 1 Benzylic O 1 Benzylic P3 3 Vinyl methyl Q~ 1 Aliphatic Broad hump T] 1 Aliphatic R3 Singlet 3 Acetyl methyl Sa Broad singlet, S = ~ 1 3 Vinyl methyl U Doublet doublet doublet, J = -17, 10, 3.5 1 Aliphatic V3 Doublet, J = 6.0 3 Aliphatic methyl W3 Doublet, J = 6.5 3 Aliphatic methyl Singlet 1~C satellite of TMS
B. I n t e r p r e t i n g t h e C o u p l i n g C o n s t a n t s T h e coupling c o n s t a n t s i n d i c a t e the a r r a n g e m e n t of the p r o t o n s in the molecule. T h e m a g n i t u d e is correlated with the n u m b e r of i n t e r v e n i n g bonds a n d the stereochemistry. A s s i g n m e n t is m a d e from charts '2 a n d t a b l e s 7-9,~-~6 a n d model compounds. G e m i n a l c o u p l i n g s across a n sp 3 c a r b o n
I (H--C--H)
I
Aksel A. Bothner-By, Advan. Magn. Resonance 1, 161 (1965). ~5R. C. Cookson, T. A. Crabb, and J. J. Hudec, Tetrahedron, Suppl. 7, 355 (1966). ~ R. C. Cookson and T. A. Crabb, Tetrahedron 24, 2385 (1968).
[19]
P M R SPECTROSCOPY OF ANTIBIOTICS
399
are all negative except in epoxides and ethyleneimines and are usually around - 1 0 to - 2 0 and ~-1 to -t-6 Hz, respectively, with a few special exceptions. 7 T h e y increase algebraically (less negative) with ~r character of the adjacent atom, amount of ~r-orbital overlap, increasing electronegativity of the other substituents on the carbon and decreasing electronegativity of substituents on an adjacent carbon. Geminal couplings across an sp 2 carbon
ql
(H--C--H) are usually around zero, ± 2 Hz. Vicinal coupling across three single bonds ( H - - C - - C - - H ) varies from 0 to -~12 (few are found that are less than 2) and depends mostly on the dihedral angle. Minor dependence on the electronegativity and on the orientation of the substituents is usually ignored unless precise dihedral angles are sought. Small rings with abnormal bond angles and bond lengths are special cases. This is one of the most powerful stereochemical determinations used in organic chemistry. 57-~9 For cyclohexanes that can ring convert and for molecules free to rotate, the couplings are a weighted average of those in the various rotamers, and thus they may change with solvent if different conformers are favored. Remember to use the average of the couplings from the individual dihedral angles, not the coupling from an averaged dihedral angle. An appraisal of the bulk of the substituents may allow selection of a preferred conformation, in which case ring substituent configuration or t h r e o - e r y t h r o type configuration problems can be solved. When one or more of the carbons has an external unsaturation
H
H--C--C--H (e.g., aldehydes, olefins, etc.) the vicinal coupling is considerably smaller, 0-6 Hz. Vinyl couplings ( H - - C ~ C - - H ) depend on the geometry and the electronegativity of the substituents. J t .... is usually + 1 2 to + 1 8 and J¢i8 is usually + 7 to + 1 1 . Those t h a t fall near the middle of the two ~Tj. E. Pike, M. A. Rebenstorf, G. Slomp, and F. A. MacKellar, J. Org. Chem. 28, 2499 (1963). 5sH. Booth, Applications of 1H nuclear magnetic resonance spectroscopy to the conformational analysis of cyclic compounds, in "Progress in NMR Spectroscopy" (J. W. Emsley, J. Feeney, and L. H. Sutcliffe, eds.), Vol. 5. Pergamon, Oxford, 1969. 59F. A. L. Anet and R. Anet, Configuration and conformation by NMR, in "Determination of Organic Structures by Physical Methods" (17. C. Nachod and J. J. Zuckerman, eds.), Vol. 3. Academic Press, New York, 1971.
400
METHODS FOR THE STUDY OF ANTIBIOTICS
[19]
ranges can be unequivocally assigned using the additivity rules based on electronegativity. 8o.81 Aromatic couplings do not v a r y much unless there is a nitro group on the ring. Long-range couplings 62,6~ through 4 or 5 bonds are usually small (--3 to ~ 1 ) but can be very important to bridge a break in an otherwise continuously coupled hydrogen chain. T h e y v a r y with geometry, planar zigzag forms having the largest couplings. The magnitude is also increased when sp 2 carbons are involved. Make a list of the possible types of couplings which these J values represent and the conformations or configurations which fit. C. Trial S t r u c t u r e s
Write a skeleton of carbons and hydrogens which fits the coupling data. Add the necessary functional groups dictated by the list of hydrogen types. Add the remaining undetected atoms of the molecular formula in all the possible ways to fit MS, UV, and I R data. It is always helpful to propose some structure on paper--ridiculous as it m a y seem--and then consider how it could be modified to make it fit the data better. If left with more than one possibility further studies are undertaken. VI. Further Studies
Sometimes the interpretation is stalled because some multiplets cannot be analyzed or an unexpected anisotropy or van der Waals effect64 has shifted a signal out of its expected position and it has been assigned incorrectly. If the problem is modified in a known way by altering the spectrum or the molecule and a solution now becomes possible, extrapolation could furnish the solution to the original problem. A. C h a n g i n g t h e S p e c t r u m
The spectrum can be simplified by substituting deuterium for hydrogen, removing it from the spectrum. Exchangeable hydrogens such as OH or N H can be identified by adding a drop of D20 to a sample in a nonD20 solvent. After thorough mixing the exchangeable hydrogens are T. Schaefer, Can. J. Chem. 40, 1 (1962). G. Allen, D. J. Blears, and K. H. Webb, J. Chem. Soc. (London) 1965, 810 (1965). M. Barfield, J. Chem. Phys. 41, 3825 (1964). S. SternheU, Rev. Pure Appl. Chem. 14, 15 (1964). M. J. Stephen, Mol. Phys. 1, 223 (1958).
[19]
P M R SPECTROSCOPY OF ANTIBIOTICS
401
nearly completely replaced with deuterium, removing their signals from the spectrum. With water-miscible solvents a new HOD signal appears in its usual place in the spectrum. Amide hydrogens are slow to exchange and may require several hours of standing (with occasional mixing) for completion. When geldanamycin acetate (II) was treated this way the O CHsO~'~
H 0
CH3
CHs
(I) R = H, Geldanamycin (II) R = COCHs, Geldanamycin acetate
amide hydrogen signals (Y_~ and X) had nearly disappeared from the spectrum in 20 min and completely disappeared in 1 hr greatly facilitating the interpretation of the DE multiplets. The HOD line appeared as a small sharp singlet midway between multiplets F and G. Protons of alcohols and amines can also be identified by the shift that is caused by adding a dropper full of hydrochloric acid vapors collected from the head space in the acid reagent bottle. 3-~ Raising the sample temperature shifts the signals of exchangeable OH or NH protons upfield faster than other signals and switching to an anisotropic solvent (discussed under solvents) scrambles the spectrum providing another chance at analysis of the multiplets. A recent technique for simplifying the spectrum uses a paramagnetic organometallic complex (III) as a shift reagent. * When added to the Rl R2
R3
O ~ R
I
2
o
Rs----~
~M /
R,
O~
~--RI
R2
R3
>=o Z I"-o
R 1 = --C(CHs) s or C(CDs) 3 R~ = --C(CHs) s o r C(CDs) s or - - C F 2 - - C F 2 - C F 3 Rs = - - H o r - - D M = Eu, P r , Yb, o r other r a r e e a r t h
(m)
402
METHODS FOR THE STUDY OF ANTIBIOTICS
[19[
sample it complexes with electron-rich functions on the molecule (CO--NH > - - N H > --OH > C-~-O > - - 0 - - > --CN), and by a pseudocontact mechanism it changes the shielding (AS) of nearby protons in an amount depending on their distance (R) and angle of deviation from the coordination axis (8): A S = K [ ( 3 cos 2 0 - - 1 ) / R 3 ] , where K is a scalar constant for the particular experiment including the binding ability of the reagent, its concentration, etc. The shifts in the spectrum can be paramagnetic (downfield) or diamagnetic (upfield) depending on 0 and the metal of the reagent. The desired effect is to increase the separation of the signals in order to make them less overlapped and more firstorder. The method works best in nonpolar solvents and with substrates that have a dominant binding site and therefore does not always work
R = -- C ( C H s ) s o r -- C F 2 - C F z - - C F 3
(iv) on antibiotics. If a chiral shift reagent (IV) is used enantiomers shift differently and can therefore be distinguished because the complex is diasterotopic. ~5 Computer programs are available 6~ which test the molecular geometry of proposed structures by computing the agreement factor between the observed and calculated shifts. This procedure is very useful when choosing from more than one proposed structure. A subtle change in the spectrum brought on by the nuclear Overhauser effects can sometimes be used to confirm the close proximity of protons across space. The test depends on internuclear relaxation, not on spin coupling. A weak radiation is applied to a given proton at its resonance frequency which partially saturates that proton's resonance signal changing its Boltzmann distribution. If a second proton is nearby across space and therefore depends on the perturbed proton for its relaxation, it too will experience a disruption of its Boltzmann distribution with the result that its resonance intensity will grow as much as 50%. The effect falls off rapidly with distance (1/R 6) and is a specific test for hydrogens in an eclipsed 5- or 6-position. R. E. Davis, M. R. Willcott, III, R. E. Lenkinski, W. yon E. Doering, and L. Birladeanu, J. Amer. Chem. Soc. 95, 6846 (1973).
[19]
P M R SPECTROSCOPY OF ANTIBIOTICS
403
TABLE III SHIFTS RESULTING FROM ACETYLATION OF GELDAN:kMYCIN
Proton
Shift, A~
Proton
Shift, A~
A X M B C D E Y2 F G Ha I Ja
+0.38 +0.09 -0.14 -0.04 -0.06 --0.67 --0.13 +0.19 +1.31 +0.04 --0.07 ? 0.01
K3 L N O
+0.05 -0.16 +0.02 -0.11
P3
--0.02
Q
T Ra $3 U Va Wa
?
--0.14 Absent -0.01 -0.26 -0.19 --0.01
The use of higher Larmor frequency has already been noted, and the N M R of other nuclei, such as 13C or 15N, may provide the needed information to solve the P M R spectrum. In the case of geldanamycin acetate (II) the first spectra were observed at 60 MHz. Comparison of the 60 and 100 MHz spectra helped identify the multiplets.
B. Changing the Sample For antibiotics a very useful reaction is acetylation or carbamation of the sample. The alcohols and amines can be characterized from the resulting shifts noting that secondary carbino166 or carbamine 32 hydrogens shift downfield much more than primary ones. The acetyl methyls can usually be counted, and the polarity may be diminished enough to allow additional observations in a less polar solvent. The carbamation reaction can be carried out in the N M R cell by the addition of trichloroacetyl isocyanate reagentY In the geldanamycin example, the acetate (II) was prepared with acetic anhydride and pyridine and the shifts recorded in Table I I I helped identify the molecular structure. Other reactions such as ketone derivatization, hydrogenation, or borohydride reduction have been used to help interpret spectra. In the geldanamycin case the methanolysis product (V) was prepared from (I) with C. R. Narayanan and M. R. Sarma, Tetrahedron Lett. 1553 (1968). 8, V. W. Goodlett, Anal. Chem. 37, 431 (1965).
404
METHODS FOR THE STUDY OF ANTIBIOTICS
[20]
CHsO~H o
~/ HaC/~ C
"VO
~'~NH~ CHaO"
O CH3 CHa C ~
H
3
0
OH
~ OCONH2
(V) p o t a s s i u m c a r b o n a t e in refluxing m e t h a n o l - c h l o r o f o r m ( 1 : 1 ) . T h e P M R s p e c t r u m of (V) h a d the I, Q, a n d T m u l t i p l e t s u n c o v e r e d , a n d a large upfield shift of the a r o m a t i c p r o t o n signal (M) showed t h a t the a m i d e m u s t h a v e been a t t a c h e d ortho to it.
[20] The Use of 13C Labeling in the Study of Antibiotic Biosynthesis By NORBERT NEUSS I. II. III. IV.
Introduction . . . . . . . . . . . . . . . Instrumental Requirements . . . . . . . . . . . Satellite Method . . . . . . . . . . . . . . Experimental Conditions . . . . . . . . . . . . A. Preliminary 14C Experiment . . . . . . . . . . B. Natural Abundance CMR Spectrum . . . . . . . C. Selection of an Appropriate 13C-Enriched Precursor . . D. Consideration of Nuclear Overhauser Effect (NOE) . . E. Conditions of Labeling . . . . . . . . . . . V. CMR in Biosynthetic Studies of Antibiotics . . . . . . VI. Biosynthesis of fl-Lactam Antibiotics . . . . . . . . A. Synthesis of Model Compounds and 1'C Precursors . . B. Fermentation, Labeling, and Isolation . . . . . . . C. Recording of the Spectra . . . . . . . . . . . D. Assignments of Chemical Shifts in the CMR Spectra . . E. Determination of Incorporation Levels of 13C Precursors F. Discussion and Conclusions . . . . . . . . . .
. . . . . .
. . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
404 405 406 406 407 407 407 407 407 408 410 411 411 414 415 417 418
I. I n t r o d u c t i o n T h e i n t r o d u c t i o n of a n t i b i o t i c s into the t h e r a p y of b a c t e r i a l i n f e c t i o n s p r o m p t e d chemical i n v e s t i g a t i o n s in m a n y l a b o r a t o r i e s t h r o u g h o u t the
404
METHODS FOR THE STUDY OF ANTIBIOTICS
[20]
CHsO~H o
~/ HaC/~ C
"VO
~'~NH~ CHaO"
O CH3 CHa C ~
H
3
0
OH
~ OCONH2
(V) p o t a s s i u m c a r b o n a t e in refluxing m e t h a n o l - c h l o r o f o r m ( 1 : 1 ) . T h e P M R s p e c t r u m of (V) h a d the I, Q, a n d T m u l t i p l e t s u n c o v e r e d , a n d a large upfield shift of the a r o m a t i c p r o t o n signal (M) showed t h a t the a m i d e m u s t h a v e been a t t a c h e d ortho to it.
[20] The Use of 13C Labeling in the Study of Antibiotic Biosynthesis By NORBERT NEUSS I. II. III. IV.
Introduction . . . . . . . . . . . . . . . Instrumental Requirements . . . . . . . . . . . Satellite Method . . . . . . . . . . . . . . Experimental Conditions . . . . . . . . . . . . A. Preliminary 14C Experiment . . . . . . . . . . B. Natural Abundance CMR Spectrum . . . . . . . C. Selection of an Appropriate 13C-Enriched Precursor . . D. Consideration of Nuclear Overhauser Effect (NOE) . . E. Conditions of Labeling . . . . . . . . . . . V. CMR in Biosynthetic Studies of Antibiotics . . . . . . VI. Biosynthesis of fl-Lactam Antibiotics . . . . . . . . A. Synthesis of Model Compounds and 1'C Precursors . . B. Fermentation, Labeling, and Isolation . . . . . . . C. Recording of the Spectra . . . . . . . . . . . D. Assignments of Chemical Shifts in the CMR Spectra . . E. Determination of Incorporation Levels of 13C Precursors F. Discussion and Conclusions . . . . . . . . . .
. . . . . .
. . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
404 405 406 406 407 407 407 407 407 408 410 411 411 414 415 417 418
I. I n t r o d u c t i o n T h e i n t r o d u c t i o n of a n t i b i o t i c s into the t h e r a p y of b a c t e r i a l i n f e c t i o n s p r o m p t e d chemical i n v e s t i g a t i o n s in m a n y l a b o r a t o r i e s t h r o u g h o u t the
[20]
13C LABELING IN ANTIBIOTIC SYNTHESIS
405
world concerned with elucidation of biosynthetic pathways of this important class of natural products. These studies consisted of addition of simple 14C-labeled substrates such as acetate, amino acids, shikimic acid, during the fermentation. After the completion of the feeding experiment, the metabolite was isolated and the labeling pattern was established by suitable chemical operations. The latter procedure can often be time consuming and, in the majority of cases, involves a bond-specific, multistep degradation process on limited amounts of material followed by laborious purification in order to achieve a constant level of radioactivity. In addition, some degradative procedures cannot be applied to complex molecules. Many of these problems can be avoided by the use of 13C-labeled precursors followed by examination of 13C NMR spectrum of the isolated 13C-enriched compound, and a comparison of that spectrum with a natural abundance CMR spectrum of an unprecursed metabolite. The ~'~C NMR or CMR spectroscopy is a powerful technique which has become extremely useful in recent years in structure elucidation and other facets of organic chemical research. Three excellent reviews on this subject have been published recently, 1-3 and therefore we shall only briefly discuss some of the general aspects of this technique.
II. Instrumental Requirements Unlike carbon-14, carbon-13 is a stable, magnetic isotope of carbon; therefore, no precautions are required in handling 13C-containing materials. Its nucleus is similar to that of hydrogen in that it has a spin of 1/,/2 and can be subjected to resonance in a strong magnetic field. Hence, a basic knowledge of proton spectroscopy can be transferred to CMR. The major difficulty with recording CMR spectra is the low natural abundance of 13C (only 1.1%) and a smaller magnetic moment than that of the protons. These two properties result in a considerably reduced sensitivity of detection of resonance (5700-fold). In other words, if a 10-rag sample were the lower limit of sample size for proton spectroscopy using a very widely available 60 MHz spectrometer, a 57-g sample would be required for carbon spectroscopy. Fortunately, a number of developments have helped solve this problem. These include: 1. The new type high field spectrometer of high stability which permits the use of a larger size sample (2 ml) in a 15 mm tube in conjunction 1F. A. Anet and G. C. Levy, Science 180, 141 (1973). 2j. B. Grutzner, Lloydia 35, 375 (1972). 3j. B. Stothers, Appl. Spectrosc. 26, 1 (1972).
406
METHODS FOR THE STUDY OF ANTIBIOTICS
[20]
with a computer capable of adding weak N M R signals with a net result of increase in intensity relative to noise signal. 2. The introduction of proton noise decoupling which results in the appearance of carbon-13 frequencies as sharp singlets rather than multiplets due to ~3C-1H spin-spin coupling. 3. The use of the pulsed Fourier transform which permits approximately a 10-fold increase in sensitivity by averaging, for instance, 100 spectra in the time necessary for the recording of a conventional spectrum. III. Satellite Method
One more aspect of 13C-labeling must be considered. In some instances, particularly when the 13C-label is contained in a carbon-bearing hydrogen atom, the spin-spin coupling between IsC nuclei and directly bonded protons produces "satellite" signals on either side of the main proton resonances in the proton magnetic resonance spectrum. These coupling constants range from 125 to 250 Hz depending on the carbon hybridization and substituents, and can be detected with a 100 MHz spectrometer available in many laboratories. In case of substantial enrichment in 13C and availability of 30-50-mg samples of a small molecular weight substance (e.g., l~C-labeled valine) a spectrum is of a quality permitting a detailed interpretation of coupling constants and degree of incorporation. To illustrate this type of spectrum we refer the reader to characteristic bands in the N M R spectrum of (2RS, 3R)-[4-13C]valine (Fig. 4). In cases of a small degree of incorporation, a C-1024 time averaging computer for multiscan averaging is required in conjunction with a 100 MHz spectrometer. Thus, during the study of biosynthesis of griseofulvin, 4 fusaric acid, 5 and piericidin A 6 the satellite signals from enriched sites in these two antibiotics were observed and used to establish their biosynthetic origin with enrichment as low as 2% and sample size ranging from 25 to 100 mg. IV. Experimental Conditions
Certain conditions have to be fulfilled for a valid experiment in the elucidation of biosynthetic pathway using a 13C precursor and CMR spectroscopy of the enriched metabolite. 4 M. Tanabe and G. Detre, J. Amer. Chem. Soc. 88, 4515 (1966). D. Desaty, A. G. McInnes, and L. C. Vining, Can. J. Biochem. 46, 1293 (1968). eM. Tanabe and H. Seto, J. Org. Chem. 3,5, 2087 (1970).
[20]
13C
LABELING
IN
ANTIBIOTIC
SYNTHESIS
407
A. Preliminary 14C Experiment It is absolutely necessary to check in a preliminary carbon-14 study the degree of incorporation of precursors to ascertain a sufficient level for 13C detection. In most cases 1% incorporation will be satisfactory. This prerequisite is necessitated by the ability to distinguish differences in peak intensity between the labeled compound and its natural ~3C abundance reference. At present this limit is a peak ratio of 1.2:1. In a case of utilization of 90% ~3C-enriched precursor, 0.2% incorporation may be detected.
B. Natural Abundance CMR Spectrum For a complete analysis of a natural abundance CMR spectrum of a metabolite to be studied, it is sometimes necessary to synthesize appropriate model compounds or derivatives in order to eliminate ambiguous assignments.
C. Selection of an Appropriate 1*C-Enriched Precursor Several simple 13C-enriched compounds are commercially available, and others can be synthesized according to the need, using standard procedures described for the synthesis of 14C-enriched compounds.
D. Consideration of Nuclear Overhauser Effect (NOE) The process of proton decoupling results in the increase of certain peaks (NOE). In order to avoid a misinterpretation of such an increase as incorporation of 13C from the precursor, it is advisable to run the natural abundance CMR spectrum of the metabolite and that of the enriched metabolite to be tested, under similar conditions (concentration, instrument setting, solvents, etc.); great care has to be taken to establish as accurately as possible the extent of NOE, generated by proton decoupling or Fourier transform technique. There are also some ways to minimize NOE.
E. Conditions of Labeling Finally, it should be obvious to anyone engaged in the technology of fermentation that conditions used in producing the 13C-labeled metabolite should closely mimic those used in the preparation of metabolite in
408
METHODS FOR THE STUDY OF ANTIBIOTICS
[20]
the regular fermentation run as well as 1~C feeding experiment preceding the 13C experiment. V. C M R in Biosynthetic Studies of Antibiotics The great value of CMR in the study of biosynthetic pathways was recognized early in the development of the technique ~ however, the problem of sensitivity delayed the use of the method until 1970, when biosynthetie formation of radicinin, 8 a microbial metabolite from Stenphylum radicinum was elucidated using the solvent peak as an internal lock. In this important modification, it is possible to switch from noise decoupling to single-frequency or continuous-wave (CW) decoupling when the decoupler is tuned to the exact frequency for irradiation of the solvent protons. Under such conditions most 13C-signals arising from CH3, CH~, or CH groups with proton shifts slightly removed from the solvent proton shifts, give characteristic close-spaced multiplets, thus permitting appropriate assignments. Until the introduction of this modification, biosynthetic investigations of antibiotics relied solely upon the presence of 13C-satellite in proton spectra to study the locus of 13C-label (see above). The summary of biosynthetic studies using the continuous wave (CW) as well as Fourier transform technique is shown in Table I. In all these studies, feeding of the enriched 13C-labeled precursor during the fermentation was followed by the isolation of the metabolite and comparison of its CMR spectrum with that of the natural abundance CMR of the metabolite. Since these experiments were published either as communications to the editor or abstracts of papers given at meetings, the details of experimental conditions and particularly spectra themselves, are not readily available. These considerations, as well as the uniqueness of the principle tested, prompted us to use as an example of a thorough biosynthetic study using CMR spectroscopy, the recent elueidation of some steps in the biosynthetic formation of fl-lactam antibiotics, cephalosporin C and penicillin V2 -12 7j. B. Stothers, Quart. Rev. 19, 144 (1965). 8 M. Tanabe, tI. Seto, and L. F. Johnson, J. Amer. Chem. Soc. 92, 2157 (1970). N. Neuss, C. tt. Nash, P. A. Lemke, and J. B. Grutzner, J. Amer. Chem. Soc. 93, 2337 (1971). ~oN. Neuss, C. tt. Nash, P. A. Lemke, and J. B. Grutzner, Proc. Roy. Soc. Ser. B. 179, 335 (1971). ~ N. Neuss, C. H. Nash, J. E. Baldwin, P. A. Lamke, and J. B. Grutzner, J. Amer. Chem. Soc. 95, 3797 (1973). ~2j. E. Baldwin, J. LSliger, W. Rastetter, N. Neuss, L. L. Huckstep, and N. DeLaHiguera, J. Amer. Chem. Soc. 95, 3796 (,1973).
[20]
13C LABELING IN ANTIBIOTIC SYNTHESIS
409
TABLE I SUMMARY OF BIOSYNTHETIC LABELING STUDIES OF ANTIBIOTICS BY C M R (UNTIL NOVEMnER 1973)
Compound
Precursor
Asperlin Cephalosporin C
[2-13C]Acetate ~ [1-13C]- and [2-1~C]Acetate ~ DL-[1-1~C]- and [2-1~C]d (2RS,3R)-[4-~SC]valine ~ (2S,3S)-[4-13C] Valine I [1-~3C]Acetate b [1-1aC]Propionate, [4-13C]methionineg [1-1~C]Glucosamine h [1-13C]Acetate~ (2S,3S)-[4- ~3C]Valine / (2RS,3R)-[4-~3C]Valine ~ oI~Tryptophan-alanine-[3- ~3C]j [1-1~C], [2-13C], and [3-1aC]Propionate [I-13C]- and [2-13C]Acetatek [1-1~C] and [2-1aC]Acetate~ [1-~3C]Propionate "~ [1-13C]Propionate and [1-~3C]butyraie ~
Cephalosporin C Chlorothricin Geldanamycin Neomycin Nybomycin Penicillin N Penicillin V Pyrrolnitrin Rifamycin Showdomycin Streptovaricin X-537A
M. Tanabe, T. Hamasaki, l). Thomas, and L. F. Johnson, J. Amer. Chem. Soc. 93, 273 (1971). b D. J. Hook, C. J. Chang, H. G. Floss, E. W. IIagaman, and E. Wenkert, Ab,~lr. Pap. Amer. Chem. Soc., 166 M E D I , p. 68 (1973). c N. Neuss, C. H. Nash, P. A. Lemke, and J. B. Grutzner J. Amer. Chem. Soc. 93, 2337 (1971). N. Neuss, C. H. Nash, P. A. Lemke, and J. B. Grutzner, Proc. Roy. Soc. Ser. B 179, 335 (1971). N. Neuss, C. H. Nash, J. E. Baldwin, P. A. Lemke, and J. B. Grutzner, J . . - i ~ e r . Chem. Soc. 95, 3797 (1973). / H. Khtender, C. H. Bradley, C. J. Sih, P. Fawcett, and E. P. Abraham, J. Amer. Chem. Soc. 95, 6149 (1973). g R. l). Johnson, A. Haber, and K. L. Rinehart, Jr., Abstr. Pap. Amer. Chem. Sot., 166 Orgn, p. 124 (1973). h S. T. Truitt, M. Taniguchi, J. M. Malik, R. M. Stroshane, and K. L. Rinehart, Jr., Abstr. Pap. Amer. Chem. Soc. 166 M E D I , p. 69 (1973). W. M. J. Knoll, R. J. Huxtable, and K. L. Rinehart, Jr., J. Amer. Chem. Soc. 95, 2703 (1973). i L. L. Martin, C. J. Chang, and H. G. Floss, J. Amer. Chem. Soc. 94, 8942 (1972). k R. J. White, E. Martinelli, G. G. Gallo, C. Lancini, and P. Beynon, Na/,r~ (London) 243, 273 (1973). z R. J. Suhadolnik and E. F. Elstner, in It. G. Floss, LIoydia 35, 399 (1972). " B. Milavetz, K. Kakinuma, and K. L. Rinehart, Jr., J. Amer. Chem. Soc. 95, 5793 (1973). '~ J. W. Westley, D. L. Pruess, and R. G. Pitcher, Chem. Commun. 161 (1972).
410
METHODS FOR T H E STUDY OF ANTIBIOTICS
[20]
H2N\ /SH e CH-(CH2L-CONHCN-CHzCHICH3)2 OOC/ 'J CO-NH-CH-COOH ~ -(~-AM INOADIPOYL)CYSTEINYLVALINE
o / CH-ICH2I~CONH o
ooc
0//
~
c.u o.
c.,
Q,.;,~N,C/..~ CH3 J COOH
N'~CH 3 COOH PENAM
O~
" ~ xCH2OAC COOH CEPHEM
FIG. 1. Proposed formation of penam and cephem antibiotics. VI. Biosynthesis of •-Lactam Antibiotics In spite of extensive effort over the past two decades, the detailed biosynthetic pathway to the fl-lactam antibiotics, penicillin, and cephalosporin, is still unknown. TM The evident relationship of these antibiotics, known as the penam and 3-cephem derivatives, to the amino acids L-valine and L-cysteine has been established by appropriate 14C incorporation experiments. 14-17 It has been postulated that both these systems could be derived from a common intermediate, an a,fl-dehydrovaline derivative of the tripeptide, 8-(a-aminoadipyl)cysteinylvaline, which after ring closure gives rise to the cephem and penam ring systems (Fig. 1). We decided to use valine with a chiral isotopic '3C-label at the 4-position as a probe for the stereochemical fate of this isopropyl group during conversion to the fl-lactam products. The synthesis of such a precursor required a considerable amount of effort. Therefore, a number of experiments had to be undertaken to reconfirm ~4C experiments and establish appropriate labeling and fermentation conditions suitable for the final experiment with chiral valine. B y way of example, details of these experiments are given below since they are typical for the study of biosynthesis using l~C-labeled precursors and C M R spectroscopy. ,sp. A. Lemke and D. R. Brannon, in "Cephalosporins and Penicillins" (E. H. Flynn, ed.), p. 370. Academic Press, New York, 1972. 14H. R. V. Arnstein and M. E. Clubb, Biochem. J. 65, 618 (1957). i, It. R. V. Arnstein and P. T. Grant, Biochem. J. 57, 353, 360 (1954). 1~S. C. Warren, G. G. F. Newton, and E. P. Abraham, Biochem. J. 163, 902 (1967). 1, E. P. Abraham, G. G. F. Newton, and S. C. Warren, I. A. M. Symp. Appl. Microbiol., Tokyo, 6, 79 (1964).
[20]
laC LABELING IN ANTIBIOTIC SYNTHESIS
411
A. Synthesis of Model Compounds and 13C Precursors The first prerequisite for these studies was fulfilled by the data available from the earlier investigations 14-17 with radioactive precursors, indicating sufficient degree of incorporation for CMR spectroscopy. The localization of the 13C-label was possible only after complete assignment of all 13C-frequencies of 16-carbon atoms in cephalosporin C (I). In order to accomplish this task it was necessary to examine CMR spectra of appropriate model substances. These included: DL-a-aminoadipic acid ethyl amide (II), eephalexin (liD, 3 methyl-7-(2-phenoxyacetamido)-3eephem (IV), and 7-aminocephalosporanic acid iV) (Fig. 2). From precursors to be used in this study [1J3C]- and [2-13C]sodium acetate were commercially available, and contained 62-68% of 13C. DL-[1-13C]-, and ~L-[2-13C]valine were prepared using KI~CN and [2-~'~C]glycine (both containing 62-63% of ~C, respectively), according to procedures described for the synthesis of analogous l~C-labeled compounds. (2RS, 3R)-[4-~'~C]Valine was prepared by reductive opening of optically pure (+)-trans-([1-~C]methyl-2-cyelopropane carboxylic acid ethyl ester giving rise to (3S)-[4-1~C]3-methylbutyric acid ethyl ester. The free acid was brolninated and subjected to aminolysis. The final purification by ion exchange chromatography gave pure (thin-layer chromatography, mass spectrometry, and amino acid analysis) (2RS, 3R)-[4"C]valine (Fig. 3). The estimated isotopic purity was 92% as estimated from the ratios of methyl frequencies containing no ~3C, but two ~CH:~. Chemical shifts and coupling constants in the NMR spectrum (Fig. 4) were as follows: NMR (D._,O)8 1.42 m (J['3C-'H] = 126 and JI'H'H] = 6.9 Hz), 8 2.72 m (J[~C-'H] ~ 1 0 Hz), and ~ 4.04 m (J ["~C-'H] -- 4.2 and J [H-'H] = 4.2 Hz). In addition, the 1~C and ~C isopropyl multiplets showed an isomeric difference of 0.05 ppm arising from the DL center. The estimated optical purity of the chiral center, based on the starting material and its optically active transformation products, was 100%)'-' For comparison the NMR spectrum of commercially available DLvaline is shown in Fig..5.
B. Fermentation, Labeling, and Isolation Submerged cultures of Cephalosporium acremonium, a superior antibiotic-producing mutant, M8650-3 is were grown at 25 ° on a rotary shaker (250 rpm) in a complex medium. 19 Cephalosporin C was labeled with is D. W. D e n n e n and D. D. Carver, Can. J. Microbiol. 15, 175 (1969). 29p. A. Lemke and C. H. Nash, Can. J. Microbiol. 17, 255 (1971).
412
METHODS FOR THE STUDY OF ANTIBIOTICS
0//
[20]
" ~ "~R3 R2
o,, R1 = -C-CH2-CH2-CH2-CH 10 11
12
13 14~00~,.,
Cephalosporin C Na-salt
(z)
15 0 u R2 = COONa; R 3 = CH2OC-CH3 16 17 18 19
o II C-CH2-CH2-CH2-CH C H 3 _ C H 2 _ N H / l O 11 2 1'
2'
12
13 141COOe 15
at-
(]z)
3"
o II 11
I
PT1 = - C - C H
4"
10 / H 2
Cephalexin
R2 = COOH; R3= CH 3 16 17
o
2~
R1 = - C - C H 2 - O lO 11
3'
3-Methyl-7;2-phenoxy acetamido)3-cephem
(~)
R2 = H; R3= CH 3 17
R1 = H; O R2 = COOH; R3 = CH2OC-CH 3 16 17 18 19
7-Amino cephalosporanic acid ( 7 A C A ) (~)
FIG. 2. Cephalosporin C (sodium salt) and model substances. Reproduced by permission of the Royal Society of London.
[20]
lsC LABELING IN ANTIBIOTIC SYNTHESIS H5C200C
tC'"
H
"'C\
413
CH3
NH3
13CH3
13CH3
(+) trans
KOH /
t PCI3JBr2 COOH
CH3 H
\CH----~C ~ NH 2
NH3
COOH
\ CH I Br
13CH3
CH3
"%C j,H \13CH3
FIG. 3. Synthesis of chiral valine (2-RS, 3R)-[423C]valine).
H3C,,,,/H "C-CH-COOH '3CH3 NH2
FIG. 4. Nuclear magnetic resonance spectrum of (2RS, 3R)-[4-1"~C]valinc in DuO.
! s.5
~:o
,~'.o
,,,,,~
3'.o
2'.o
,Io
o'.5
FIG. 5. Nuclear magnetic resonance spectrum of commercial DL-valine in D:O.
414
METHODS FOR THE STUDY OF ANTIBIOTICS
[20 i
the appropriate 13C-precursor; namely, [1-~C]sodium acetate and [2-~3C]sodium acetate (Merck, Sharp and Dohme, Canada, 62-63 atom % of ~3C), DL- [ 1-13C ] valine, DL- [2-~3C] valine and, in the last experiment, ( 2 R S , 3R)-[4,~3C]valine as follows: a stock solution was prepared by dissolving 20 mg of each precursor in 1 ml of water and sterilizing by membrane filtration. One milliliter of this solution was added to 70 ml of the whole culture in individual flasks during the period of rapid synthesis of the antibiotic; i.e., after 46, 54, 66, 78, and 90 hr of incubation. Fermentation broth was collected by filtration after 115 hr. Cephalosporin C was purified and crystallized as the sodium salt. 2° Similar conditions were used in submerged cultures of P e n i c i l l i u m chrysogenum21; penicillin V was purified and crystallized as the potassium salt.
C. Recording of the Spectra C M R spectra of the metabolites were recorded at 25.2 MHz on the Varian XL-100-15 spectrometer with a computer of average transients (c.a.t.; this method involves repeated scanning and summing of the spectrum in a computer, the ultimate result being great improvement of signal-to-noise ratio), using 12-mm tubes and the signal from a concentric tube containing acetone (d6) as lock (Fig. 10). The C M R spectra of compounds (IV) and (V) were recorded at 15.1 M H z using the Fourier transform technique 22 (Table II). Chloroform was used as solvent and internal reference for (IV), and aqueous sodium bicarbonate (3%) for compound (V). A variety of proton decoupling techniques were used to simplify spectra accumulation and peak assignments. Chemical shifts and intensity data were measured with full proton noise decoupling. -~,-~4 18C-Spectral assignments were simplified by utilizing single-frequency and off-resonance proton decoupling techniques. 25,26 All chemical shifts and intensities were obtained from ca. 1 N aqueous solutions containing ~ 10% dioxane as internal reference except for com2op. W. Trown, E. P. Abraham, G. G. F. Newton, C. W. Hale, and G. A. Miller, Biochem. J. 84, 157 (1962). 21p. A. Lemke, C. H. Nash, and S. W. Pieper, J. Gen. Microbiol. 76, 265 (1973). :2 A. Allerhand, D. W. Cochran, and D. Doddrell, Proc. Nat. Acad. Sci. U.S. 67, 1093 (1970). 23F. J. Weigert, M. Jautelat, and J. D. Roberts, Proc. Nat. Acad. Sci. U.S. 60, 1152 (196S). 2, L. F. Johnson and M. E. Tare, Can. J. Chem. 47, 63 (1969). ~5M. Tanabe, T. Hamasaki, D. Thomas, and L. F. Johnson, J. Amer. Chem. Soc. 93, 273 (1971). 26j. D. Roberts, F. J. Weigert, J. I. Kroschwitz, and H. J. Reich, J. Amer. Chem. Soc. 92, 1338 (1970).
[2ol
13C
415
LABELING IN ANTIBIOTIC SYNTHESIS
TABLE II 13C CHEMICAL SHIFTS OF CEPHALOSPORIN MODELS AND p H ])EPEN1)ENCE OF RESONANCES IN CEPHALOSPORIN C(I)
Assignment"
II~
C-2 C-3 C-4 C-6 C-7 C-8 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C11 C2' C3' C4'
IIP
------11.0* 163.0 175.1 167.8 135.8 14.6" ---. . 157.7 181.9 ---
164.5 68.4 49.9 135.4 133.9 25.1 12.9 133.9 -. . --19.5 174.8 -. . 63.6 61.3* 63.6* 62.1
IV c
Vd
164.7 73.9 75.8 136.1 134.3 28.9* 22.9* 125.4 -. . ---171.9 --
167.0 75.8 60.4 130.0" 133.7" 29.0* ------
24.0* 128.0 17.6 172.0 -. . ---
34.5 77.4 62.3 69.8
I p H 6.0
I p H 3.0
167.8 74.5 58.5 135.2 133.2 24.3 12.0 158.1 172.6 163.1 137.9 14.1 20.6 127.8 14.6 173.2 -. . . ---
167.3 69.5 60.1 134.9 133.0 23.8 12.2 158.0 172.3 163.0 138.4 15.1 21.6 128.0 14.8 172.8
--
T h i s n u m b e r i n g s y s t e m is d i f f e r e n t f r o m t h e a c c e p t e d n u m b e r i n g s y s t e m for t h i s class of c o m p o u n d s a n d is u s e d u n i q u e l y for a n e a s y i d e n t i f i c a t i o n of c h e m i c a l shifts. E n t r i e s w i t h a s t e r i s k s d e n o t e r e s o n a n c e s w h e r e t h e a s s i g n m e n t m a y p o s s i b l y be i n v e r t e d . b S p e c t r a were r e c o r d e d a t 25.2 M H z on t h e V a r i a n X L - 1 0 0 - 1 5 u s i n g 12-Into t u b e s a n d t h e s i g n a l f r o m a c o n c e n t r i c t u b e c o n t a i n i n g acetone-d6 as lock. All c h e m i c a l s h i f t s a n d i n t e n s i t i e s were o b t a i n e d f r o m ca. 1 M a q u e o u s s o l u t i o n s c o n t a i n i n g ~ 1 0 % d i o x a n e as i n t e r n a l reference. S h i f t s are g i v e n in p a r t s p e r m i l l i o n r e l a t i v e to CS2 a n d h a v e u n c e r t a i n t i e s of a b o u t +_0.1 p p m . e S p e c t r u m recorded a t 15.1 M H z u s i n g t h e F o u r i e r t r a n s f o r m t e c h n i q u e (A. Allerh a n d , D. W. C o c h r a n , a n d D. Doddrell, Proc. Nat. Acad. Sci. U.S., 67, 1093 (1970). C h l o r o f o r m w a s u s e d as s o l v e n t a n d i n t e r n a l reference. S p e c t r u m r e c o r d e d a t 15.1 M H z u s i n g t h e F o u r i e r t r a n s f o r m t e c h n i q u e as a b o v e . A q u e o u s s o d i u m b i c a r b o n a t e s o l u t i o n (3 %) w a s t h e s o l v e n t . pounds
(IV)
relative
to carbon
D.
and
Assignments The
(V)
of Chemical
identification
cal shifts
and
(see above).
an
Shifts
are given
disulfide and have uncertainties
Shifts in the CMR
of the 13C-label was made
increased
intensity
of the
in parts
of about
per million
_0.1
ppm.
Spectra by characteristic
signal
over
that
chemi-
of natural
416
METHODS FOR THE STUDY OF ANTIBIOTICS NH~ ]
0 ,,
/ S ~ _
~CH-(~)H2-(~H2-g2 - C-NH~
[20]
~'t o
o
FIG. 6. Assignments of '3C frequencies. Encircled numbers: 2, 6, 7, 11, 14, proton
decoupling frequencies; 12, 13, chemical shifts and model substances; 3, 4, 17, 19, multiplicity, chemical shifts, and model substances. NH~ 0 ( ,, i s . . CH-CH2-CH2-CH2-~.-NH-r-----T ~ t e ~ L l ! CH20 -CH3 00Na
FIG. 7. Assignments of [~3C]carbonyl frequencies. Encircled numbers: 10, model (7-aminoeephalosporanic acid), literature, CH31sCOONa; 8, 16, pH dependence; 15, 18, CH314COONa, CI-I318COONa.
abundance (1.1% of 1~C). This type of comparison required structural assignments of 16 carbon frequencies in the natural abundance proton noise decoupled 13C-spectrum of cephalosporin C. The assignment of the resonances was based on off-resonance and single-frequency proton decoupling experimentsY 5,26 The carbon signals were divided into groups according to the number of directly bonded protons, selected in accordance with the reported proton spectra of cephalosporin C derivatives, ~7 thus providing an unambiguous assignment of each of the proton-bearing carbons C-2, C-6, C-7, C-11, and C-14. Although C-12 and C-13 could not be differentiated by this technique, their resonances were readily identified on the basis of chemical shifts and appropriate models. 22 Frequencies of C-3, C-4, C-17, and C-19 were located from their multiplicites, chemical shifts, and model systems.28,~9 Initial assignment of the five carbonyl carbon frequencies (C-8, C-10, C-15, C-16, C-18) was accomplished using model systems, the shifts reported by others, and the known pH dependence of the carboxyl groups 22,2s (Figs. 6 and 7). The pH dependence of the chemical shift of C-15 and C-16 marked these resonances as carboxyl carbons which could then be differentiated using the model systems (Table II). The remaining carbonyl carbons, C-8, C-10, and C-18, were tentatively identified from model studies. .,TR. D. G. Cooper, P. V. Demarco, C. F. Murphy, and L. A. Spangle, J. Chem. Soc. C, 340 (1970). :~ E. Lippmaa, T. Pehk, K. Anderson, and C. Rappe, Org. Magn. Res. 2, 109 (1970). -~G. B. Savitsky, P. D. Ellis, K. Namikawa, and G. E. Maciel, J. Chem. Phys.
49, 2395 (1968).
[20]
13C LABELING IN ANTIBIOTIC SYNTHESIS NH3÷ 0 [ , CH-CH%'-CI~-CH2-C~NH~ 14 13 12 I1 I0 9 ~/ 15
0~
~b
417
S 1
2
4~
CH20~ CH3
C-10
C-15 C 18
C 16
C-8
]2i0
14~ ~4 6
2(~6
24 3
ppm ,elative in CS2 ]0% dioxane as int ~ef
FIG. 8. A portion of CMR spectra of CH~l"COONa-labeled cephalosporin C (Na salt) (top; c.a.t, output reduced by a factor of 2; 50 scans), and of unlabeled antibiotic in D20 (bottom: c.a.t, output normal; 50 scans). Reproduced by permission of the Royal Society of London. The 13C incorporation data provided convincing proof of the assignment of these carbonyl carbons. When CH313COONa was employed as the precursor, the same relative incorporation levels were obtained for the adjacent carbonyt positions C-10, C-15, and C-18, respectively (Fig. 8). The proton-bearing carbons, C-11, C-14, and C-19, have been unequivocally identified by decoupling techniques. B y utilizing cephalosporin C derived from 13CH3COONa, the different (see Fig. 9) degrees of 13C incorporation at each of these positions were established with an unambiguous assignment of resonances as well as confirmation of assignments of C-8 and C-16 chemical shifts (Table II). E. D e t e r m i n a t i o n of Incorporation Levels of 13C Precursors The integrated intensity data for cephalosporin generated from labeled and unlabeled precursors were obtained from spectra recorded on the same day with the same instrumental settings using samples of the same concentration. T h e y are accurate to -+-0.2. The signal from acetone (d6) provided a convenient check on relative intensities between samples. In order to obtain percent incorporation, the values in columns 4 and 5 in Table I I I tabulation have to be corrected according to the formula: % incorporation -- 1 0 0 / 6 2 ( / - - 1), where l is the observed intensity. The intensity data are listed in Table III.
418
METHODS FOR THE STUDY OF /~NTIBIOTICS
H3•
0 m CH-CHz-CH2-CH2-C-NH ~ u I t3 TZ . 10 t'
o
E~
~
S 1
"(
.
"c.,oc-c,~ ~OONa~1
C-17 L
127.8
[20]
C-7 L
133.2
/I
18 t9
II
C-6
C-14
i
L
135.2
137.9
p#m relative to CS2 10% dioxane as int. rel.
Fro. 9. A portion of CMR spectra of I'CH3COONa labeled cephalosporin C (sodium-salt) (top: c.a.t, output reduced by a factor of 2; 50 scans) and of unlabeled antibiotic in D20 (bottom: c.a.t, output normal; 50 scans). Reproduced by permission of the Royal Society of London. F. Discussion and Conclusions It has been shown earlier that Cephalosporium grown in the presence of [1-14C]sodium acetate produced cephatosporin C labeled in the aeetoxy group as well as in the C-153° and C-10 of the D-a-aminoadipoyl side chain. These results were confirmed under our experimental conditions. Labeling cephalosporin C using [2-13C]sodium acetate resulted in distribution of the label in C-14, C-13, C-12, and C-11 of the D-a-aminoadipoyl side chain. Based on the increase of intensities of the corresponding 1~C frequencies in the C M R spectrum of cephalosporin C (Fig. 9) the amount of incorporation in C-13, C-12, and C-11 of the aminoadipoyl side chain appears to be about one-half of that at C-14. Therefore, the carbon skeleton of the D-a-aminoadipoyl side chain is presumably formed via condensation of a-ketoglutarate and acetyl coenzyme A. The amount of the label dispersion mentioned above could, in fact, be expected to arise from cycling of the tricarboxylic acids via the Krebs cycle2 ~ The results of [13C] acetate incorporation are summarized in Table III. Earlier studies of the incorporation 32 of DL-[1-1~C]valine into cephalosoThis numbering system of the a-AAA side chain is different from the accepted numbering system for this class of compounds and is used uniquely for an easy identification of chemical shifts. .~1H. R. Mahler and E. H. Cordes, Biol. Chem., p. 525 (1966). ~P. W. Trown, B. Smith, and E. P. Abraham, Biochem. J. 86, 284 (1963).
[20]
13C
LABELING IN ANTIBIOTIC SYNTHESIS
419
TABLE III INCORPORATION OF 13C-LA.BELED ACETATES AND 13C CHEMICAL SHIFTS OF CEPHALOSPORIN C a * CH313C00Na • NH3(~
13CH3C00Na 0
H
H
14CH--CH~--CH2--CH2--C--NH ~
0
' ~t
~
~CH20CCH3
/
COONa
Relative intensities ~
Assignmentb
Chemical shifts c
Normal abundance
13CH3COO-Na+ labeled
CH313COO-Na + labeled
C-2 C-3 C-4 C-6 C-7 C-8 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19
167.8 74.5 58.5 135.2 133.2 24.3 12.0 158.1 172.6 163.1 137.9 14.1 20.6 127.8 14.6 173.2
1.0 1.0 1.2 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.8 1.0 1.0 0.8
1.0 1.2 1.2 0.8 1.0 1.0 1.2 2.0 1.8 1.8 4.6 1.0 0.8 1.0 1.0 3.6
1.0 1.0 1.2 0.8 1.0 1.4 2.2 1.0 1.2 1.2 1.0 5.0 1.0 0.8 3.6 0.8
a
Reproduced by permission of the Royal Society of London. b This numbering system is different from the accepted numbering system for this class of compounds and is used uniquely for an easy identification of chemical shifts. cSpectra were recorded at 25.2 MHz on the Varian XL-100-15 using 12 m m tubes and the signal from a concentric tube containing acetone (d6) as lock. All chemical shifts and intensities were obtained from ca. 1 M aqueous solutions containing ~ 1 0 % dioxane as internal reference. Shifts are given in ppm relative to CS2 and have uncertainties of about _0.1 ppm. ,1These are measured relative intensities from the spectra run under essentially identical instrumental conditions and accurate to + 0.2. The signal from acetone (d6) provided a convenient check on relative intensities between samples. In order to obtain percent incorporation, the figures in cohlmns 4 and 5 should be corrected according to the formula: % incorp. = 100/62(l - 1) where / i s the observed intensity.
420
METHODS FOR THE STUDY OF ANTIBIOTICS
[20]
COQNa
C-10
C-15 £:-18
C-16
C-8
1210
L I 14 1 14.6
2(~ 6
24.3
ppm relative to CS2
10% dioxane as int. ref
FzG. 10. A portion of CMR spectrum of DL-[1-"C]valine-labeled cephalosporin C, sodium salt, in D:0. c.a.t, output reduced by a factor 2; 50 scans. Reproduced by permission of the Royal Society of London.
sporin C revealed a considerable dilution of the molar radioactivity. However, about 90% of the radioactivity in the antibotic was shown to be located in the valinyl moiety of cephalosporin C. Labeling our culture with DL-[l-lSC]valine resulted in localization of the label specifically in the C-16 carboxyl (Fig. 10). When we used DL-[2-1~C]valine, the label in cephalosporin C was found exclusively in C-4 (Fig. 11). Comparison of the proton noise decoupled CMR spectrum of cephalosporin C precursed with (2RS, 3R)-[4-1~C]valine with that of unlabeled antibiotic showed a 5-fold enhanced intensity of the C-2 resonance without any other detectable change in the spectrum (Fig. 12). When the C M R spectrum of penicillin V grown using the same precurNH~
0
I s ca-ca2-cH~-CH~-~-NH~'~,
141 13
co¢'
•
12
II
IO
i"
i"
.L~L,~.~.
o
"-~
"1
o
~,o,c.-c.,
.coo,.
C-4
58.5
C-3
p~l relative to CS2 10% dioxlne as int. ref,
74.5
Fz~. 11. A portion of CMR spectrum of DL-[22"C]valine-labeled cephalosporin C, sodium salt, in D~O. c.a.t, output reduced by a factor of 2; 50 scans. Reproduced by permission of the Royal Society of London.
[20]
13C LABELING IN ANTIBIOTIC SYNTHESIS
NH3 ~
421
0
14CHCH~CHzCHzCHN ~ ~6 IS 2 ~ 1 13 12 i1 1o 3 coo' ~ ' 4 ~ 15 O~ "~ 17CHzOCOCH3 COONa 18 I~ 16
Fro. 12. A portion of CMR spectra of chiral valine-labeled (top; 108 scans) and unlabeled (bottom; 51 scans) cephalosporin C in D~O with 10% dioxane as internal standard.
O H II ~ O-CH2-C-Hkl ~ O//
,3-CH 3
a
H - S :
,,,,CH o.
N ~ / \CH 3 COOK
-CH 3
FIG. 13. A portion of CMR spectra of chiral valine-labeled (top; 64 scans) and unlabeled (bottom; 64 scans) penicillin V in D:O with 10% dioxane as internal standard. sor was c o m p a r e d w i t h t h a t of u n l a b e l e d m e t a b o l i t e , ~3 it i n d i c a t e d a 1.7 ___ 0.2-fold e n h a n c e d i n t e n s i t y of t h e fl-CH3 r e s o n a n c e a t C - 2 w i t h o u t a n y o t h e r d e t e c t a b l e c h a n g e s in t h e s p e c t r u m (Fig. 13). T h u s in b o t h .~3R. A. Archer, R. D. G. Cooper, P. V. Demarco, and L. R. F. Johnson, Chem. Commun. 1970, 1291 (1970).
422
METHODS FOR T H E STUDY OF ANTIBIOTICS
[20]
cases a highly stereospecific incorporation of the precursor has been observed (Figs. 14 and 15). To illustrate some of the points in regard to experimental conditions, particularly the occurrence of NOE, the Fourier transform spectra of cephalosporin C (natural abundance and enriched metabolite) are shown in Figs. 16 and 17. lSCO0° I
0 II
--= --=S
CH(CH2) 3CHN ' = " ' ~ ~ ~ "~1" 141312" lo .L2,5 3~r'l,I nPnPu NH~ 0 "/ / 1~- is is 3 °
COONa
*CH 3
-'~ 0c,'r~
H2N~H ~' Z~H"CH3
.13C
1COOH
Fro. 14. Incorporation of (2RS, 3R)-[4-"C]valine into cephalosporin C. O H H II -=- "= S O-CH2-C_HN = _--
*' t,n 3
| COOK * 4
H2N'~ ~
H
'I
P. chrysogenum
2~H ~CH3
* 13C
COOH
Fro. 15. Incorporation of (2RS, 3R)-[4-"C]valine into penicillin V. C3 C18 C15]
H~ I
ClO'
o
. H
'4C]H-CHz-CH2-CHz-~-NH~ ~ 0 ] ,3 ,2 ,, to g 1 ~ ..~L COO~ O~ 4~ CHzOCCHz
C8
I c,61
c4
"
C~. \
176.8
i
I
186.9
137
I
117.1
97.2
,coo,. c~,
Cl,
C8
C12
.17,.,qc;II
c13 ..k
t
I
I
i
I
77.3
57.4
37.5
17.6
0
PPM
Fzo. 16. Fourier transform natural abundance CMR spectrum of cephalosporin C in D20/dioxane (NT 51, C ,-- 1 M).
[20]
423
13C L A B E L I N G IN ANTIBIOTIC SYNTHESIS
NH3 o H H I ,* :- --- $ 14CH-CH2-CHz-CHz-C-NH~--~e ~ ' ~-~= ~ ~ o oo ~ ~ 4 CHzOCCH3
C2
COON| C3 I: I
C18 C15 s e.,lnl/ C8 -"/~" I ~ I' ~
C4 C !
I ~ 1
I
I
I
I
176.8
156.9
137
117.1
C17 ! C7 i C6 C14 i , I j :1
6
I
: C12 C13 i /C19 Cll t ' l l
;
I
97.2 72.3 PPM
,J
_~
I
L
I
5 .4
37.5
17.6
0
FIG. 17. Fourier transform CMR spectrum of '~C-labeled cephalosporin C in D,.O/dioxane (NT 235, C ~- 0.7 M). Assuming that, under fermentation conditions used for cephalosporin production, only L-valine is being utilized, the incorporation of DL-[1JsC]- and DL-[2-13C]valine is about 16% (Figs. 10 and 11). In the case of incorporation of (2RS, 3R)-[4J3C]valine, the intensity of the C-2 signal in the CMR spectrum corresponds to 8.6% of incorporation (Fig. 12). These differences in incorporation of differently labeled valines correspond in fact to dilution of the label with unlabeled valine in order to maintain similar conditions during labeling of the fermentation. The increase of the fl-CH8 resonance in the spectrum of penicillin V corresponded to only 1.6% of incorporation (Fig. 13). This low degree of incorporation is presumably due to a smaller amount of (2RS, 3R)-[4l~C]valine used in this fermentation experiment (one-fifth of the amount used in the fermentation of cephalosporin C). An unequivocal decision in regard to the rate of incorporation of our valine precursors cannot be reached owing to a number of complicating factors that have to be considered in the biosynthesis of Cephalosporium and Penicillium. For example, one could mention the different rate uptake of D- and L-isomers of valine 16 and selective inhibition of incorporation of L-valine into cephalosporin C. 17 Confirmation of this stereospecific incorporation of (2RS, 3R)-[413C] valine was furnished by a disclosure from another laboratory, :~~where the amino acid of the opposite chirality, (2S, 3S)-[4-'3C]valine, was prepared by a completely different route and used in the fermentation of Cephalosporium. The metabolites produced were isolated, and their CMR 34H. Kluender, C. H. Bradley, C. J. Sih, P. Fawcett, and E. P. Abraham, J. Amer. Chem. Soc. 95, 6149 (1973).
424
METHODS FOR THE STUDY OF ANTIBIOTICS
[20]
CH3 H,~"',,,,,Ca3
HN. 2~,,~H,, (2S,5S)-[4-13C] I CDOH
Valine
~~,NCH30~/ cDH ~ C .H2OCO3CH NHCOR PEN N
NHCOR CEPHC
H2N\ R= e /CH-(CH2)3OOC
Fro. 18. Incorporation of (2S, 3S)-[4-13C]valine into cephalosporin C in penicillin V. spectra indicated the location of the l~C-label in this case in the a-CH~ of penicillin N and in the exocyclic methylene, C-17 of cephalosporin C (Fig. 18). It should be pointed out that the decision in regard to the locus of the label in all cases mentioned above could be reached in just one spectral measurement. These experiments clearly illustrate a big advantage of the 13C technique over the classical 14C-radiotracer methodology. It is easy to visualize complicated degradation methods that would have been required to locate the label by the latter, standard method. It is obvious that in this particular instance those efforts would have been futile. Biogenetic consequences of the stereospecific incorporation of (2RS, 3R)-[4-18C]valine into cephalosporin C and penicillin V (Figs. 14 and 15) are of significance. The outcome of the experiment points to the possible derivation of these two important antibiotics from a common a,fldehydrovaline derivative of tripeptide (Fig. 1). It is quite possible that the ring closure is preceded.by the existence of a three-point attachment 35 between the enzyme and the symmetrical substrate involving only one methyl group, with the other available for transformation necessary for the ring closure. Finally, these experiments disprove a proposal suggesting the formation of 6-aminopeniciltanic acid from acetone, glycine, and cysteine2 6 The introduction of 13C-labeling represents an important new step in s5 A. G. Ogston, Nature (London) 162, 963 (1948).
36I. R. Shimi and G. M. Imam, Arch. Microbiol 60, 275 (1968).
[20]
13C LABELING IN ANTIBIOTIC SYNTHESIS
425
the elucidation of biosynthetic pathways of natural products. However, as with other techniques, the method has some drawbacks and pitfalls. The reader should carefully consider experimental conditions of 13Clabeling and accuracy of spectral measurements in order to take full advantage of this new methodology.
Acknowledgments The author gratefully acknowledges Nancy DeLaHiguera's review of the bibliography.
[21]
429
BIOSYNTHESIS OF GUANIDINATED INOSITOL
[21] P a t h w a y s o f B i o s y n t h e s i s o f t h e G u a n i d i n a t e d Inositol M o i e t i e s of S t r e p t o m y c i n a n d B l u e n s o m y c i n
By JAMES B. WALKER Knowledge concerning enzymic reactions involved in the biosynthesis and metabolism of a given antibiotic can be utilized in rational approaches to (a) the design of fermentation media for increased antibiotic production, (b) mutant selection procedures for obtaining superior producing strains, (c) the directed biosynthesis of structural analogs of the antibiotic, (d) tile determination of factors governing differentiation to the antibiotic-synthesizing state, and (e) the determination of selection pressures which resulted in the evolutionary acquisition of such novel biosynthetic capabilities. NH2
NH2.
C ---NH2 , NH
H2N
3
C=~O I O
H2N
3
6
6
tt
OH
O 4t
HsC\CH2
O 1p
4'
/
H3C\CH2
0
1p
/
0
.
HO
.
.
.
,,k HO
(i)
(ii)
Fie. 1. Structures of dihydrostreptomycin (I) and bluensomycin (or glebomycin) (lI). Dihydrostreptomycin is N-methyl-~-L-glucosamine(1-o2)-a-h-dihydrostreptose(1 --> 4)streptidine. Streptomycin has an aldehyde group rather than a hydroxymethyl group at position 3',,. Bluensomycin is N-methyl-a-L-glucosamine(1---> 2)a-L-dihydrostreptose(1--->4)bluensidine. Mild acid hydrolysis of these compounds gives streptidine or bluensidine, respectively, plus the disaccharide, dihydrostreptobiosamine (DSBA). Bluensidine has also been called glebidine. None of the components of these antibiotics has so far been found elsewhere in nature.
430
ANTIBIOTIC BIOSYNTHESIS
[21]
. fP
+~ 0 0
0
-x~_ 0
~ 0
o~
~, 0
"
~-_~~ T
II ~
+~ fill
.,~
~-~
~
~-~-~---~ ~ II ~
0
,.,.,
.
0-0 ~l~l
0
~ o m
Z
~- - ~ o ~ 0
"~ "
*~
I
o--.,~o
I
I
.o
.~
[21]
BIOSYNTHESIS OF GUANIDINATED INOSITOL
431
The following group of articles is concerned with enzymes involved in biosynthesis by Streptomyces strains of the guanidinated inositol moieties of the streptomycin family of antibiotics, including dihydrostreptomycin (I) and bluensomycin (II), whose structures are given in Fig. 1.1 Studies in vivo on the biosynthesis of streptomycin by intact mycelia 2 have provided important guideposts for enzymic studies with cell-free extracts. ~,4 Results of the latter enzymic studies are summarized in Fig. 2. Two analogous sequences of 5 enzymic reactions each, operating in series, appear to be involved in biosynthesis of the streptidine moiety of dihydrostreptomycin (I) from myo-inositol (V). Each sequence converts a hydroxyl group to a guanidino group and involves, in order, a dehydrogenation (C and H ) , transamination (D and I), phosphorylation (E and J), transamidination (F and K), and dephosphorylation (G and N). For certain pairs of corresponding reactions in the two sequences, it is known that different enzymes are involved, although some have overlapping substrate specificities. Only one of the above two sequences appears to be operative in strains that synthesize bluensomycin (II). 4 Descriptions of the individual enzymic reactions in the following articles will follow the suggested biosynthetic sequence, starting with reaction C. Emphasis will be placed in these articles on the preparation and detection of various intermediates and their analogs and on detailed descriptions of several alternative assays for each enzyme. Radiochemical assays involving trace amounts of substrates and products have dominated these early enzymic studies, but it is anticipated that assays can subsequently be developed that utilize, e.g., ninhydrin and Sakaguchi reactions or dansylation of intermediates containing amino, guanidino, a n d / o r keto groups. 1Published and previously unpublished work reported in these articles was supported by the Robert A. Welch Foundation and the National Institutes of Health. 2 A. L. Demain and E. Inamine, Bacteriol. Rev. 34, 1 (1970). 3j. B. Walker, Lloydia 34, 363 (1971). 4j. B. Walker, J. Biol. Chem. 249, 2397 (1974).
FIG. 2. Current concept of enzymic steps involved in biosynthesis of the streptidine moiety of dihydrostreptomycin (I) by Streptomyces humidus ATCC 12760 and the bluensidine moiety of bluensomycin (II) by S. glebozus ATCC 14607, starting from glucose-6-P (III). Extracts of S. glebos~s catalyze reactions C, D, E, F, G, and K. The step at which the carbamoyl group is added has not been established. Enzyme G dephosphorylates substrate molecules which have escaped carbamoylation. Enzymes H and I are apparently missing in S. glebosus. Abbreviations: DSBA, dihydrostreptobiosamine; KGAM, a-ketoglutaramate; Orn, ornithine; Pyr, pyruvate; NDP-sugar, nucleosidediphosphate sugar.
432
ANTIBIOTIC BIOSYNTHESIS
[21]
MOBILITIES OF VARIOUS BIOSYNTHETIC INTERMEDIATES AND ANALOGS Relative H V E mobility d
R/'
HC1 elution from Dowex 50 c o l u m n ~
1D-1-Amino-1-deoxy-scyllo-inositol-6-P 1-Amino- 1-deoxy-scyllo-inositol-4-P
0.04
0.5 N
0.0
O. 10
O. 5 N
- O. 3
d- O. 9
1D-1,3-Diamino-l,3-dideoxy-scyUo-
0.13
1.0 N
--0.5
+0.9
0.08
2.0 N
-0.5
d-l.0
0.17 0.21 0.20
0.5 N 1.0 N 2.0 N
- 0.3 --0.9
+0.2 -+0.1
0.43
2.5 N
--
--
0.33
2.0 N
- 0.9
+0.2
1 D- 1,3-Diguanidino- 1,3-dideoxy-scylloinositol-6-P
0.40
2.0 N
- 0.8
- 0.5
1D-1,3-Diamino-l,2,3-trideoxy-scyllo-
0.25
1.0 N
--
--
0.39 0.59 0.83
0.5 N 1.0 N 2.5 N
-- 1 . 2 - 1.1 - 1.8
-- 0 . 2 - 1.0 - 1.0
0.83
2.5 N
- 1.8
-- 1 . 0
0.92 0.71 Streaks
5.0 N 1.0 N 1.0 N
- 2.0 -- 1.1
-- 1.7 ---
Streaks
H~O
- 0.3
--
Compounda
p H 3.6 p H 10.4 + I. 0
inositol-2-P
1D-1,3-Diamino-l,3-dideoxy-scylloinositol-6-P
1-Guanidino-l-deoxy-scyllo-inositol-4-P 2-Guanidino-2-deoxy-neo-inositol-5-P 1D-1-Amino-3-guanidino-l,3-dideoxy-
scyllo-inosi tol-6- P 1D- 1-Amino-3-guanidino-1,2,3-trideoxy-
scyllo-inositol-6-P 1D-1-Guanidino-3-amino-l,3-dideoxy-
scyllo-inositol-6-P
inositol-6-P
Aminodeoxy-scyllo-inositol Guanidinodeoxy-scyUo-inositol 1D-1-Amino-3-guanidino-1,3-dideoxy-
scyllo-inositol 1D- 1-Guanidino-3-amino-l,3-dideoxy-
scyllo-inositol 1,3-Diguanidino-l,3-dideoxy-scylla-inositol 2-Guanidino-2-deoxy-neo-inositol 1D- 1-Guanidino- 1-deoxy-3-keto-scylloinositol
Keto-scyllo-inositol
Cyclitol n o m e n c l a t u r e according to I U P A C t e n t a t i v e rules [Eur. J. Biochem. 5, 1 (1968)], except t h a t a keto s u b s t i t u e n t is so designated r a t h e r t h a n n a m i n g c o m p o u n d as a derivative of cyclohexanone. b Ascending paper c h r o m a t o g r a p h y , 80% p h e n o l - 2 0 % H20, N H 4 O H a t m o s p h e r e . T h i s v o l u m e [24]. c M i n i m u m concentration of HC1 required to elute c o m p o u n d adsorbed on a column containing Dowex 50 (H +) X 8 cation exchange resin, 200-400 mesh, in the stepwise sequence: H20, 0.5 N HC1, 1.0 N HC1, 2.0 N HC1, 2.5 N HC1, 5.0 N HC1. This v o l u m e [23]. Mobilities relative to distance traveled b y a pieric acid m a r k e r d u r i n g highvoltage paper electrophoresis at t h e indicated p H . T h i s v o l u m e [22]. A m i n u s sign indicates m i g r a t i o n toward t h e n e g a t i v e electrode, a n d a positive sign indicates m i g r a t i o n toward t h e positive electrode. Mobilities v a r y s o m e w h a t w i t h point of application a n d t e m p e r a t u r e .
myo-INOSITOL:NAD + 2-OXIDOREDUCTASE
[22]
433
Mobilities of certain of the biosynthetic intermediates and their analogs are summarized in the table ; these properties can be utilized for both separation and identification. The enzymes themselves emerge from a Sephadex G-100 column in the following approximate order: streptomycin-6-P phosphatase (N), glutamine: keto-scyllo-inositol aminotransferase (D), alanine: 1D-l-guanidino-l-deoxy-3-keto-scyUo-inositol aminotransferase (I), inosamine-P amidinotransferase (F, K), streptomycin 6-kinase (0), guanidinodeoxy-scyllo-inositol-4-P phosphatase (G), and inosamine kinase(s) (E, J). Except for enzymes N and O, most of the enzymes appear to be sulfhydryl enzymes. Although commercial strains of Streptomyces selected by sequential mutation procedures for high productivity might well have higher levels of certain of these biosynthetic enzymes, all the procedures to be described employ the more generally available Streptomyces strains from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852.
[22] m y o - I n o s i t o l : N A D + 2 - O x i d o r e d u c t a s e By
JAMES B . WALKER
OH
OH
+ tt
H
NAD+ ~
0
OH m yo - Inositol
+
NADH
+
(1)
tt
OH Keto- s c y l l o - ino sitol (scyllo-inosose ; myo-inosose-2)
When Streptomyces hygroscopicus ]orma glebosus ATCC 14607 (S. glebosus) is grown with myo-inositol as a major energy source, myoinositol 2-dehydrogenase activity 1 is induced, and activity can be readily measured in crude extracts spectrophotometrically at 340 nm by conventional procedures, in either direction. 2 However, most streptomycin-producing strains cannot utilize myo-inositol as a major energy source, and in such strains myo-inositol 2-dehydrogenase activity has not yet been i E C 1.1.1.18. F o r the e n z y m e f r o m A e r o b a c t e r 2 j . B. Walker, u n p u b l i s h e d data.
a e r o g e n e s , see this series, Vol. 5 [36].
myo-INOSITOL:NAD + 2-OXIDOREDUCTASE
[22]
433
Mobilities of certain of the biosynthetic intermediates and their analogs are summarized in the table ; these properties can be utilized for both separation and identification. The enzymes themselves emerge from a Sephadex G-100 column in the following approximate order: streptomycin-6-P phosphatase (N), glutamine: keto-scyllo-inositol aminotransferase (D), alanine: 1D-l-guanidino-l-deoxy-3-keto-scyUo-inositol aminotransferase (I), inosamine-P amidinotransferase (F, K), streptomycin 6-kinase (0), guanidinodeoxy-scyllo-inositol-4-P phosphatase (G), and inosamine kinase(s) (E, J). Except for enzymes N and O, most of the enzymes appear to be sulfhydryl enzymes. Although commercial strains of Streptomyces selected by sequential mutation procedures for high productivity might well have higher levels of certain of these biosynthetic enzymes, all the procedures to be described employ the more generally available Streptomyces strains from the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852.
[22] m y o - I n o s i t o l : N A D + 2 - O x i d o r e d u c t a s e By
JAMES B . WALKER
OH
OH
+ tt
H
NAD+ ~
0
OH m yo - Inositol
+
NADH
+
(1)
tt
OH Keto- s c y l l o - ino sitol (scyllo-inosose ; myo-inosose-2)
When Streptomyces hygroscopicus ]orma glebosus ATCC 14607 (S. glebosus) is grown with myo-inositol as a major energy source, myoinositol 2-dehydrogenase activity 1 is induced, and activity can be readily measured in crude extracts spectrophotometrically at 340 nm by conventional procedures, in either direction. 2 However, most streptomycin-producing strains cannot utilize myo-inositol as a major energy source, and in such strains myo-inositol 2-dehydrogenase activity has not yet been i E C 1.1.1.18. F o r the e n z y m e f r o m A e r o b a c t e r 2 j . B. Walker, u n p u b l i s h e d data.
a e r o g e n e s , see this series, Vol. 5 [36].
434
ANTIBIOTIC BIOSYNTHESIS
[22]
detected in crude extracts by a spectrophotometric assay at 340 nm, even when assayed in the equilibrium-favored reverse direction. Such an assay might be feasible with more purified preparations, when such preparations become available. Since it is not yet known whether the same gene codes for both the biosynthetic and catabolic myo-inositol 2-dehydrogenase activities, two procedures will be described below for detection of biosynthetic myo-inositol dehydrogenase activity in extracts of mycelia grown under conditions in which the myo-inositol catabolic pathway does not appear to be induced. Both assay methods employ radioactive substrates and involve transamination of the keto-scyllo-inositol formed in Eq. (1) to give aminodeoxy-scyllo-inositol (coupling of reactions C and D). s Components of the incubation mixtures are usually separated by high voltage paper electrophoresis at pH 3.6 and counted. Alternatively, separations can be performed with very small Dowex 50 (H ÷) columns, as described for larger columns.4
Assay Methods
Method 15 myo_[U_14C]inosito1 + NAD+ c_~keto_scyllo_[U_14C]inositoI + NADH -{- H+ (2) D
Keto-scyllo-[U-~4C]inositol -}- L-glutamine---> aminodeoxyoscyllo-[U-14C]inositol -~ a-ketoglutaramate (3) Principle. This method is based upon conversion of labeled myo-inositol, having no net charge, to labeled aminodeoxy-scyllo-inositol (pK~ 7.6), having one positive charge at pH 3.6, in a coupled enzyme assay. Reagents Extract of Streptomyces hygroscopicus ]orma glebosus ATCC 14607
(S. glebosus) myo-[U-14C]Inositol, ca. 200 Ci/mole, from New England Nuclear (myo-[U-3H]inositol would also be satisfactory, but not myo- [2-SH ] inositol) NAD ÷, 3 mM Potassium phosphate buffer, 100 mM, containing 13 mM EDTA and 8 mM pyridoxal-P, pH 7.4. L-Glutamine, 40 mM ' This volume [21]. 4This volume [23]. ~J. B. Walker, ]. Biol. Chem. 249, 2397 (1974).
[22]
myo-INOSITOL:NAD + 2-OXIDOREDUCTASE
435
Procedure. The complete incubation mixture contains: myo-[U14C]inositol, 5 ~1 (e.g., 68,000 cpm) ; phosphate buffer, EDTA, and pyridoxal-P, 5 ~1; NAD ÷, 5 ~1, glutamine, 5 ~1, and dialyzed S. glebosus extract, 10 ~1. After incubation in a stoppered 13 X 100 mm test tube at 35 ° for 145 min, 10-~1 aliquots are spotted at the 30-cm mark and the components separated by high-voltage paper electrophoresis at pH 3.6 (see below). Nondialyzed extracts can also be employed; NAD and amino donor requirements are more pronounced with dialyzed extracts. '~ Growth of Mycelia. Well sporulated slants of S. glebosus, as well as most other strains of Streptomyces employed in these studies, are obtained by growth at room temperature on 1% maltose-0.5% Tryptone-0.2% yeast extract-l.5% agar-tap water. After incubation for 4-10 days at room temperature, a 1-cm square of surface growth and conidia is transferred with a stiff iron wire loop to a 2-liter Erlenmeyer flask containing 500 ml of autoclaved medium composed of 0.05% glucose-l% Soytone-l% Tryptone-0.2% yeast extract-0.03% K2HPQ-tap water. To assure dispersal of eonidia and mycelial fragments, the agar-square inoculum is often rubbed vigorously with the loop against the inside of the flask below the surface of the medium. After growing on a New Brunswick Psychrotherm rotary shaker at 240 rpm for 2 or 3 days at 26 °, or on a similar shaker at room temperatures of 24-28 °, mycelia are harvested by suction filtration through Whatman No. 1 qualitative filter paper (12.5 em diameter) on a Biichner funnel, pressed dry between paper toweling for 5-10 min under a 5-1b weight, rolled together into a cylindrical pad, wrapped with Saran wrap, weighed, frozen, and then stored in capped jars in a freezer for subsequent use. Many of the idiophase enzymes from various strains to be described in subsequent sections remain active in frozen pads for more than a year. Higher activities of most of these enzymes are usually obtained when large inocula from fresh, well sporulated slants are employed, using slants which had previously been inoculated heavily with fresh conidia. Rapid initial growth from many disseminated loci (e.g., viable conidia~ favors subsequent high specific activity of these idiophase enzymes. Since idiophase enzyme '~ctivities usually reach a peak after the rapid growth phase, G it is recommended that flasks inoculated at the same time from a given slant be harvested after both 2 days and 3 days of growth to assure getting at least one pad with optimal activity. Yields of 6-15 g of frozen mycelia are obtained per flask, depending upon conditions. In the case of S. glebosus, addition of 1% myo-inositol to the above growth medium causes induction later in the growth phase of high levels of a number of enzymes 6j. B. Walker and V. S. Hnilica, Biochim. Biophys. Acta 89, 473 (1964).
436
ANTIBIOTIC BIOSYNTHESIS
involved- in catabolism 2-dehydrogenase2
of
myo-inositol,
[22]
including
myo-inositol
Preparation o] Extracts. Extracts of frozen mycelial pads are prepared by sonication with a Branson sonifier of batches of 6 g of mycelia dispersed in 12 ml of deionized water contained in a wide-mouthed glass tube, 25-ml capacity, surrounded by an ice-water bath. Sonication is performed i n 1-min segments separated by recooling periods, during which time the suspension is stirred with a thermometer until the temperature is lowered to 5-10% The total time of sonica~ion optimal for a given enzyme should be determined empirically; 3-6 min is satisfactory for most purposes. Sonicates are then centrifuged at 30,000 g for 20 min at 2 °, and the supernatant solutions are stored frozen. Dialyzed S. glebosus extracts for use in studies for which reaction D activity must be retained are prepared by adding to 4 ml of the above supernatant solution 0.3 ml of a solution containing 0.7 M potassium phosphate, 0.3 M EDTA, and 0.04 M pyridoxal-P, pH 7.6. The resulting solution is pipetted into cellophane dialysis tubing, immersed in 4 liters of cold deionized water, dialyzed for 16 hr at 4 °, and stored frozen. High-Voltage Paper Electrophoresis Technique. Separations are performed on buffer-saturated Whatman No. 1 filter paper with a Savant horizontal plate apparatus (46-cm plate size). A refrigerated solution of ethylene glycol and water is circulated through the hollow metal supporting plate during the approximately 90-min run. One plastic sheet (Savant) insulates the filter paper sheet from the grounded aluminum cooling plate, and another plastic sheet is placed on top of the filter paper; a heavy square of thick plate glass rests on the top plastic sheet during a run. Each electrode vessel contains 1 liter of buffer. The filter paper sheet is 57 cm long and has previously been ruled with a hard graphite pencil so as to have 2 to 6 parallel and separated tracks, each 3.7 cm wide. Each track on that 46-cm portion of the paper which will rest on the cooling plate is marked off in 46 numbered 1-cm segments, with the No. 1 segment nearest the negative electrode. Unruled track space (2.5 cm wide) is provided at each side of the paper for the reference compound. The unruled 5.5-cm portions at each end of the filter paper serve as wicks when immersed, right after sample application, in the buffer vessels containing the electrodes. Before a run is started, the filter paper sheet is dipped in buffer contained in a wide pan, quickly placed in position on top of the lower plastic sheet, blotted as dry as possible with paper toweling and aligned in final position before samples are applied. At the start of a run a small drop of a saturated solution of picric acid is applied as a mobility reference marker at the 10-cm segment level on each edge of the paper. After the 10-td sample aliquots and the picric
[22]
myO-INOSITOL : N A D + 2-OXIDOREDUCTASE
437
acid markers are spotted, the paper ends are immersed in buffer, and the upper plastic sheet and then glass plate are placed on top and the cooling water circulation is started. The power supply normally is set at 1500 V. The run is usually terminated after the yellow pierie acid spots have migrated from 11 to 18 era. A razor blade is used to sever tile wet filter paper next to the buffer vessels, and the sheet is quickly hung by one (anode) end on a horizontal glass rod, employing stainless steel clamps, Excess buffer is blotted from the extreme lower (eathodet end of the hanging paper sheet. The sheet is dried at room temperature in a hood. Strips are cut from the dried paper, spliced end-to-end with Scotch transparent tape, with suitable lengths of filter paper strips (1.5-em wide) as spacers, and run through a Nuclear Chicago radioactive strip counter. Quantitative data are obtained by cutting numbered l-era segments from tile strips, placing the two halves of each segment flat on the bottom of a scintillation bottle containing 5 ml of diluted Liquifluor solution in toluene, and counting with a liquid scintillation system. Two buffers have been employed in our electrol)horetic separations: (a) an acid buffer in which amino and guanidino groups each have a single positive charge, and i)hosphate ester groups have a single negative charge; and (b) a basic buffer in which guanidino groups have one positive charge, amino groups have no charge, and phosphate ester groups have two negative charges. These buffers are prepared as follows, using glass-distilled water. (a) Acid buffer (pH ca. 3.6): 26 g of ammonium formate and 17.4 ml of 88% formic acid are dissolved in 4 liters of water. (b) Basic buffer (pH ca. 10.4): 72.6 g of glycine and 32.8 g of sodium hydroxide pellets arc dissolved separately and made up to 4 liters with water. Buffers are stored in plastic bottles. Each batch can t)e used for many runs before replacement; after each run the buffer solutions are returned to the bottle. The approximate relative mobilities of certain of the biosynthetic intermediates are given elsewhere. 3 Mobilities vary somewhat with the point of sample application.
Method II 5 myo-Inositol + NA1) + ~ keto-scgllo-inositol + N A I ) t t + H + •
4
(4)
D
Keto-sc?tllo-inositol + l-ammo- l-deoxy-scyllo-[ l-1 C]inositol --~ aminodeoxy-scyllo-inositol + l-keto-scyllo-[l-l~C]inositol
(5)
Principle. In this method a high concentration of nonlabeled myoinositol is employed to help overcome the unfavorable equilibrium of reaction C. The nonlabeled keto-scyllo-inositol formed in Eq. (4) serves
438
ANTIBIOTIC BIOSYNTHESIS
[22]
as acceptor of an amino group from labeled aminodeoxy-scyllo-inositol in an exchange reaction catalyzed by transaminase D. Labeled aminodeoxy-scyUo-inositol with one positive charge at pH 3.6 is converted to labeled keto-scyllo-inositol with no net charge.
Reagents Extract of S. glebosus ATCC 14607 1-Amino-l.deoxy-scyllo- [ 1-14C] inositol, 3.5 Ci/mole, from California Bionuelear Corp., Sun Valley, (scy!lo-inosamine, Cat. 72673). Labeled compound can also be prepared as described in Method I and isolated by ion-exchange chromatographyA Potassium phosphate buffer, 100 mM, containing 13 mM EDTA, and 8 mM pyridoxal-P, pH 7.4 myo-Inositol, 110 mM NAD ÷, 3 mM
Procedure. The complete incubation mixture contains: 1-amino-1deoxy-scyUo-[1-1~C]inositol, 5 ~l (e.g., 68,000 cpm); phosphate buffer, EDTA, and pyridoxal-P, 5 ~l; myo-inositol, 5 ~1; NAD ÷, 5 ~l; and nondialyzed or dialyzed extract of S. glebosus (see Method I), 10 ~l. After incubation in are spotted at voltage paper formed in Eq. the extract
a stoppered 13 X 100 mm test tube at 35 °, 10-/A aliquots the 30-cm segment and the components separated by highelectrophoresis at pH 3.6. The labeled keto-scyllo-inositol (5) is transformed to negatively charged metabolites when is prepared from mycelia previously induced with
myo-inositol. Properties myo-Inositol 2-dehydrogenase activity has only recently been detected in cell-free extracts of Streptomyces, 5 and extensive studies have not yet been made. Biological Distribution. myo-Inositol 2-dehydrogenase activity has also been detected in cell-free extracts of S. bikiniensis ATCC 11062, a streptomycin-producing strain, employing Method II. In addition to the occurrence of this enzyme in streptomycin producers, it is anticipated that strains of Streptomyces which can utilize myo-inositol as a carbon source would also have myo-inositol 2-dehydrogenase activity induction by substrate. It is not yet known whether the inducible dehydrogenase of S. glebosus is identical with the myo-inositol dehydrogenase involved in biosynthesis of bluensomycin or streptomycin.
KETO-scyllo-INOSITOL AMINOTRANSFERASE
[23l
439
Purification. Purification has not yet been performed, so it is not known whether enzymes C and D are physically associated. The assays of Methods I and II are adequate for detection and preliminary substrate specificity studies, but they would not be useful for purification purposes unless a preparation of purified transaminase D is available. If the inducible and biosynthetic myo-inositol 2-dehydrogenase of S. glebosus are identical, a spectrophotometric assay based on either the forward or reverse reactions of Eq. (1) would be the method of choice for purification studies. Specificity. D-chiro-Inositol and L-chiro-inositol are not effective substrates. In crude extracts N A D P + has less than 10% of the activity of N A D t In the coupled assay of Method I, aminodeoxy-scyllo-inositol is a better amino donor than L-glutamine, streptamine (1,3-diamino-l,3dideoxy-scyllo-inositol) is as effective as a-glutamine, and L-alanine has only slight activity at high concentrations. The foregoing amino donor specificities, of course, are a property of transaminase D, not the 2-dehydrogenase. I t appears that enzymes C and H are different enzymes?
[23] L-Glutamine:
Keto-scyllo-inositol A m i n o t r a n s f e r a s e ~ By JAMES B. WALKER HeC--CH 2
tt
O II
OH
O
I C--NH Ctt 2 2 I O + CH~ I HC,--NH:
@
H OH
Keto-s c y l l o inositol
o/~C" O-
L -
Glutamine
0 II
OH
~ N
tt2
D
+ HO
OH
Aminodeoxy-
s c y l l o -inositol ( s c y l l o -inosamine)
C--NH2 I CHz CH2
(1)
E
o/~C'~o-
a-Ketoglutaramate
This reaction is unusual among reactions involving glutamine in that the a-NH._, group, rather than the amide group, is transferred, the first
1EC 2.6.1.50.
KETO-scyllo-INOSITOL AMINOTRANSFERASE
[23l
439
Purification. Purification has not yet been performed, so it is not known whether enzymes C and D are physically associated. The assays of Methods I and II are adequate for detection and preliminary substrate specificity studies, but they would not be useful for purification purposes unless a preparation of purified transaminase D is available. If the inducible and biosynthetic myo-inositol 2-dehydrogenase of S. glebosus are identical, a spectrophotometric assay based on either the forward or reverse reactions of Eq. (1) would be the method of choice for purification studies. Specificity. D-chiro-Inositol and L-chiro-inositol are not effective substrates. In crude extracts N A D P + has less than 10% of the activity of N A D t In the coupled assay of Method I, aminodeoxy-scyllo-inositol is a better amino donor than L-glutamine, streptamine (1,3-diamino-l,3dideoxy-scyllo-inositol) is as effective as a-glutamine, and L-alanine has only slight activity at high concentrations. The foregoing amino donor specificities, of course, are a property of transaminase D, not the 2-dehydrogenase. I t appears that enzymes C and H are different enzymes?
[23] L-Glutamine:
Keto-scyllo-inositol A m i n o t r a n s f e r a s e ~ By JAMES B. WALKER HeC--CH 2
tt
O II
OH
O
I C--NH Ctt 2 2 I O + CH~ I HC,--NH:
@
H OH
Keto-s c y l l o inositol
o/~C" O-
L -
Glutamine
0 II
OH
~ N
tt2
D
+ HO
OH
Aminodeoxy-
s c y l l o -inositol ( s c y l l o -inosamine)
C--NH2 I CHz CH2
(1)
E
o/~C'~o-
a-Ketoglutaramate
This reaction is unusual among reactions involving glutamine in that the a-NH._, group, rather than the amide group, is transferred, the first
1EC 2.6.1.50.
440
ANTIBIOTIC BIOSYNTHESIS
[23]
such reaction observed in a prokaryoteY Meister and co-workers had earlier discovered an L-glutamine:a-ketoacid aminotransferase in mammalian liver; they found that such transaminations are made relatively irreversible by either spontaneous cyclization of a-ketoglutaramate or deamidation by an amidase to form a-ketoglutarate. ~
Assay M e t h o d
Principle. Transaminations involving pyridoxal-P as coenzyme are the sum of two half-reactions. One of the half-reactions of Eq. (1) is Eq. (2), which can be assayed as indicated in Eq. (3). The amino group of labeled aminodeoxy-scyllo-inositol is transferred to keto-scyllo-inositol, giving labeled keto-scyllo-inositol. Reactants and products are usually separated by high voltage paper electrophoresis. Alternatively sepaD
Keto-scyllo-inositol + enzyme-pyridoxamine-P aminodeoxy-scyllo-inositol + enzyme-pyridoxal-P (2) D Keto-scyllo-inositol + aminodeoxy-scyllo-[14C]inositol --~ aminodeoxy-scyllo-inositol + keto-scyllo-[14C]inositol (3) rations can be performed with small Dowex 50 (H ÷) columns. This assay can be used for both crude and purified preparations; purified preparations must contain pyridoxal-P at all times to retain activity.
Reagents Extract of Streptomyces hygroscopicus ]orma glebosus ATCC 146074 1-Amino-l-deoxy-scyllo- [1-14C]inositol, 3..5 Ci/mole, from California Bionuclear Corp., Sun Valley, Calif. (scyllo-inosamine[1-1~C], Cat. No. 72673). Labeled compound can also be prepared as described below. Potassium phosphate buffer, 100 raM, containing 13 mM EDTA and 8 mM pyridoxal-P, pH 7.4 Keto-scyllo-inositol, 20 m M (freshly prepared solution), from Sigma (myo-inosose-2, Cat. No: I 5375). This compound can also be prepared from myo-inositol by either catalytic oxidation or fermentation with Acetobacter suboxydans ATCC 621. ~ J. B. Walker and M. S. Walker, Biochemistry 8, 763 (1969). s See this series, Vol. XVIIA, addendum to article [136]. This volume [22]. T. Posternak, Biochem. Prep. 2, 57 (19527
[23]
KETO-seyllo-INOSITOL AMINOTRANSFERASE
441
Procedure. The complete incubation mixture contains: 1-amino-1deoxy-scyllo-[14C]inositol, 5 tL1 (e.g., 68,000 cpm); phosphate buffer, EDTA, and pyridoxal-P, 5 t~l; keto-scyllo-inositol, 5 ~l; and nondialyzed or dialyzed extract of S. glebosus, l0 t~l. After incubation in a stoppered 13 X 100 mm test tube at 35 °, a 10-~1 aliquot is spotted at the 30-cm segment and the components separated by high-voltage paper electrophoresis at pH 3.6, as described earlier. ~ The labeled amino donor has one positive charge (pK,, = 7.6) at pH 3.6, whereas the labeled product is uncharged (see Table I of Walker 6 for mobil[ties). The extract should be prepared from cells not induced with myo-inositol, to prevent catabolism of keto-scyllo-inositol. Preparation o] Labeled Aminodeoxy-scyllo-inositol. Any of several methods can be employed. For example, 1-amino-l-deoxy-scyllo-[114C]inositol can be chemically synthesized from glucose and ['4C]nitromethane, as the penultimate intermediate in an established procedure for synthesis of myo-[2-14C]inositol. 7 In our early work, labeled aminodeoxy-scyllo-inositol and its 4-phosphate, along with other ring-labeled precursors of the streptidine moiety of streptomycin, were prepared by feeding a pulse of myo-[14C]inositol to a culture of S. griseus ATCC 12475 and extracting the mycelia. ~ However, the most efficient and dependable conversion of myo-[l~C]inositol to aminodeoxy-scyllo[14C]inositol employs the coupled enzymic procedure described in Method I of an earlier article, ~ with reagent volumes suitably scaled-up for preparative purposes. Labeled substrate and labeled product can be readily separated by ion-exchange column chromatography (see below). Unconverted myo-[~4C]inositol can be isolated and used again to increase the ultimate yield. When extracts of certain streptomycin-producing strains are used as the source of enzymes C and D, the desirability of adding excess myo-inositol 2-dehydrogenase (Sigma Cat. No. I 5503) should be explored in a preliminary microassay.
Isolation o] Various Labeled Biosynthetic Intermediates by Ion-Exchange Column Chromatography. Samples are deproteinized, if necessary, with the minimal required amount of trichloroacetic acid and centrifuged to remove precipitated macromolecules. The precipitate is washed with water and centrifuged again. The combined supernatant solutions are applied to the top of a glass column, 1 cm X 22 cm, containing Dowex 50W (H +) X8 cation exchange resin (Bio-Rad analytical grade), 200-400 mesh. (The resin is used as purchased without treatment, except that the column is washed with 500 ml of glass-distilled water shortly before This volume [21]. G. I. Drummond,J. N. Aronson, and L. Anderson,J. Org. Chem. 26, 1601 (1961). J. B. Walker and M. S. Walker, Biochemistry 6, 3821 (1967).
442
ANTIBIOTIC BIOSYNTHESIS
[23]
use.) The column is washed with water, and successive stepwise elutions are performed, employing in sequence 0.5 N, 1.0 N, 2.0 N, 2.5 N, and 5.0 N concentrations of HC1. Approximately 80-120 ml total volume of water or eluent are employed at each step. Column fractions are 3-4 ml each (timed collection, 5-6 rain/tube) ; 5 ~l from each tube are spotted on a separate filter paper square. The paper is dried with a hot air blower, placed flat on the bottom of a scintillation bottle containing 5 ml of Liquifluor, and the sample is counted. Column fractions containing the desired labeled component are pooled in a ceramic evaporating dish, which is placed in a large vacuum desiccator adjacent to two petri plates containing pellets of CaCl~ and NaOH, respectively; the desiccator is carefully evacuated with a vacuum pump. The next day the dry fractions are taken up in a small volume of water with the aid of Pasteur pipettes and stored frozen. Most intermediates, except keto derivatives, are relatively stable for years in the frozen state. The elution behavior of various intermediates, using this protocol, are summarized in the table2 Tris buffer should not be used in an incubation when the compound to be prepared would be eluted in the 1.0 N HC1 fraction. Properties
Biological Distribution. L-Glutamine:keto-scyllo-inositol aminotransferase has also been found in S. bikiniensis ATCC 11062, S. griseus ATCC 12475, and S. ornatus ATCC 23265. In streptomycin producing strains, L-glutamine:keto-scyllo-inositol aminotransferase can be distinguished from L-alanine: 1D-l-guanidino-l-deoxy-3-keto-scyUo-inositol aminotransferase 9 by separation of the two enzymes on a Sephadex G-100 column, as well as by differential heat inactivation of the latter enzyme2 Specificity. 1° From stability studies (see below), pyridoxal-P appears to be a required cofactor. L-Glutamine appears to be the physiological amino donor. Very high concentrations of L-alanine and L-glutamate have slight donor activity. Aminodeoxy-scyllo-inositol and 1D-4-amino-4deoxy-myo-inositol are excellent amino donors. Streptamine (1,3-diamino-l,3-dideoxy-scyllo-inositol) and 2-deoxystreptamine are also good amino donors; it is not known which one of their two amino groups undergoes transamination. Relatively inactive amino donors include: L-asparagine, L-aspartate, D-alanine, D-glutamine, L-isoglutamine, L-glutamic-7-hydroxamate, glycine, 2-amino-2-deoxy-neo-inositol, and 2-amino-2-deoxy-myo-inositol. Among the amino acceptors, 1D-4-keto-myo-inositol is an excellent substrate. This compound can be prepared as the racemic mixture [myo9This volume [27]. loj. B. Walker, Lloydia 34, 363 (1971).
[23]
KETO-scyllo-INOSlTOL AMINOTRANSFERASE
443
inosose-4(6)] by heating myo-inositol with fuming nitric acid in a platinum crucible. 5 Pyruvate and ~-ketoglutarate at high concentrations can also serve as amino acceptors. This latter property is of practical interest, since labeled keto-scyllo-inositol can be prepared from labeled aminodeoxy-scyllo-inositol by transamination with pyruvate, followed by successive passage of the reaction mixture through Dowex 50 (H +) and Dowex 1 (C1-) columns. Labeled keto-scgllo-inositol appears in the water washes from both columns. It should be noted that keto-inositols are very unstable in alkaline solutions. Other Assays. Several additional assay procedures can be envisioned. For example, either of the two coupled enzyme assays previously described ~ could be modified by adding an excess of myo-inositol 2-dehydrogenase (Sigma Cat. No. I 5503) in order to make the transamination step rate-limiting. Alternatively, keto-scyllo-inositol (Sigma Cat. No. I 5375) could be transaminated with uniformly labeled L-['~C]glutamine, and the labeled a-ketoglutaramate formed could be measured by its decarboxylation in the presence of eerie sulfate2 On occasion the aminodeoxy-scyllo-inositol formed in Eq. (1) has been measured in a multistep assay by its conversion to 1-[~4C]guanidino-l-deoxy-scgllo-inositot 4-phosphate. Stability. After mycelial extracts of S. bikiniensis have been dialyzed overnight at 4 ° in the absence of pyridoxal-P, the aminotransferase activity of Eq. (1) cannot be detected, even when pyridoxal-P is included in the incubation mixture. Aminotransferase activity can be retained if pyridoxal-P is present during dialysis." Presence of pyruvate during dialysis spares much of the pyridoxal-P requirement. Loss of activity during dialysis is not prevented by the presence of pyridoxamine-P, pyridoxal, or L-alanine. Presumably these results reflect a need to keep this cofactor in the aldehyde form for optimal binding through Schiff base formation with a lysine residue at the active site. Furthermore the apoforms of certain pyridoxal-P dependent enzymes have recently been shown to be susceptible to degradation by specific proteolytic enzymes in other organisms, and this might be applicable in Streptomyces, especially since these bacteria have very active proteolytic enzymes. S. bikiniensis glutamine: keto-scyllo-inositol aminotransferase is relatively resistant to heating at 55 ° for 5 rain, in contrast to L-alanine: 1D1-guanidino-l-deoxy-3-keto-scyllo-inositol aminotransferase," which is inactivated by this treatment2 °
444
ANTIBIOTIC BIOSYNTHESIS
[24]
[24] A T P : I n o s a m i n c P h o s p h o t r a n s f e r a s c ( s )
By
1
JAMES B . WALKER
OH
OH
+ MgATP
~ MgADP +
H
4
i
(i)
z Osp OH
OH
Aminodeoxyscyllo- inositol ( s cyllo- inosamine)
'
7~+ NH~
NH,+
I C:NH +
NH~
I__,
+
(2) HO ~
H OH
OPO~-
1 D- 1-Guanidino- 3-amino1, 3- dideoxy-s cyllo - inositol
(N-amidinostreptamine) OH
OH
+ HO
2-
~
MgATP
~ MgADP
+
6
s
(3)
2-OsP
Deoxystreptamine
Equation (1) is a reaction involved in biosynthesis of both the bluensidine moiety of bluensomycin and the streptidine moiety of streptomycinY-4 Equation (2) is a reaction involved in biosynthesis of the streptidine moiety of streptomycin 5 (for biosynthetic pathway see an earlier 1EC 2.7.1.65. ~J. B. Walker and M. S. Walker, Biochemistry 6, 3821 (1967). 3j. B. Walker, L l o y d i a 34, 363 (1971), 4j. B. Walker, J. Biol. C h e m . 249, 2397 (1974). 5j. B. Walker and M. S. Walker, Biochem. Biophys. Res. C o m m u n . 26, 278 (1967).
[24]
ATP :INOSAMINE PHOSPHOTRANSFERASE(S)
445
articleG). In Eq. (3) 2-deoxystreptamine or streptamine serves as an analog of a physiological phosphate acceptor. 2 Of all the enzymatic activities known to be involved in the biosynthesis or enzymic modification of the streptomycin family of antibiotics, the activities of Eqs. (1) through (3) are the most difficult to demonstrate consistently in cell-free extracts. Optimal conditions for growing and extracting mycelia to give reproducible, active inosamine kinase preparations have not yet been established.
Assay Methods Method I E
Aminodeoxy-scyllo-[~4C]inositol + MgATP ---> 1-amino-l-deoxy-scyllo-[14C]inosit.ol-4-P + MgA1)P (4) J
lD-l-[~4C]Guanidino-3-amino-l,3-dideoxy-scyllo-inositol + MgATP --~ 11)-1-[14C]guanidino-3-amino-1,3-dideoxy-scyllo-inositol-6-P + MgAI)P (5) Principle. Labeled phosphorylated product is separated from labeled substrate by paper chromatography or high-voltage paper electrophoresis and counted. L-Ornithine is included in the incubation mixture to inhibit subsequent transamidination of the phosphorylated inosamine product. Reagents Extract of Streptomyces bikiniensis ATCC 11062 1-Amino-l-deoxy-scyllo- [ 1-1'~C]inositol, 3.5 Ci/mole, from California Bionuclear Corp., Sun Valley, Calif. (scyllo-inosamine[1-14C], Cat. No. 72673). Labeled compound can also be prepared as described in an earlier article. 7 1D-l- [ 14C] Guanidino-3-amino-l,3-dideoxy-scyllo-inositol (N-amidinostreptamine), prepared as described ~ Tris C1, 0.5 M, containing 40 mM MgCl~, pH 7.4 L-Ornithine, 80 mM 2-Mercaptoethanol, 0.3 M ATP, 36 mM, pH 6.8-7.0
Procedure. The complete incubation mixture contains: either aminodeoxy-scyllo- [ 1~C] inositol or 1D- 1- [1~C] guanidino-3-amino- 1,3-dideoxyThis volume [21]. 7This volume [23]. 8This volume [27].
446
ANTIBIOTIC BIOSYNTHESIS
[24]
scyllo-inositol, 5 td; Tris-Mg buffer, 5/zl; ornithine, 5 ~1; mercaptoethanol, 1 tL1; ATP, 5/zl; and dialyzed extract of S. bikiniensis, 10 t~l. After incubation in a stoppered 13 X 100 mm test tube at 35 °, 10 ~l are spotted (a) at the 22-cm segment and the components separated by high-voltage paper electrophoresis at pH 3.6, 9 or (b) on a paper chromatogram and developed with ammoniacal phenol (see below). Mobilities are given in the table of an earlier article2 Growth of Mycelia. The procedures for maintaining slants and growing and harvesting mycelia of S. bikiniensis are similar to those described for S. glebosus2 However, most work involving S. bihiniensis has utilized mycelia grown for 2-3 days on a medium of 2% peptone-0.2% yeast extract-tap water. Preparation of Extracts. Extracts of S. bikiniensis, as well as other streptomycin-producing strains, can be prepared by sonication, as described2 However, another extraction procedure, which requires no special equipment and can be readily scaled up to process large quantities of mycelia, can be employed for extraction of many enzymes, including aminotransferases, kinases, amidinotransferase, and phosphatases. This latter method utilizes hydrolysis of a portion of the peptidoglycan cell wall with lysozyme plus EDTA. Most strains of Streptomyces form a DNA gel which prevents separation by centrifugation during extraction with lysozyme; this gel can be dissolved by adding a small amount of deoxyribonuclease and Mg 2÷. For some reason lysozyme extracts of S. bikiniensis do not form this gel. In this procedure, frozen 2- to 3-day mycelial pads of S. bikiniesis are shaved with scissors into a beaker and extracted at room temperature for 1 hr with 3 volumes of 100 mM potassium phosphate containing 13 mM EDTA and 1 mg/ml crystalline egg-white lysozyme, pH 7.4. The mixture, immersed in a water bath, is stirred either continuously or intermittently during cell-wall digestion. The resulting suspension is centrifuged for 30 min at 30,000 g and 2 °. Except for the case of aminotransferases, 7,8 the supernatant solution is dialyzed overnight at 4 ° against 4 liters of 3 mM Tris C1, pH 7.4, to which 0.1 ml of mercaptoethanol has been added. The dialysis tubing had previously been treated with EDTA and stored in deionized water at 4 ° . The dialyzed solution is stored in the freezer, where it is stable for several months. Paper Chromatography. The RI values listed in the table of an earlier article 6 were obtained by ascending paper chromatography with Whatman No. 1 filter paper, developed with a solvent of 80% phenol-20% H_~O, NH40H atmosphere. Paper is cut in sheets 23 cm wide and 27 cm long, This volume [22].
[24]
ATP :INOSAMINE PHOSPHOTRANSFERASE(S)
447
and ruled in pencil as follows: (a) vertically, 5 lines, 5 cm apart, with margins of 1.5 cm at each side; (b) horizontally, 25 lines, 1 cm in width, starting 2 cm from the bottom. Samples are spotted with a 5-td micropipette, which is rinsed 3 times with water between samples, in the center of each of the bottom 1-cm segments. Samples are dried with a portable hair drier, and additional aliquots are applied as desired in 5-td increments. The paper is rolled into a cylinder, edges not touching, and stapled at two places, 7 cm from each end. Separation is performed in a cylindrical glass chromatography jar, 6 inches X 12 inches (e.g., Corning 431428) with ground top lightly greased and covered with a weighted round glass plate during a separation. To conserve developing solvent, an inverted shallow petri dish cover is placed on the bottom of the jar, and a petri dish cover (100 mm }( 20 mm) containing 80% phenol-20% H20 to a depth of less than 1 cm is placed on top of the inverted dish. After 0.8 ml of concentrated NH40H is added by pipette along the inside wall of the jar, the bottom end of the paper cylinder is placed in the phenol, and the cover is placed on top of the jar. The solvent usually reaches the top of the paper within 16 hr at room temperature. The paper is then removed from the jar, and its lower end is placed on paper toweling for 10 rain. The paper is then clamped, lower end up, with a stainless steel clamp to a horizontal glass rod suppogted by a ring stand, and dried in a hood. Drying takes several hours at room temperature, or less if portable hair dryers are used to blow heated air on the suspended sheet. The horizontal 1-cm segments along each path are cut into 3 pieces, placed flat on the bottom of a bottle containing 5 ml of Liquifluor scintillation fluid in toluene, and counted with a liquid scintillation system.
Method II Inosamine -~- MgATP E MgADP + inosamine-P (6) F Inosamine-P + L-[guanidino-l~C]arginine--~ L-ornithine + N-[14C]amidinoinosamine-P (7)
Principle. This assay involves coupling of ATP:inosamine phosphotransferase with L-arginine:inosamine-P amidinotransferase. TM The inosamine substrate of Eqs. (1), (2), or (3) is not labeled; label is introduced into the final product from commercially available L-[guanidino14C] arginine. Labeled product is separated by paper chromatography and counted. Some dephosphorylation of the amidinated product can occur if phosphatases are present. 10This volume [25].
448
ANTIBIOTIC BIOSYNTHESIS
[24]
Reagents Dialyzed extract of S. bik~iniensis ATCC 11062 (see Method I) Tris C1, 0.5 M, containing 40 mM MgCI~, pH 7.4 ATP, 36 mM, pH 6.8-7.0 2-Mercaptoethanol, 0.3 M L- [Guanidino-~4C] arginine, 10-40 Ci/mole, 33 t~Ci/ml Inosamine derivative, 5 mM (aminodeoxy-scyllo-inositol, monoamidinated streptamine, streptamine, or 2-deoxystreptamine)
Procedure. The complete incubation mixture contains: inosamine derivative, 5 t~l; [guanidino-~4C]arginine, 5 ~l; Tris-Mg, 5 ~l; mercaptoethanol, 1 ~l; ATP, 5 ~l; and dialyzed extract of S. bikiniensis, 10 ~l. After incubation in a stoppered 13 X 100 mm test tube at 35 °, 10-~l aliquots are spotted on a paper chromatogram, and the components are separated with ammoniacal phenol (Method I) and subsequently counted. Mobilities of the expected products ~ are given in Table I of an earlier article2 For purification purposes, an excess of purified inosamine-P amidinotransferaseTM would have to be added if this assay is employed. Preparation o] Aminodeoxy-scyllo-inositol. Any one of several procedures can be employed. For example, aminodeoxy-scyllo-inositol can be prepared from bluensomycin by mild acid hydrolysis to give bluensidine (1D-l-O-carbamoyl-3-deoxy-3-guanidino-scyllo-inosi~ol). Alkaline hydrolysis of bluensidine gives aminodeoxy-scyllo-inositol. 11 Alternatively, the hydrolysis could be performed in reverse order. Alkaline hydrolysis of bluensomycin gives deamidinodecarbamoylbluensomycin, which would give aminodeoxy-scyllo-inositol on subsequent acid hydrolysis. If bluensomycin is not available, aminodeoxy-scyllo-inositol can be chemically synthesized either from myo-inositol by way of an azido de- , rivative or from keto-scyllo-inositol (myo-inosose-2, Sigma Cat. No. I 5375) by reduction of its oxime with sodium amalgam22 Preparation o] Streptamine and Streptidine ]rom Dihydrostreptomyc/n. Mild acid hydrolysis of dihydrostreptomycin gives streptidine 13,~4 (1,3-diguanidino-l,3-dideoxy-scyllo-inositol), which can be converted to streptamine (1,3-diamino-l,3-dideoxy-scyUo-inositol) by alkaline by1~B. Bannister and A. D. Argoudelis,J. Amer. Chem. Soc. 85, .119 (1963). 12L. Anderson and H. A. Lardy, J. Amer. Chem. Soc. 72, 3141 (1950). ~' R. L. Peck, R. P. Graber, A. Walti, E. W. Peel, C. E. Hoffhine,Jr., and K. Folkers, J. Amer. Chem. Soc. 68, 29 (1946). ~ M. S. Walker and J. B. Walker, J. Biol. Chem. 241, 1262 (1966}.
[24]
A T P : INOSAMINE PHOSPHOTRANSFERASE(S)
449
drolysis. 1~,15 A covered beaker containing 107 g of dihydrostreptomycin sulfate dissolved in 360 ml of 1 N H~S04 is placed in a 37 ° water bath. The reaction mixture is incubated for 2 days, and the crystals of streptidine sulfate are collected by suction filtration with a sintered glass filter, washed twice with a small amount of cold water, washed with acetone, and dried. Yield: 47 g of crude streptidine sulfate. Several successive recrystallizations are perfo¢med by dissolving, e.g., 108 g of crude strew tidine sulfate in 1600 nfl of boiling water slightly acidified with H2S04, placing the solution in the refrigerator overnight, and collecting, washing, and drying the crystals as before. Streptamine sulfate is prepared by refluxing for 24-48 hr a mixture of 25 g of streptidine sulfate and 1250 ml of a saturated aqueous solution of barium hydroxide. Then 1 N H..SO~ is slowly added with stirring to the hot mixture until the pH stabilizes at 5-6 (about 415 ml required). The mixture is filtered while hot, and the filtrate is cooled. To the chilled filtrate 0.4 volume of acetone is slowly added, and the suspension is filtered under suction. The precipitate is washed three times with a total of 30 ml of cold water, washed with acetone, and dried. Yield: 11.5 g of streptamine sulfate. Preparation o] Monoamidinated Streptamines. To a covered beaker is added 8.1 g of streptamine sulfate and 5.2 g of barium hydroxide in 125 ml of hot water. The mixture is digested on a steam bath for 5 min, filtered by suction, and the barimn sulfate precipitate is washed with hot water. The filtrate plus washings are evaporated by boiling to a volume of 25-50 ml, and the beaker is placed in a hood. Powdered S-methylisothiouronium sulfate is added, with stirring, in portions over several days; 2 g are added initially, with a total of 2 g more added over several days. After 6 days the mixture is neutralized with 2 N H..,SO~. Fractional precipitation with increasing amounts of acetone give several precipitates, which are then assayed for guanidino compounds by the Sakaguchi reaction. A late-precipitating acetone fraction usually contains monoamidihated streptamines relatively free from streptamine and streptidinc. Preparation o] 2-Deoxystreptamine ]rom Kanamycin. ~,~6 To 5 g of kanamycin sulfate (Schwarz/Mann) in 10 ml of hot water is slowly added, with stirring, 2.2 g of barium hydroxide. After 15 min, the mixture is centrifuged. A saturated aqueous solution of barium hydroxide is slowly added, with stirring, to the supernatant solution plus washing until no more barium sulfate precipitate is formed. After centrifugation, the 1~R. L. Peck, C. E. Hoffhine, Jr., E. W. Peel, R. P. Graber, F. W. Holly, R. Mozingo, and K. Folkers, J. Amer. Chem. Soc. 68, 776 (1946). 1~M. J. Cron, D. L. Johnson, F. M. Palermiti, Y. Perron, H. D. Taylor, D. F. Whitehead, and I. R. Hooper, J. Amer. Chem. Soc. 80, 752 (1958).
450
ANTIBIOTIC BIOSYNTHESIS
[24]
supernatant solution is refiuxed for 75 min with an equal volume (ca. 25 ml) of concentrated HC1. The hydrolyzate is decolori~ed with acidwashed charcoal and filtered; the filtrate is concentrated in a vacuum desiccator over NaOH pellets. Ethanol is added slowly to the concentrated solution until turbidity appears. On chilling, an oil is formed which is converted to a :precipitate by the addition of methanol. The precipitate is washed twice with cold 75% methanol, then acetone, and dried. Yield: 560 mg of deoxystreptamine dihydrochloride.
Properties
Purification. Inosamine kinase activities, concentrated by treatment of an extract with Mn 2÷ and precipitation with (NH4)2S0~, can be separated from most other enzymes involved in streptidine biosynthesis by (a) Sephadex G-100 column chromatography, 17 (b) batch treatment with DEAE-cellulose, is or (c) DEAE-cellulose column chromatography, as described. 1° A mercaptan should be present at all stages of purification. Biological Distribution. Inosamine kinase activities are believed to occur in all strains of Streptomyces which synthesize the streptomycin family of antibiotics, s° However, as stated earlier, optimal conditions of growth and enzyme extraction to obtain consistently active preparations have not yet been determined. Specificity. There are two separate specificity problems. One concerns whether more than one enzyme is responsible for catalyzing Eqs. (1), (2), and (3) ; if more than one enzyme is involved, each enzyme presumably has its own characteristic substrate specificity. At present it can only be stated that streptamine is also an excellent phosphate acceptor. 2 Guanidinodeoxy-scyllo-inositol and streptidine ~8 are not phosphate acceptors, nor is the enantiomer of the acceptor of Eq. (2). At higher concentrations it appears that a number of isomeric inosamines can accept a phosphate group and subsequently be transamidinated when crude extracts are the source of enzymes. These findings must, however, be checked with more purified enzyme preparations, because of the probable presence of epimerases in crude extracts. In crude extracts ATP and dATP can serve as phosphate donors, whereas CTP, UTP, dTTP, and G T P are inactive. 2,~ Either Mg 2÷ or Mn 2÷ is required as cofactor. Assignment of the phosphate group para to the amino group which is subsequently to be transamidinated is not based on direct evidence. The possibility that the phosphate group is initially esterified at another position 1~A. L. Miller and J. B. Walker, J. Bacteriol. 99, 401 (1969). 18j. B. Walker and M. S. Walker, Biochim. Biophys. Acta 148, 335 (1967).
[25]
L-ARGININE :INOSAMINE-P AMIDINOTRANSFERASE(S)
451
and transferred to the para position by a phosphomutase has not been ruled out, but is considered unlikely?" Alternate Assay. With purified enzyme preparations, a radiochemical assay can be developed which utilizes [V-32P]ATP as the phosphate donor. Small columns containing Dowex 1 (C1-) resin can be used; labeled product is not adsorbed on such columns, whereas labeled substrate and inorganic phosphate are adsorbed. This rapid assay is also useful for determining acceptor specificity, since candidate substrates are not usually available in labeled form. Inhibition. Sulfhydryl reagents such as p-chloromercuribenzoate and formamidine disulfide inhibit these kinase activities. Purified enzyme preparations are stabilized by the addition of serum albumin and mercaptans. 1~M. S. Walker and J. B. Walker, J. Biol. Chem. 246, 7034 (1971).
[2 5] L - A r g i n i n e : I n o s a m i n e - P A m i d i n o t r a n s f e r a s e (s)
By JAMES B. WALKER H
L -
NH~ F, K _
Arginine +
L-Ornithine +
2-OsP OH R = (a) - - O H ;
(c)
- 0 - C(=
--- ~3
(1) OH
(d) --
(b) --NH-- C(=NH+)NI-I, ;
60~ R z-Osp
NH~
N H 2 ; and their corresponding 2-deoxy derivatives
O)NIi~;
L-Canavanine -[- L-ornithine ~- L-canaline + L-arginine
(2)
L-Arginine + N H ~ O H - * L-ornithine -k H 2 N - - C ( = N H 2 + ) N H - - O H
(3)
Prior to the discovery of this enzyme in extracts of mature mycelia of certain Streptomyces, 2,3 the only known amidinotransferases were those involved in the biosynthesis of creatine and certain other phosphagens in higher animals. 4,5 Rapid and convenient colorimetric assays have been developed for amidinotransferases which are based on reactions re1See also this series, Vol. 17A [146]. 2 j. B. Walker, J. Biol. Chem. 231, 1 (1958). s M. S. Walker and J. B. Walker, J. Biol. Chem. 241, 1262 (1966). 4 j. B. Walker, in "The Enzymes" 3rd ed. (P. Boyer, ed.), Vol. 9, p. 497. Academic Press, New York, 1973. E. Grazi and F. Conconi, this series, Vol. 17A [145].
[25]
L-ARGININE :INOSAMINE-P AMIDINOTRANSFERASE(S)
451
and transferred to the para position by a phosphomutase has not been ruled out, but is considered unlikely?" Alternate Assay. With purified enzyme preparations, a radiochemical assay can be developed which utilizes [V-32P]ATP as the phosphate donor. Small columns containing Dowex 1 (C1-) resin can be used; labeled product is not adsorbed on such columns, whereas labeled substrate and inorganic phosphate are adsorbed. This rapid assay is also useful for determining acceptor specificity, since candidate substrates are not usually available in labeled form. Inhibition. Sulfhydryl reagents such as p-chloromercuribenzoate and formamidine disulfide inhibit these kinase activities. Purified enzyme preparations are stabilized by the addition of serum albumin and mercaptans. 1~M. S. Walker and J. B. Walker, J. Biol. Chem. 246, 7034 (1971).
[2 5] L - A r g i n i n e : I n o s a m i n e - P A m i d i n o t r a n s f e r a s e (s)
By JAMES B. WALKER H
L -
NH~ F, K _
Arginine +
L-Ornithine +
2-OsP OH R = (a) - - O H ;
(c)
- 0 - C(=
--- ~3
(1) OH
(d) --
(b) --NH-- C(=NH+)NI-I, ;
60~ R z-Osp
NH~
N H 2 ; and their corresponding 2-deoxy derivatives
O)NIi~;
L-Canavanine -[- L-ornithine ~- L-canaline + L-arginine
(2)
L-Arginine + N H ~ O H - * L-ornithine -k H 2 N - - C ( = N H 2 + ) N H - - O H
(3)
Prior to the discovery of this enzyme in extracts of mature mycelia of certain Streptomyces, 2,3 the only known amidinotransferases were those involved in the biosynthesis of creatine and certain other phosphagens in higher animals. 4,5 Rapid and convenient colorimetric assays have been developed for amidinotransferases which are based on reactions re1See also this series, Vol. 17A [146]. 2 j. B. Walker, J. Biol. Chem. 231, 1 (1958). s M. S. Walker and J. B. Walker, J. Biol. Chem. 241, 1262 (1966). 4 j. B. Walker, in "The Enzymes" 3rd ed. (P. Boyer, ed.), Vol. 9, p. 497. Academic Press, New York, 1973. E. Grazi and F. Conconi, this series, Vol. 17A [145].
452
ANTIBIOTIC BIOSYNTHESIS
[25]
lated to the arginine:ornithine exchange reaction 6 catalyzed by these enzymes. 2 Either one of the assays indicated in Eqs. (2) 2'7 and (3) 2,8 can be utilized (a) for detecting strains of Streptomyces capable of synthesizing the streptomycin family of antibiotics, and (b) for studying the mechanisms of controls which govern differentiation of these strains to the antibiotic-synthesizing state after a phase of rapid vegetative growth. In this article assays will be described based on each of the above equations. It is not yet known whether one enzyme9 or two different enzymes1° catalyze reactions F and K in the reaction scheme shown in an earlier article2 ~
Assay Methods Method 18,9 Principle. This assay is based on Eq. (1). L-[Guanidino-l*C]arginine donates its labeled amidino group to a natural or synthetic inosamine-P derivative, and the labeled product is separated by paper chromatography and counted. 1~ This method, unlike the other methods to be described, is specific for inosamine-P amidinotransferase. Reagents Dialyzed extract of mature mycelia of Streptomyces bikiniensis ATCC 11062 or other strain of Streptomyces which synthesizes one of the streptomycin family of antibiotics L- [Guanidino-14C] arginine, 12-30 Ci/mole Tris C1,0.5 M, containing 10 mM EDTA, pH 7.4 2-Mercaptoethanol, 0.3 M Solution containing chemically phosphorylated inosamine derivative or hot water extract of mature mycelia of a producing strain grown in presence of 0.5 % myo-inositol
Procedure. The complete incubation mixture contains: labeled arginine (33 gCi/ml), 5 gl; Tris-EDTA, 5 t,1; mercaptoethanol, 1 t*l; phosphorylated inosamine derivative, 10 gl; and dialyzed extract of S. bikiniensis or S. glebosus ATCC 14607, 10 ~1. After incubation in a stoppered ej. B. Walker, J. Biol. Chem. 221, 771 (1956). J. B. Walker, Biochim. Biophys. Acta 73, 241 (1963). s j. B. Walker, d. Biol. Chem. '235, 2357 (1960). ~J. B. Walker, J. Biol. Chem. 249, 2397 (1974). lo L. C. Pla, Biochim. Biophys. Acta 242, 541 (1971). 11This volume [21]. ~2This volume [24].
[25l
L-ARGINI NE :INOSAMINE-P AMIDINOTRANSFERASE (S)
453
13 X 100 mm test tube at 35 °, a 10-t,1 aliquot is spotted on a paper chromatogram, and the components are separated with ammoniacal phenol and counted. 12 Mobilities of expected products are given in Table I of an earlier article. 11 Excessive amounts of inorganic phosphate markedly lower the Rr values. The presence of EDTA inhibits action of phospharases on the reactants, and in the case of chemically phosphorylated acceptors chelates inhibitory Ba 2÷. Streptomyces Extracts. Mycelia are grown and harvested as described. 13 Dialyzed extracts can be prepared from sonicates TM or from lysozyme extracts22 Nondialyzed supernatant solut!ons from sonicates can be utilized if desired as the source of both amidinotransferase and phosphorylated acceptors2 In such cases the addition of carbamoyl-P to the incubation mixture often markedly improves the yield of labeled product. 9 Preparation o] Natural Acceptors. Mycelia of a strain capable of synthesizing streptomycin or bluensomycin are grown (with 0.5 to 1.0% myo-inositol included in the growth medium), harvested, and stored as described. 1~ Freshly harvested, or frozen, mycelia are added to an equal weight of hot water contained in a centrifuge tube immersed in a boiling water bath. The mixture is stirred for 7 to 10 rain with a glass stirring rod, then cooled and centrifuged. The supernatant solution is stored in the freezer. The hot-water extracts with the highest levels of acceptors are determined by assay with an active dialyzed amidinotransferase preparation (Method I).
Preparation of Chemically Phosphorylated Inosamine Derivatives. 3 The nonspecific procedure of Plimmer and Burch TM is employed to produce mixtures of phosphorylated isomers of a given inosamine derivative. The crude reaction mixtures after removal of most phosphate as the barium salt, are used as sources of amidino acceptors, without further purification. Amidinotransferase is believed to react with only one phosphorylated isomer in each case. Inosamine derivatives which can be phosphorylatcd to give amidino acceptors include aminodeoxy-scyllo-inosamine, streptamine, 2-deoxystreptamine, 2-amino-2-deoxy-neo-inositol, streptidine, and 2-deoxystreptidine. Commercially available antibiotics can also be directly utilized. For example, phosphorylation of dihydrostreptomycin gives a mixture which includes the correct streptidine-P isomer, and phosphorylation of kanamycin gives a mixture which includes the correct 2-deoxystreptamine isomer; in both instances hydrolysis of glycosidic bonds accompanies phosphorylation. Amidinotransferase plus a-[guani13This volume [22]. 1~R. H. A. Plimmer and W. J. N. Burch, Biochem.J. 31,398 (1937).
454
ANTIBIOTIC BIOSYNTHESIS
[25]
dino-14C]arginine react with the correct isomer in a mixture, and product labeled in the guanidino group can then be separated on a Dowex-50 (H ÷) column 15 (see Table I of an earlier article11). In a typical preparations, 2-5 g of the inosamine derivative are added to a 50-ml round-bottom flask containing 10 ml of concentrated H3P04. Then 5 g of powdered P205 are quickly added, with minimal exposure to the atmosphere, and a loosely stoppered reflux condenser is immediately joined to the flask. The mixture is heated on a steam bath for 6 hr and left at room temperature overnight. A hot saturated solution of Ba (OH)2 is then slowly added to the mixture, with thorough stirring, to give a slightly alkaline pH. The completely neutralized mixture is filtered, and the precipitate is washed several times with hot water. The original filtrate and each successive wash are separately frozen for subsequent assay for amidino acceptor activity. NazS04 is added to filtrates, if necessary, to precipitate any Ba 2+ remaining in solution. These preparations should be assayed in the presence of EDTA.
Method IP Principle. This assay is based on Eq. (2). Canavanine is a naturally occurring analog of arginine, so this reaction is analogous to the arginine: ornithine exchange transamidination catalyzed by both animal glycine amidinotransferase 6 and bacterial inosamine-P amidinotransferase. ~ The arginine formed is assayed by any of several modifications of the Sakaguchi reaction. If desired, canaline can be removed as it is formed by formation of a ketoxime with acetone to make this readily reversible reaction unidirectional/ It should be noted that streptomycin, bluensomycin, and their guanidinated precursors also react with the Sakaguchi reagent, so dialyzed extracts or purified enzyme preparations must be used. Reagents Dialyzed extract of mature mycelia of S. bikiniensis ATCC 11062 Potassium phosphate, 1 M, pH 7.5 L-Canavanine sulfate, adjusted to pH 7.5 with KOH, 0.25 M L-Ornithine.HC1, 0.1 M Acetone, 20% (v/v) in water Trichloroacetic acid, 30% (w/v) in water Sodium hydroxide, 10% (w/v) in water ~-Naphthol, 1 mg/ml, in 95% ethanol ~ This volume [23].
[25]
L-ARGININE :INOSAMINE-P AMIDINOTRANSFERASE(S)
455
Urea, 20% (w/v) in water Clorox, 50% (v/v) in water
Procedure. The complete incubation mixture contains, in a final volume of 1.0 ml: canavanine, 0.2 ml; phosphate, 0.1 ml; ornithine, 0.2 ml; acetone solution, 0.1 ml; and dialyzed extract, After incubation in a stoppered 13 X 100 mm test tube at 35% the mixture is treated with 0.3 ml of trichloroacetie acid solution. After 10 rain the mixture is centrifuged, and an aliquot of the supernatant solution is added to a 20 X 150 mm test tube and made up to 5.0 ml with water; 1 ml of NaOH solution and 1 ml of a-naphthol solution are added and mixed thoroughly. After 5 min, the Clorox solution is added, and the tube is shaken immediately after addition; 60 sec later 2 ml of urea solution are added with shaking. Absorbancy at 540 nm is measured against a control consisting of the complete mixture minus ornithine. Method l i p Principle. This assay, the standard method used in our laboratory, is based on Eq. (3) and serves equally well as an assay for animal glycine amidinotransferase,s Hydroxylamine reacts with the enzyme-amidine ("active urea") intermediate 2,4 to give hydroxyguanidine, which is measured as its colored complex with pentacyanoaminoferrate. Crude extracts can be assayed provided that arginase activity is low, since ornithine strongly inhibits hydroxyguanidine formationJ Hydroxylamine reacts with esters and certain other acyl derivatives often present in crude extracts to form hydroxamates, which give a similar color with pentacyanoaminoferrate. Excessive amounts of EDTA or mercaptans can interfere with the color development. Reagents Extract of Streptomyces bikiniensis ATCC 1106212 L-Arginine.HC1, 1 M L-Ornithine.HC1, 0.6 M NH.,OH • HC1, neutralized with cold 2 M KOH, 2 M, stored frozen Potassium phosphate buffers, 1 M, pH 7.4 and pH 7.0, respectively Acetone Na3[Fe(CN)~NH~], 1% in water, aged at least 1 day (Fisher Cat. No. S-659) Hydroxyguanidine hemisulfate • H.20, 300 t~g/ml (Eastman Cat. No. 9241). Synthesis of this compound was described previously in this series. 1 Trichloroacetic acid, 30% (w/v) in water
456
ANTIBIOTIC BIOSYNTHESIS
[25]
Procedure. The complete incubation mixture contains, in a final volume of 1.0 ml: arginine, 0.1 ml; NH~OH, 0.3 ml; phosphate buffer, pH 7.4, 0.1 ml; and enzyme solution. For a blank, 0.1 ml of ornithine is substituted for arginine. For a standard, 0.5 ml of hydroxyguanidine is substituted for the enzyme solution. After incubation in a stoppered 13 X 100 mm test tube at 37 °, the reaction is stopped with 0.4 ml of trichloroacetic acid, and 1.0 ml of water is added. After 10 min the mixture is centrifuged in the same test tube, and a 1.5-ml aliquot of the supernatant solution is pipetted into another 13 X 100 mm test tube, followed by addition of 0.5 ml of water, 2.0 ml phosphate buffer, pH 7.0, 0.3 ml acetone, and 0.3 ml pentacyanoaminoferrate. The mixture is rapidly stirred, and after 10 min the absorbance is read at 480 nm. The color does not form if excess NH20H is not removed as the ketoxime of acetone. Definition of Unit and Specific Activity. A unit of amidinotransferase is defined as the amount that catalyzes the formation of 1 t~mole of hydroxyguanidine per hour in the assay described in Method III. Specific activity (units/mg of protein) is based on measurement of protein by the Lowry method? 6 Purification Procedure Growth and Extraction of Mycelia. Mycelia of S. bikiniensis ATCC 11062 are grown at 24-28 ° for 2 days on a medium of 2% peptone-0.2% yeast extrac~tap H20 and harvested as described. 1~ Frozen mycelial pads are extracted with lysozyme-EDTA as described. 12 The maximal amount of MnCl~ 'solution which can be employed without precipitating amidinotransferase is determined with an aliquot of the supernatant solution. Treatment with Mn 2÷ and (NH4)2SO~. To 70 ml of the supernatant solution is added, dropwise with stirring, 7 ml of 10% MnCl~ • 4H~O in 0.1 M phosphate buffer, pH 7.4. After 20 min the mixture is centrifuged. To 70 ml of the supernatant solution is added 70 mg of L-arginine.HCl and 7 tzl of mercaptoethanol; 17 g of powdered (NH~)2SO4 is next added in small portions, with stirring, over a 35-min period, with the pH maintained near 7.0 by cautious addition of dilute NH4OH. After 20 min the precipitate is removed by centrifugation. To 76 ml of the supernatant solution is added 15.5 g of powdered (NH4)2SO~, following the same procedure, but this time saving the precipitate. The precipitate is dissolved in 3 ml of 0.1 M phosphate buffer, pH 7.4, containing EDTA, 5 mg/ml, and dialyzed overnight against 4 liters of 1 mM phosphate ~60. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
[25]
L-ARGI NINE :INOSAMINE-P AMIDINOTRANSFERASE (S)
457
PURIFICATION OF INOSAMINE-P AMIDINOTRANSFERASF
Step
Total activity (units)
Total protein (rag)
Lysozyme extract Mil s+ s u p e r n a t a n t solution (NH4)~SO4 fraction, 4 0 - 7 0 % DEAE-cellulose, 0.3 M NaC1 eluate
672 490 367 280
840 350 204 28
Specific activity Recovery (units/mg) (%) 0.8 1.4 1.8 10.0
100 73 55 42
buffer, pH 7.4, containing 200 mg of EDTA and 0.1 ml of mercaptoetltanol. DEAE-CelIulose Chomatography. DEAE-eellulose (medium mesh, 92 mEq/g) is washed twice with 1 N NaOH and then water. The adsorbent is treated with 1 N HC1, stirred under suction until bubbles stop evolving, and washed with water. Fines are decanted at each step. A 58 X 2-cm column is packed and equilibrated with 10 mM potassium phosphate buffer, pH 7.0, containing EDTA, 0.5 mg/ml, and mercaptoethanol, 0.1 ~l/ml. The dialyzed (NH4)2SO~ fraction, 5.5 ml, containing 204 mg protein, is added to the column, followed by 70 ml of equilibration buffer; 6-ml fractions are collected. The protein is eluted with one column volume of 0.3 M NaC1 in the same buffer. Amidinotransferase is eluted with 0.3 M NaC1, separating it from ATP:inosamine phosphotransferasp, 12 which is eluted with 0.2 M NaC1, and from ATP:streptomycin 6-phosphotransferase, ~7 which is eluted with 0.6 M NaC1 in buffer. The purification procedure is summarized in the table.
Properties Biological Distribution. Amidinotransferase specific activity increases 50-fold after the phase of rapid growth in strains of Streptomyces which produce the streptomycin family of antibiotics, 18 including S. grise'us strains ATCC 12475, 10137, 11429, 11984, and 27001; S. humidus ATCC 12760; S. griseocarneus ATCC 12628; S. galbus ATCC 14077; S. ornatus ATCC 23265; and a bluensomycin-producing strain, S. hygroscopicus forma glebosus ATCC 14607. 9 Specificity and Alternative Assays. In addition to the specificities shown in Eqs. (1), (2), and (3), inosamine-P amidinotransferase can readily amidinate 2-amino-2-deoxy-neo-inositol-5-P, which surprisingly differs in configuration from the normal acceptors of Eq. (1) in positions ~TThis volume [49]. lsj. B. Walker and V. S. Hnilica, Biochim. Biophys. Acta 89, 473 (1964).
458
ANTIBIOTIC BIOSYNTHESIS
[25]
1, 2, 4, and 5. Evidently the relationship between positions 3 and 6 are most important for acceptor activity. Inosamine-P amidinotransferase, like glycine amidinotransferase, also amidinates the ornithine analogs glycylglycine and 1,4-diaminobutylphosphonic acid. 19 Inosamine derivatives must be phosphorylated to serve as substrates, and the phosphate must be esterified with the hydroxyl group para to the amino group to be transamidinated~°; derivatives phosphorylated in the ortho position are not active. 21 When L-[guanidino-~4C]arginine and amidinotransferase are incubated with either chemically phosphorylated streptidine, or with streptidine-P synthesized within mycelia following the addition of 1 mg/ml of streptidine sulfate to a mature culture of almost any strain of Streptomyces 3-10 hr prior to harvest, label is introduced by an exchange reaction into the 3-guanidino moiety of streptidine-6-P2 However, derivatives of streptidine-6-P in which position 4 is substituted with a bulky group, as in streptomycin-6-P, are not substrates for amidinotransferase. Since most amidino donors can couple with most amidino acceptors, a variety of additional assays can be employedA9 However, canavanine:NH20H transamidination is obscured by a nonenzymic reaction between these two compounds. When arginine is the donor, the ornithine formed can be measured by the method of Chinard. 5 Inhibitors. L-0rnithine is the most potent competitive inhibitor. Product inhibition by ornithine particularly interferes with kinetic studies, except in the case of Method II. If necessary, ornithine can be removed by enzymic conversion to citrulline in the presence of added carbamoyl-P and ornithine carbamoyltransferase. Strong inhibition occurs with the sulfhydryl reagents, formamidine disulfide. 2HC1 (freshly prepared aqueous solution, kept slightly acid at all times), cystine, cystamine, and p-chloromercuribenzoate,is
1,j. B. Walker, Lloydia 34, 363 (1971). =oM. S. Walker and J. B. Walker, J. Biol. Chem. 046, 7034 (1971). ~lj. B. Walker and M. Skorvaga,J. Biol. Chem. 248, 2441 (1973).
[26]
GUANIDINODEOXY-8eyIIo-INOSITOL-4-P PHOSPHOHYDROLASE
459
[26] 1-Guanidino-l-deoxy-scyllo-inositol-4-P Phosphohydrolase B y JAMES B. WALKER NH
OH
2
NH
~=NH +
2
L
H + H20
Mg2+ G ~ HPO~- +
2-OsPO ~
I~H
(1)
H OH
OH
This enzyme participates in the biosynthesis of streptomycin 1-3 but is apparently not required for biosynthesis of bluensomycin, although it is present in bluensomycin producers. 4 Transamidination products '~' substituted at position 5 with a guanidino group (streptidine-P) or an O-carbamoyl group (bluensidine-P) are not good substrates.
Assay Method Principle. 1- [ ~4C] Guanidino- 1-deoxy-s cgllo-inositol-4-P is incubated with an enzyme preparation, and the components of the reaction mixture are separated by paper chromatography. This assay can be used for both crude and purified preparations. Reagents Extract of mature mycelia of Streptomyces bikiniensis ATCC 11062, or other strain which synthesizes one of the streptomycin family of antibiotics
1- [~4C ] Guanidino-l-deoxy-scyllo-inositol-4-P Tris C1, 0.5 M, containing 40 m M MgCl~, pH 7.4 2-Mercaptoethanol, 0.3 M
Procedure. The complete incubation mixture contains: 1-[14C]guanidino-l-deoxy-scyllo-inositol-4-P, 10 t~l; Tris C1-Mg, 5 ~l; mercaptoethanol, 1 ~l; and enzyme preparation, 10 ~l. After incubation in a stoppered 13 }( 100 mm test tube at 35 °, a 10-t~l aliquot is spotted, and the compo1j. B. Walker and M. S. Walker, Biochem. Biophys. Res. Commun. 26, 278 (1967). 2 M. S. Walker and J. B. Walker, J. Biol. Chem. 246, 7034 (1971). 3j. B. Walker, Lloydia 34, 363 (1971). 4j. B. Walker, J. Biol. Chem. 249, 2397 (1974). This volume [251.
460
ANTIBIOTIC BIOSYNTHESIS
[25]
nents are separated by paper chromatography with ammoniacal phenol and counted2 For mobilities, see the table of a previous articleY Preparation o] 1-[14C]Guanidino-1-deoxy-scyllo-inositol-4-P. The simplest procedure is to grow mycelia of S. bikiniensis ATCC 11062, S. griseus ATCC 12475, or other streptomycin producing strain as described 6 with 0.5% myo-inositol added to the growth medium. (Do not use S. glebosus ATCC 14607.) Frozen mycelial pads are sonicated as described, 8 and 10-~l aliquots of the nondialyzcd supernatant solutions from various pads are incubated with L-[guanidino-~4C]arginine as described with a dialyzed extract in Method I of a previous article2 However, here the supernatant solutions are utilized as a source of both inosamine-P amidinotransferase and amidino acceptors. Generally two peaks of radioactive products will appear on ammoniacal phenol paper chromatograms2 The peak at an Ry of 0.17 is 1-[~4C]guanidino-l-deoxy-scyllo-inositol-4-P. The broad peak centering around R~ 0.40 is streptidine-P. It is advisable to assay a number of different mycelial pads, so that the best preparation can be utilized for a suitably scaled-up incubation. The labeled products from a scaled-up incubation are separated on a Dowex 50 (H ÷) column2 1-[~C]Guanidino-l-deoxy-scyllo-inositol-4-P is eluted with 0.5 N HC1, and labeled streptidine-P is eluted with 2.0 N HC1 (Table I). 7 Alternatively, chemically phosphorylated aminodeoxy-scyllo-inositol can be employed as the amid no acceptvr ~ with a dialyzed extract of a streptomycin or bluensomycin producer as the source of amidinotransferase, and the labeled product is isolated by the same procedure.
Separation o] 1-Guanidino-l-deoxy-scyllo-inositol-4-P Phosphatase ]rom Streptomycin-6-P PhosphataseY Both of these phosphatase activities can be readily demonstrated in dialyzed lysozyme extracts ~ or sonicates s of frozen mycel~al pads of streptomycin producing strains. The following procedure is typical, but not necessarily optimal. All operations are carried out at 4 ° or less. Frozen pads of mature mycelia of S. bikiniensis ATCC 11062, 18.7 g, are sonicated with an equal weight of water in a beaker for a total of 15 rain in 30-sec bursts, and centrifuged 20 min at 30,000 g. To 20 ml of supernatant solution is slowly added, with stirring, 6.7 ml of 10% MnCl_~ • 4H~O in 0.1 M Tris C1, pH 7.4. After 20 rain the mixture is centrifuged. Powdered (NH4)~S04 is added to the supernatant solution to bring it to 20% of saturation. After 15 min the mixture is centrifuged. The supernatant solution is slowly brought to 65% saturation with powdered (NH~):SO~ while the pH is adjusted to neutralThis volume [24]. 'This volume [21]. 8This volume [22]. 9This volume [23].
[26]
GUANImNODEOXY-scyllo-INOSITOL-4-PPHOSPHOHYDROLASE
461
ity with 1 M NH4OH. After 15 rain the mixture is centrifuged, and the precipitate is taken up in 5 ml of 0.1 M potassium phosphate buffer, pH 7.4, containing 10 mM EDTA, and dialyzed for 2 days against 4 liters of 1 mM phosphate buffer, pH 7.4, containing 0.1 mM EDTA and 0.1 ml of mercaptoethanol, with one change in external medium. A 0.5-ml aliquot of the dialyzed preparation, containing 17.5 mg of protein (Lowry), is diluted with 1 ml of 0.1 M Tris C1, pH 7.4, and applied to a Sephadex G-200 column, 3 cm )< 67 cm, previously equilibrated with 10 mM Tris C1, pH 7.4, plus mercaptoethanol (10 t~l/10 ml). The column is eluted at the rate of 1 drop per 37 sec with the equilibrating buffer; 2.5-ml fractions are collected, and 1.5 mg of bovine serum albumin is added to odd-numbered tubes, starting with tube 61, to preserve activity of guanidino-deoxy-scyllo-inositol-P phosphatase. The fractions are stored frozen. Streptomycin-6-P phosphatase activity 1° is almost completely excluded and is eluted between tubes 45 and 60 (peak at 53), with the bulk of the protein. Guanidinodeoxy-scyllo-inositol-P phosphatase activity is eluted in a broad peak centering on tube 111.
Properties
Biological Distribution. This enzyme apparently occurs in all strains of Streptomyces which produce the streptomycin family of antibiotics, 5 including bluensomycin producers. 4 It is not yet known whether the similar enzyme detected in S. hygroscopicus ]orma glebosus ATCC 14607 differs in its molecular properties from the corresponding enzyme in streptomycin-producing strains. Specificity. 1-Guanidino-l-deoxy-scyllo-inositol-4-P phosphatase acts equally well on 2-[14C]guanidino-2-deoxy-neo-inositol-5-P, ~- formed by transamidination of chemically phosphorylated 2-amino-2-deoxyneo-inositol derived from hygromycin. 1D-[l'C]Guanidino-5-amino-l,5dideoxy-scyllo-inositol-4-P, formed by transamidination of phosphorylated streptamine, can also serve as a substrate. 2 1D-1,5-diguanidino1,5-dideoxy-scyllo-inositol-4-P (streptidine-P) and 1D-l-guanidino-ldeoxy-5-O-carbamoyl-scyllo-inositol-4-P (bluensidine-P) ~ are not readily dephosphorylated. This phosphatase has an absolute and relatively specific requirement for Mg 2+. Mercaptans stimulate activity of purified preparations. Inhibitors. Sulfhydryl reagents, such as p-chloromercuribenzoate, cystamine, and freshly prepared aqueous solutions of formamidine disulfide • 2 HC1, strongly inhibit activity. lOThis volume [281.
462
[27]
ANTIBIOTIC BIOSYNTHESIS
[27] L-Alanine:lD-1-Guanidino-l-deoxy-3-ketoscyllo-inositol Aminotransferase By JAMES B. WALKER
o __A
I•H2
L-Alanine +
NH~
NI__A ?=Nil: ,
It
Pyruvate +
(1)
tt Oil
Ott 1 D-Guanidino-3amino- 1, 3-dideoxy-
scyllo- inositol
This aminotransferase 1,2 participates in the biosynthesis of streptomycin but is absent from strains which synthesize bluensomycin 3 (structure in previous article4). This aminotransferase is somewhat less specific with respect to amino donors than is L-glutamine:keto-scyllo-inositol aminotransferase, '~ but the two enzymes appear to be closely related, possibly being derived from a common ancestral gcne. :*
Assay Method
Principle. The assay is carried out in the reverse direction of Eq. (1). Amino donor labeled in the guanidino group, with two positive charges at p H 3.6, is converted to labeled product with one positive charge. Labeled substrate and labeled product are separated by high-voltage paper electrophoresis at p H 3.6 and counted. 6 This assay can be used with both crude and purified preparations; purified preparations must contain pyridoxal-P at all times to retain activity. Reagents Extract of Streptomyces bikiniensis A T C C 11062, or another strain which synthesizes streptomycin 1 D - l - [ 14C ] Guanidino-3-amino-l,3-dideoxy-scyUo-inositol 1j. B. Walker and M. S. Walker, Biochem. Biophys. Res. Commun. 26, 278 (1967) 2j. B. Walker and M. S. Walker, Biochemistry 8, 763 (1969). 3j. B. Walker, J. Biol. Chem. 249, 2397 (1974). 4This volume [21]. 5 This volume [23]. 6 This volume [22].
[27]
GUANIDINODEOXY-3-KETO-8cyllo-INOSITOLAMINOTRANSFERASE 463 Potassium phosphate, 0.2 M, containing 13 mM EDTA, pH 7.4 Sodium pyruvate, 0.15 M, freshly prepared
Procedure. The complete incubation mixture contains: 1D-I-I~4CI guanidino-3-amino-l,3-dideoxy-scyllo-inositol, 5 ~l; phosphate- EDTA, 5 ~1; pyruvate, 5 td; and extract of S. bikiniensis, 10 ~l. After incubation in a stoppered 13 X 100 mm test tube at 35 °, a 10-~l aliquot is spotted at the 34-cm segment, and components are separated by high-voltage paper eleetrophoresis at pH 3.6 and counted2 For mobilities, see the table of a previous article. ~
Preparation of ID-1-[14C] Guanidi~m-3.amino- l ,3-dideoxy-scyllo-inositol. 1-['4C]Guanidino-l-deoxy-scyllo-inositol-4-P, prepared as described, ~ is successively dephosphorylated, dehydrogenated, and transamihated (cf. Fig. 2 of a previous article *) during a single incubation with a nondialyzed supernatant solution of a sonicate of S. bikiniensis, prepared as described2 The complete incubation mixture contains: 1-[~C]guanidino-l-deoxy-scyllo-inositol-4-P, 0.72 ml (5.3 X 10'~ cpm); 0.5 M Tris C1, pH 8.8, 0.3 ml; 0.2 M L-alanine, 0.2 ml; and supernatant solution from sonieate of S. bikiniensis, 0.72 ml. The mixture is incubated 3 hr at 35 ° in a 100-ml beaker, with occasional swirling to assure sufficient aeration. (The dehydrogenation step will not proceed with this volume of solution in a small test tube where the depth of solution limits availability of oxygen.) The mixture is treated with 0.2 ml of 30% trichloroacetic acid and centrifuged. Labeled components in the supernatant solution plus washing are separated on a column containing Dowex 50 (H+I as described ~ (see the table~). Unchanged labeled substrate, as well as labeled intermediates, can be reclaimed during the same column isolation procedure. Occasionally the incubation mixture has been fortified with pyridoxal-P; this is essential for purified preparations, but can often be omitted when crude sonicates are the source of enzyme. More often, the labeled substrate is preincubated with Escherichia coli alkaline phosphatase (Sigma type III) to increase the final yield.
Preparation o] 1D-I- [~4C] Guanidino:-l-deoxy-3-keto-scyllo-inositol. This compound can be prepared by suitably scaling up the quantities used in the assay procedure, and subsequently separating the labeled components on a Dowex 50 (H +) column (the table~). Unreacted substrate can be reclaimed. Tris buffer should not be used, because it is eluted by 1.0 N HC1 along with the desired product. Separation o] the Two Inosamine Transaminases. 2 L-Glutamine:ketoscyllo-inositol aminotransferase5 can be prepared free from the aminotransferase activity described in this article by (a) use of S. glebosus This Yolume [26].
464
ANTIBIOTIC BIOSYNTHESIS
[27]
ATCC 14607 as the enzyme source a (b) treatment of an extract of S. bikiniensis at 55 ° for 5 min, 8 or (c) separation of both aminotransferases on a Sephadex G-100 column. For separation of the two aminotransferases, either sonicates or lysozyme extracts of S. bikiniensis can serve as starting material. All procedures are performed at 4 ° or less. To 30 ml of a lysozyme extract 9 of S. bikiniensis is added 0.3 ml of neutralized pyridoxal-P (20 mg/ml), and then 2 ml of 10% MnCl~ • 4H20 is added slowly with stirring. After 20 rain the suspension is centrifuged. Powdered (NH4)..SO,, 12 g, is slowly added with stirring to 27 ml of the supernatant solution; the solution is kept neutral by the careful addition of 2 M NH4OH. After 20 min the suspension is centrifuged, and the precipitate is taken up in 8 ml of 0.1 M potassium phosphate buffer, pH 7.4, containing 10 mM EDTA and 1 mg pyridoxal-P per milliliter. The solution is dialyzed for several hours; 1 ml of this preparation, containing 19.6 mg of protein (Lowry), is applied to a Sephadex G-100 (40-120 ~m) column, 2 X 54 cm, which had previously been equilibrated with 1 mM potassium phosphate containing per milliliter 0.1 mg of EDTA and 0.1 mg of pyridoxal-P, pH 7.4. Fractions of 2.4 ml (40 drops) are collected, 1 drop per second. Protein is first detected in tube 19. L-Glutamine:ketoscyllo-inositol aminotransferase activity is eluted first in a peak centering on tube 25, whereas L-alanine:lD-l-guanidino-l-deoxy-3-keto-scylloinositol aminotransferase activity is ¢luted in a peak centering on tube 28; there is thus some overlap, but tubes can be selected which have essentially only one of tho two activities. -° Properties
Biological Distribution. L-Alanine: 1D-l-guanidino-l-deoxy-3-ketoscyllo-inositol aminotransferase occurs in S. bikiniensis ATCC 11062, S. griseus ATCC 12475, S. ornatus ATCC 23265, and presumably other strains which synthesize streptomycin. This enzyme appears to be absent from S. hygroscopicus ]brma glebosus ATCC 14607, which synthesizes bluensomycin2 Specificity and Alternate Assays. From enzyme stability studies pyridoxal-P appears to be a required cofactor. At a concentration of 2 mM, L-alanine is the most active amino donor among the amino acids tested, followed by L-glutamate, and then L-glutamine. The corresponding Damino acids are inactive. When L-glutamine is the amino donor the reaction should be relatively irreversible because of cyclization or enzymic deamidation of the a-ketoglutaramate formed? There is evidence that s j. B. Walker, Lloydia 34, 363 (1971). gThis volume [24].
[28]
STREPTOMYCIN-6-P PHOSPHOHYDROLASE
465
at high concentrations keto-scyllo-inositol can serve as an amino acceptor, and a large number of other compounds can serve as amino donors. This enzyme catalyzes a very active exchange transamination between 1D-1-gu anidino-3-amino- 1,3.dideoxy.scyllo-inositol and 1D-l- [ 14C] guanidino-l-deoxy-3-keto-scyllo-inositol, ~,~ and this reaction can serve as an alternate assay. 1L-l-[14C]Guanidino-3-amino-l,3-dideoxy-scyllo-inositol, prepared by enzymic dephosphorylation of the transmidination product with streptamine-P as amidino acceptor, 1° cannot serve as amino donor in the reverse direction of Eq. (1). Stability. This aminotransferase is unstable to dialysis in the absence of pyridoxal-P and cannot be significantly reactivated by the subsequent addition of pyridoxal-P. Pyruvate spares the pyridoxal-P requirement during dialysis. Pyridoxamine-P, pyridoxal, and L-alanine do not protect this enzyme during dialysis. The enzyme from S. bikiniensis is inactivated by heating at 55 ° for 5 min, even in crude extracts. lOThis volume [25].
[28]
Streptomycin-6-P Phosphohydrolase
By JAMES B. WALKER I~N--C--~NH+ I
NH H
NH
O
[\ ,
HsC
.N/ l
[
~\ CH.OH
NH~ I
/I
I
Ix, O H
/[
Dihydrost repto my cin~ 6- P
I Dihydrostreptomycin
+
HPO~-
This enzymic reaction is believed to be the final step in streptomycin biosynthesis (Fig. 2 of a previous article1). Evidence that this reaction is the final step is derived primarily from the observations that streptomycin-6-P accumulates in the culture medium of (a) a particular strain of Streptomyces gri, eus (HUT 6037) grown in the presence of 5% glucose, 2 and (b) all streptomycin producers tested, following addition of This volume [21]. : R. Nomi, O. Nimi, T. Miyazaki, A. Matsuo, ami H. Kiyohara, Agr. Biol. Chem. 31, 973 (1967).
[28]
STREPTOMYCIN-6-P PHOSPHOHYDROLASE
465
at high concentrations keto-scyllo-inositol can serve as an amino acceptor, and a large number of other compounds can serve as amino donors. This enzyme catalyzes a very active exchange transamination between 1D-1-gu anidino-3-amino- 1,3.dideoxy.scyllo-inositol and 1D-l- [ 14C] guanidino-l-deoxy-3-keto-scyllo-inositol, ~,~ and this reaction can serve as an alternate assay. 1L-l-[14C]Guanidino-3-amino-l,3-dideoxy-scyllo-inositol, prepared by enzymic dephosphorylation of the transmidination product with streptamine-P as amidino acceptor, 1° cannot serve as amino donor in the reverse direction of Eq. (1). Stability. This aminotransferase is unstable to dialysis in the absence of pyridoxal-P and cannot be significantly reactivated by the subsequent addition of pyridoxal-P. Pyruvate spares the pyridoxal-P requirement during dialysis. Pyridoxamine-P, pyridoxal, and L-alanine do not protect this enzyme during dialysis. The enzyme from S. bikiniensis is inactivated by heating at 55 ° for 5 min, even in crude extracts. lOThis volume [25].
[28]
Streptomycin-6-P Phosphohydrolase
By JAMES B. WALKER I~N--C--~NH+ I
NH H
NH
O
[\ ,
HsC
.N/ l
[
~\ CH.OH
NH~ I
/I
I
Ix, O H
/[
Dihydrost repto my cin~ 6- P
I Dihydrostreptomycin
+
HPO~-
This enzymic reaction is believed to be the final step in streptomycin biosynthesis (Fig. 2 of a previous article1). Evidence that this reaction is the final step is derived primarily from the observations that streptomycin-6-P accumulates in the culture medium of (a) a particular strain of Streptomyces gri, eus (HUT 6037) grown in the presence of 5% glucose, 2 and (b) all streptomycin producers tested, following addition of This volume [21]. : R. Nomi, O. Nimi, T. Miyazaki, A. Matsuo, ami H. Kiyohara, Agr. Biol. Chem. 31, 973 (1967).
ANTIBIOTIC BIOSYNTHESIS
466
[28]
15 mM inorganic phosphate to the medium. 3 It is believed that accumulation of streptomycin-6-P in both instances results from inhibition of Eq. (1): inhibition in the case of HUT 6037 strain by the low pH of the medium, and inhibition in streptomycin producers in general by product inhibition by the added inorganic phosphate. Streptomycin-6-P phosphatase has marked phosphotransferase activity and can be employed to transfer phosphate groups to hydroxyls adjacent to nitrogenous groups of certain aminoglycoside antibiotics and other amino alcohols. 4-6
Assay Methods Method I Principle. Dephosphorylation of [3'a-3H] dihydrostreptomycin-6-P is followed by paper chromatography. This assay can be used for both crude extracts and partially purified preparations. This assay should be employed at intervals, during purification procedures which utilize less specific assays, to be certain that the desired phosphatase is being isolated. Reagents
Extract of mature mycelia of Streptomyces bikiniensis ATCC 110627,s or other strain which synthesizes streptomycin (EDTA not present; ~ 1 mM inorganic phosphate) [3'a-3H]Dihydrostreptomycin-6-P (see below) Tris-C1, 0.5 M, containing 40 mM MgC12, pH 9.0 Procedure. 4 The complete incubation mixture contains: [3H]dihydrostreptomycin-6-P, 5 t~l (e.g., 40,000 cpm); Tris-Mg, 5 ~l; and enzyme preparation, 20 td. After incubation in a stoppered 13 X 100 mm test tube at 35 °, a 10-~l aliquot is spotted, and the components are separated by paper chromatography with ammoniacal phenol and counted, s For mobilities, see the table of a previous article. 1 Since ~H-labeled compounds cannot be counted accurately on strips at R r values greater than 0.9 because of quenching by colored decomposition products of phenol, activity is measured as decrease in counts per minute remaining in phosphorylated substrate. Similar assays can be employed with 1D-1,3114C]diguanidino-l,3-dideoxy-scyllo-inositol-6-P (streptidine-P) or 1D-l-amino-3- [1~C]guanidino-l,3-dideoxy-scyllo-inositol-6-P as substrate; in the latter cases appearance of labeled product can be readily followed. A. L. Miller and J. B. Walker, J. Bacteriol. 104, 8 (1970). M. S. Walker and J. B. Walker,J. Biol. Chem. 246, 7034 (1971). J. B. Walker and M. Skorvaga, Y. Biol. Chem. 248, 2441 (1973). ' J. B. Walker and M. Skorvaga,,/. Biol. Chem. 248, 2435 (1973). This volume [22]. SThis volume [24].
[281
STREPTOMYCIN-6-P
PHOSPHOHYDROLASE
467
Preparation of [3'~-3H]Dihydrostreptomycin-6-P. [3'a-~H]Dihydrostreptomycin (700--3000 Ci/mole) is obtained from Amersham-Searle. The complete incubation mixture contains: [3H]dihydrostreptomyein, 0.5 ml (6 X 106 cpm) ; 0.5 M Tris-C1, pH 9.0, containing 40 mM MgCl~, 0.5 ml; 36 mM ATP, pH 7, 0.5 ml; and dialyzed lysozyme extract of S. bikiniensis ATCC 11062,8 1.0 ml. After incubation in a stoppered 13 X 100 mm test tube at 35 ° for 90 min, the reaction is stopped by heating at 100 ° for 4 min and the solution is centrifuged. The supernatant solution is added to a column (1 X 25 cm) containing Bio-Rex-70 (NHg) carboxylic acid resin, 100-200 mesh. Fractions of 3 ml are collected, at a flow rate of ca. 0.6 ml/min. The column is successively eluted stepwise with a total of 60 ml of each of the following concentrations of ammonium formate: 0.1 M, 0.3 M, 0.8 M, and 2.0 M. Labeled dihydrostreptomycin-6-P is eluted in the 0.8 M fraction and unchanged dihydrostreptomycin in the 2.0 M fraction (cf. the table, in a previous article1). Tubes containing the radioactive product are combined in a porcelain evaporating dish and evaporated to dryness in a vacuum desiccator over CaCI~. Residual ammonium formate is removed in a vacuum over a shallow layer of concentrated H._.SQ in petri dishes, to which glass wool has been added to increase the surface area. The labeled compound is taken up in a small amount of water and stored frozen. Pretreatment of Bio-Rex-70 Resin. Bio-Rex-70 (Na +) is obtained from Bio-Rad. The resin, 30 g, is suspended in 800 ml of glass-distilled water, and fines are decanted. The resin is washed two times with 1 N NaOH, then water to pH 7. The resin is next washed with 1 N HC1 then water to pH 7. The resin is then washed once with methanol, washed four times with water, and suspended in 500 ml of 1 M NH40H. The mixture is stirred slowly for 4 hr and then washed with glass-distilled water to pH 7-9 and stored. Method H N
p - N i t r o p h e n y l - P + 2 - d e o x y s t r e p t a m i n e --+ p-nitrophenol + 2-deoxystreptamine-6-P
H2N--~4C --NH
NH2 L- [Guanidino - ~4C ] arginine +
HO~
(2)
[H2 opo~-
~. L-Ornithine
+
~OH
z ~ ~H2
~o~/
(3) opo~-
1 D- 1- Amino- 3guanidino-1, 2, 3trideoxy- s c y l l o inositol-6-P
468
ANTIBIOTIC BIOSYNTHESIS
[28]
Principle. Streptomycin-6-P phosphatase (N) catalyzes transfer of phosphate from dihydrostreptomycin-6-P or p-nitrophenyl-P to amino alcohols such as Tris, streptamine, or 2-deoxystreptamine~; esterification occurs adjacent to the basic group. In the first step of this 2-step enzymic assay [Eq. (2)], a solution containing streptomycin-6-P phosphatase catalyzes transfer of phosphate from p-nitrophenyl-P to streptamine or 2-deoxystreptamine. In the second step [Eq. (3)], EDTA is added to inhibit further phosphatase action, and the streptamine-P isomer shown above is transamidinated in the presence of L-[guanidino-~4C]arginine and a dialyzed extract containing inosamine-P amidinotransferase2 The labeled product is separated by paper chromatography and counted. Reagents Preparation from mature mycelia of streptomycin producing strain of Streptomyces containing streptomycin-6-P phosphatasel°; no EDTA and ( 1 mM Tris or inorganic phosphate Dialyzed extract of mature mycelia of S. glebosus ATCC 14607 ~ or S. bikiniensis ATCC 11062 s p-Nitrophenyl-P, disodium, 6 mM EDTA, 0.3 M, pH 8 Streptamine 2HC1, 120 mM, or 2-deoxystreptamine.2 HC1 85 mM, s adjusted to pH 8 L- [Guanidino-~4C] arginine (12-25 Ci/mole), 33 ~Ci/ml
Procedure. In the first step, the complete incubation mixture contains: p-nitrophenyl-P, 5 ~l; streptamine or 2-deoxystreptamine, 5 ~l; and streptomycin-6-P phosphatase preparation, ~° 10 t~l. (In later stages of purification, Mg 2÷ should be added.) After incubation in a stoppered 13 X 100 mm test tube at 35 ° for 45 min, the following are added: EDTA, 1 ~l; [l*C]arginine, 5/~l; and dialyzed extract of S. glebosus (or S. bikiniensis), l0 ~l. After further incubation at 35 ° for 45 min, a 10-~l aliquot is spotted, and the labeled components are separated on an ammoniacal phenol paper chromatogram and counted, s Mobilities of the expected products are given in Table I of a previous article. 1 Method III Principle. Streptomycin-6-P phosphatase catalyzes transfer of phosphate from p-nitrophenyl-P to both water and Tris. ~ The p-nitrophenol formed is measured at 400 nm. This rapid, but nonspecific, assay can gThis volume [25]. 1oThis volume [26].
[28]
STREPTOMYCIN-6-P PHOSPHOHYDROLASE
469
be utilized with partially purified enzyme preparations during isolation procedures. However, Method I or II should be utilized at intervals to confirm that the phosphatase being isolated is indeed streptomycin-6-P phosphatase.
Reagents Partially purified streptomycin-6-P phosphatase preparation1°; no EDTA and ( 1 mM inorganic phosphate p-Nitrophenyl-P, disodium, 10 mM Tris C1, 0.5 M, containing 10 mM MgCI~, pH 8
Procedure. The complete incubation mixture contains: p-nitrophenyl-P, 0.5 ml; Tris-Mg, 1.0 ml; and enzyme preparation plus water, 2.5 ml. After incubation at 35% the absorbance is measured at 400 nm.
Purification The procedure for separation of streptomycin-6-P phosphohydrolase activity from 1-guanidino-l-deoxy-scyllo-inositol-4-P phosphohydrolase is described in a previous article. 1° The substrate specificity of this preparation has been determined, utilizing yarious mono- and diphosphorylated streptomycin derivatives. 4-6 Nimi et al. n have described a more extensive purification procedure for what appears to be a similar enzyme from their strain of S. griseus, H U T 6037, utilizing DEAE Sephadex A-50 chromatography. The substrafe specificity of their phosphatase preparation has not been reported.
Properties Biological Distribution. Streptomycin-6-P phosphatase appears to occur primarily in mature mycelia of streptomycin producing strains of Streptomyces, such as S. bikiniensis ATCC 11062, S. griseus ATCC 12475, S. griseocarneus ATCC 12628, and S. galbu.~ ATCC 14077. Little or no activity has been detected in S. kanamyceticus ATCC 12853, S. griseus ATCC 10971, and S. ]radiae. Activity appears to be readily released into the medium in Nomi's H U T 6037 strain. 11 Specificity. In the characterization of a given phosphatase, it is essential that the spectrum of substrate specificity be determined. Even nonspecific alkaline phosphatase from Escherichia coli (Sigma type III) will dephosphorylate streptomycin-6-P, given enough enzyme and time. Streptomycin-6-P phosphatase does not transfer phosphate esterified at the 110. Nimi, H. Kiyohara, T. Mizoguchi, Y. Ohata, and R. Nomi, Agr. Biol. Ct~em. 34, 1150 (1970).
470
ANTIBIOTIC BIOSYNTHESIS
[28]
primary 3'a-hydroxymethyl position of dihydrostreptomycin, but it does transfer phosphate esterified at secondary hydroxyl groups adjacent to basic nitrogenous groups, such as occur in streptidine-6-P, streptomycin-3"-P, Tris-P, ethanolamine-P, and numerous aminocyclitol-P derivatives. ~,5 The rate of formation of p-nitrophenol from p-nitrophenyl-P is enhanced in the presence of amino alcohols which can serve as phosphate acceptors. This behavior suggests formation of a phosphoryl enzyme, with dephosphorylation the rate-limiting step. The relatively high phosphotransferase activity of this enzyme perhaps could be employed in molecular modification of antibiotics when it is desired to protect or modify a group adjacent to an amino group. Dialysis of the S. bikiniensis enzyme against EDTA decreases activity; activity can be restored by the addition of Mg ~÷. Alternative Assays. Nimi et al. 11 employ a bioassay for streptomycin released following incubation of enzyme with streptomycin-6-P for 24 hr. Other assays might include action of the enzyme on (a) [3H]dihydrostreptomycin-3.'a,6-diP 12 to give [3H]dihydrostreptomycin-3'a-P which can be separated by paper chromatography (Table I of a previous article1); or (b) dihydrostreptomycin-6-3~P to give 32Pi, which can be detected in the water wash of a small Pasteur pipette column containing Dowex 50 (H ÷) resin, whereas unreacted substrate is retained on the column. Inhibitors. Streptomycin-6-P phosphatase is not inhibited by sulfhydryl reagents, unlike 1-guanidino-l-deoxy-scyllo-inositol-4-P phosphatase, but it is inhibited by EDTA and inorganic phosphate. Certain amino alcohols inhibit formation of inorganic phosphate by competing with water for phosphate transferred from the presumed phosphoryl-enzyme intermediate. 5 Moderate (e.g., 0.03 M) concentrations of inorganic phosphate included in industrial streptomycin fermentation media might well increase the overall yield of antibiotic by decreasing autoinhibition, since streptomycin-6-P has no biological activity.:. 3 Alternatively, mutants lacking streptomycin-6-P phosphatase could be selected and employed to increase overall yield. Active antibiotic could then be generated by incubation with nonspecific alkaline phosphatase or the enzyme described here.
1, This volume [51].
[29]
~- (a-AMINOADIPYL)CYSTEINYLYALINE SYNTHETASE
471
[29] 6-(a-Aminoadipyl)cysteinylvaline Synthetase B y PATRIClA FAWCETT and E. P. ABRAHAM
-O2C. CH(NH3)(CH~)3CONH. CH(CH2SH)CO2- + HaN. CH(CH(CH3)2)CO~- --~ +
-02C. CH (NH3) (CH2)3CONHCH (CH~SH)CON HCH(CH(CH~)~)CO C A tripeptide, g-(a-aminoadipyl)cysteinylvaline, was shown by Arnstein and Morris l to be present in small amounts in the mycelium of Penicillium chrysogenum. A mixture of related peptides was obtained by Loder and Abraham 2 from the mycelium of Cephalosporium acremonium. The major component of the mixture was shown to be g-(L-a-aminoadipyl)-L-cysteinyl-D-valine. 2 Two minor components appeared to be tetrapeptides, one differing from the tripeptide in containing a glycine residue and one also in containing a fl-hydroxyvaline residue in place of valine. 2 g-(a-Aminoadipyl)cysteinylvaline synthetase, which catalyzes the formation of this tripeptide from g-(L-a-aminoadipyl)-L-cysteine and L-valine, has been found in a broken-cell system from C. acremonium2 No synthesis of the tripeptide from L-a-aminoadipic acid and a-cysteinyl-L-valine could be shown to occur. 3
Assay Method Principle. The enzyme is assayed by measurement of the incorporation of 14C from labeled valine into g-(a-aminoadipyl)cysteinylvaline in the presence of ~-(L-a-aminoadipyl)-L-cysteine. The synthesis of g-(L-aaminoadipyl)-L-cysteine has been described by Loder and Abraham. 4 The labeled product is isolated by paper electrophoresis and chromatography, after oxidation to the sulfonic acid form, and identified by comparison with an authent!c sample of the tripeptide. ~-(L-a-aminoadipyl)-L-cysteinyl-D-valine has been synthesized by Usher. ~ The disulfide of the corresponding all-L tripeptide has been synthesized by Rudinger?
1H. R. V. Arnstein and D. Morris. Biochem. J. 76, 357 (1960). 2p. Bronwen Loder and E. P. Abraham. Biochem. J. 123, 471 (1971). 3p. Bronwen Loder and E. P. Abraham. Biochem. J. 123, 477 (1971). 4p. Bronwen Loder, E. P. Abraham, and G. G. F. Newton, Biochem. J. 112, 389 (1969). J. J. Usher, unpublished experiments, 1973. "J. Rudinger, Czech. Chem. Commun. 27, 2246 (1962); personal communication (1968).
472
ANTIBIOTIC BIOSYNTHESIS
[29]
Reagents n-Valine, 0.2 M solution in water DL-[l-14C]Valine, 1.0 ~Ci/~l, 0.03 ~mole/~l $- (L-a-Aminoadipyl) -L-cysteine 4 Adenosine 5'-triphosphate disodium salt, 9 mg Phosphoenolpyruvate tricyclohexylammonium salt, 12 mg Pyruvate kinase, crystalline suspension, 10 mg/ml Enzyme, crude preparation as described below, 3 ml
Procedure. Adenosine triphosphate (9 mg) and phosphoenolpyruvate (12 mg) are weighed into the same tube, and the enzyme preparation (3 ml) is added. The pH of the solution is adjusted to 7.0 with 1 M NaOH, and pyruvate kinase (75 t~l of suspension) is added. Samples (1 ml) of this mixture are added to tubes containing $-(L-~-aminoadipyl)L-cysteine (1 mg) in 175 ~1 of water and 5 ~1 of 1 M NaHCOs. Samples of the solution of n-valine (20 t~l) and DL-[1-14C]valine (5 td) arc added to each tube and the tubes incubated at 27.5 ° for 1 hr. After incubation the mixtures are centrifuged at 20,000 g for 30 min and the supernatants removed and freeze-dried. The residues are extracted with 70% (v/v) ethanol (each 2 X 200 td) and the extracts diluted with water (2 ml) and freeze-dried. The desalted extracts are treated with a 1.5% solution of performic acid in 98% formic acid (0.2 ml) at 0 ° and the mixtures kept at 0 ° for 6 hr. After dilution with water (2 ml) the solutions are freeze-dried. Each residue is dissolved in a solution (25 ~l) of synthetic $-(L-aaminoadipyl)-L-cysteinyl-D-valine (100 ~g). Samples (5 ~l) of the resulting solutions arc spotted onto Whatman No. 1 paper and subjected to electrophoresis (70 v/cm) for 2.5 hr at pH 1.8 in 20% (v/v) acetic acid containing 2% (v/v) formic acid and then to chromatography in the second dimension in butan-l-ol:acetic acid:water (4:1:4 by volume). Under these conditions the labeled tripeptide migrates' slightly less far than glutathione sulfonic acid toward the anode and shows an R value relative to that of glutathione sulfonic acid of about 2.75. The position of the labeled tripeptide on the paper is located by radioautography 7 and the radioactive spot is counted. 7 The paper is then sprayed with ninhydrin and the identity of the labeled product with ~-(a-aminoadipyl)cysteinylvaline is confirmed by the coincidence of the radioactive and ninhydrin positive spots. B. Smith, S. C. Warren, G. G. F. Newton, and E. P. Abraham, Biochem. d. 103, 877 (1967).
[29]
~- (ct-AMINO&DIPYL)CYSTEINYLVALINE SYNTHETASE
473
Preparation Cephalosporium acremonium C91 is grown in shake-flasks in a chemically defined medium as described by Smith et alJ The mycelium is harvested by filtration 72 hr after inoculation and washed on the filter with water. It is then resuspended in 0.1 M Tris • HC1 buffer, pH 7.0, containing 0.1 M KC1, 20 mM MgSO4, and 1 mM cysteine (1 g damp-dry mycelium/3 ml), and the suspension is homogenized in a Potter-Elveh]em homogenizer with a Teflon pestle cooled in ice. The homogenate is diluted with an equal volume of buffer and a sample (20 ml) of the resulting mixture subjected to ultrasonic treatment (60 W, 20 Hz) in a jacketed glass vessel cooled at 0 ° and with a titanium probe, 1 cm in diameter, ending about 2 mm below the surface. Half of the treated suspension is centrifuged for 30 min at 20,000 g, and the supernatant is removed. The particulate fraction is washed by resuspension in the buffer (20 ml) and recentrifugation at 20,000 g. It is finally resuspended in buffer to give a volume equal to that of the suspension from which it has been obtained and used immediately after preparation. 8-(a-Aminoadipyl)cysteinylvaline synthetase is present in both the complete mixture obtained after ultrasonic treatment and in the particulate fraction, but very little is found in the supernatant fraction. The crude enzyme preparation from 1 g damp-dry mycelium synthesises about 15 nmoles of tripeptide in 1 hr under the conditions described.
Specificity $-(~-Aminoadipyl)cysteinylvaline synthetase differs from glutathione synthetase, which is found mainly in the supernatant fraction obtained by centrifugation after ultrasonic treatment of the mycelium. It catalyzes the synthesis of tripeptide from $-(L-a-aminoadipyl)-L-cysteine and L-valine, but nosynthesis has been observed from ~-(D-a-aminoadipyl)-scysteine and L-valine, from ~-(L-~-aminoadipyl)-L-cysteine and D-valine, or from L-~-aminoadipic acid and L-cysteinyl-L-valine?. ~
P. Bronwen Loder, unpublished experiments, 1971.
474
ANTIBIOTIC BIOSYNTHESIS
[30]
[30] A c y l C o A : 5 - A m i n o p e n i c i l l a n i c A c i d A c y l t r a n s f e r a s e
By STEN GATENBECK Acyl CoA + 6-aminopenicillanic acid (6-APA) --* penicillin + CoA Assay Method ~
Principle. The method is based on the determination of radioactive benzylpenicillin formed with 14C-labeled phenylacetyl CoA and 6-APA as substrates. Reagents 6-APA, O.O4M Dithiothreitol, 0.25 M Phenylacetyl-l-l~C CoA, 7 mM. The synthesis of this reagent is performed by reacting the mixed anhydride of the labeled acid and carbonic ester with CoA. Buffer solution, pH 8.4 (0.2 M Tris, 50 mM phosphate, 0.2 M NaC1, 1 mM EDTA) Benzylpenicillin, 3 mM Penicillinase
Procedure. Incubations are carried out at 30 ° in tubes containing the following mixture: Enzyme, 0.1 ml; 6-APA, 0.025 ml; dithiothreitol, 0.01 ml; phenylacetyl-l-l~C CoA, 0.025 ml (200,000 dpm); buffer, 0.1 ml. After 40 min 0.01 ml af carrier benzylpenicillin is added to each tube, and the total content is spotted on a paper chromatogram strip after adjusting the pH of the solution to 5.5. The paper strips, preimpregnated with citrate buffer, pH 5.7, are developed in ether saturated with water. The radioactive benzylpenicillin is localized on the paper strip by using a radiochromatogram scanner. The amount of benzylpenicillin formed is determined by cutting out the radioactive area from the paper chromatogram and by dipping the paper into a solution of PPO and POPOP in toluene-methanol (1 : 1) and measuring the radioactivity in a scintillation spectrometer. Definition of Unit and Specific Activity. One unit is defined as the amount of enzyme that catalyzes the formation of 1 nmole of penicillin in the above test. Specific activity is the number of units of activity per milligram of protein determined by the biuret method. 1S. Gatenbeck and U. Brunsberg, Acta Chem. Scand. 9.2, 1059 (1968).
[30]
ACYL CoA:AMINOPENICILLANIC ACID ACYLTRANSFERASE
475
Other Assay Methods. 2 Methods based on the following exchange reaction have been used for determining the enzyme activity. Acyl APA + *APA;~ acyl *APA + APA
Enzyme Purification Growth of Cells and Extract Preparation. Penicillium chrysogenum ATCC 12,687 is grown on a rotary shaker (1" stroke, 250 rpm) at 28 ° in 500-ml conical flasks each containing 150 ml of substrate of the following composition (all in grams per liter): KH2P04, 3.0; Na~SO4, 0.5; MgS04 • 7H20, 0.25; ZnS04 • 7H20, 0.02; M n S Q • H20, 0.02; Fe (NH4)_~(SO~) 2 • 6H.~O, 0.1 ; CuSO~ • 5H20, 0.005 ; CaC12 • 2H20, 0.05; yeast extract, 1.0; NH4-acetate, 3.5; NH4-1actate, 6.0; glucose, 10; lactose, 30; distilled water. The mycelia from 12 flasks are harvested and washed with buffer (0.2 M Tris, 50 mM phosphate, 0.2 M NaC1, 1 mM EDTA, pH 7.8) after 96 hr of growth. The cells are ground with sand at 4 ° for 10 min, and the cell debris is removed by centrifugation for 20 min at 30,000 g. Ammonium Sul]ate Precipitation. Solid ammonium sulfate is slowly added with stirring to the supernatant solution from the preceding step until a 0.30 saturated solution is obtained. The precipitate is collected by centrifugation (30,000 g for 20 min) and discarded. Additional solid ammonium sulfate is added to the supernatant solution until 0.50 saturation is reached. After centrifugation, the supernatant solution is discarded, and the precipitate is dissolved in 30 inl of 20 mM phosphate buffer, pH 7.8; the solution is passed through a column of Sephadex G-25, coarse. Hydroxyapatite Gel Adsorption and Elution. The eluate from the preceding step is treated with hydroxyapatite (7.0 g). The gel is washed with 20 ml of 20 mM phosphate buffer, pH 7.8, followed by 10 ml of 40 mM phosphate buffer, pH 7.8. The enzyme is solubilized with 25 ml of 70 mM phosphate buffer, pH 7.8. The volume of the enzyme solution is then reduced to 4 ml by ultrafiltration. Sephadex G-200 Column. The enzyme concentrate is placed on a sephadex G-200 column (3 X 70 cm) which has been equilibrated with a buffer of 0.2 M Tris, 50 mM phosphate, 0.2 M NaC1, 1 mM EDTA, pH 7.8. The enzyme is eluted at about 190 ml with the same buffer. The yields and the specific activities of the various fractions obtained during the purification procedure are outlined in the table. 2D. L. Pruess and M. J. Johnson, J. Bacteriol. 94, 1502 (1967).
476
ANTIBIOTIC BIOSYNTHESIS
[31]
PURIFICATION OF 6-AMINOPENICILLANIC ACID ACYLTRANSFERASE FROM
Penicillium chrysogenum
Step Cell extract (NH4)~SO4 fractionation Hydroxyapatite Sephadex G-200
Protein (mg)
Units
Specific activity (units/mg protein)
2640 144 14.4 0.7
1584 2016 252 64
0.6 14.0 17.5 91.0
Yield (%) 100 127 16 4
Properties Specificity. Fractionated enzyme preparations utilize a number of acyl-CoA derivatives although at different rates, e.g., phenoxyacetylCoA, p-methoxyphenylacetyl-CoA, octanoyl-CoA. The enzyme does not show any penicillin acylase activity. Ejlect of pH. In Tris buffer the optimum enzyme activity is approximately pH 8.5. The activity is very low at pH 7. Activators. The enzyme activity is stimulated by the presence of thiol compounds. Comments. An enzyme that catalyzes acyl group exchange between various penicillins has been described 2 under the name penicillin acyltransferase. The likely Uni Uni Uni Uni Ping Pong mechanism for the 6-APA acyltransferase reaction suggests that the two enzymes are identical.
[31] Phenacyl :Coenzyme A Ligase By
RICHARD BRUNNER a n d MAX ROHR
Phenacyl:coenzyme A ligase catalyzes the synthesis of phenacetylCoA and phenoxyacetyl-CoA according to Eq. (1). RCOOH 4- CoA ~- ATP ~- CoA-SCOR -b AMP + PP
(1)
where R is the phenacetyl or phenoxyacetyl radical, known as the acyl side chains of benzylpenicillin and phenoxymethylpenicillin. The enzyme was found to be formed in a penicillin-producing strain of Penicillium chrysogenum in the course of penicillin production experi-
476
ANTIBIOTIC BIOSYNTHESIS
[31]
PURIFICATION OF 6-AMINOPENICILLANIC ACID ACYLTRANSFERASE FROM
Penicillium chrysogenum
Step Cell extract (NH4)~SO4 fractionation Hydroxyapatite Sephadex G-200
Protein (mg)
Units
Specific activity (units/mg protein)
2640 144 14.4 0.7
1584 2016 252 64
0.6 14.0 17.5 91.0
Yield (%) 100 127 16 4
Properties Specificity. Fractionated enzyme preparations utilize a number of acyl-CoA derivatives although at different rates, e.g., phenoxyacetylCoA, p-methoxyphenylacetyl-CoA, octanoyl-CoA. The enzyme does not show any penicillin acylase activity. Ejlect of pH. In Tris buffer the optimum enzyme activity is approximately pH 8.5. The activity is very low at pH 7. Activators. The enzyme activity is stimulated by the presence of thiol compounds. Comments. An enzyme that catalyzes acyl group exchange between various penicillins has been described 2 under the name penicillin acyltransferase. The likely Uni Uni Uni Uni Ping Pong mechanism for the 6-APA acyltransferase reaction suggests that the two enzymes are identical.
[31] Phenacyl :Coenzyme A Ligase By
RICHARD BRUNNER a n d MAX ROHR
Phenacyl:coenzyme A ligase catalyzes the synthesis of phenacetylCoA and phenoxyacetyl-CoA according to Eq. (1). RCOOH 4- CoA ~- ATP ~- CoA-SCOR -b AMP + PP
(1)
where R is the phenacetyl or phenoxyacetyl radical, known as the acyl side chains of benzylpenicillin and phenoxymethylpenicillin. The enzyme was found to be formed in a penicillin-producing strain of Penicillium chrysogenum in the course of penicillin production experi-
[31]
PHENACYL :COENZYME A LIGASE
477
merits on a laboratory scale? It appeared at 2 days when penicillin production was beginning and increased markedly at 3 and 4 days, i.e., just prior to and during the stage of most rapid penicillin fermentation. The finding l-3 that mycelial extracts of Penicilliura chrysogenum contain a 6-aminopenicillanic acid acyltransferase, which catalyzes the formation of penicillins from 6-aminopenicillanic acid and the CoA derivatives of phenylacetic or phenoxyacetie acid, led to the conclusion that the final step in penicillin biosynthesis might involve the direct N-acylation of 6-aminopenicillanic acid by the CoA-activated side-chain precursors.
Assay Method
Principle. The method employed involves the measurement of the rate of formation of acylhydroxamate in the presence of excess ATP and phenylacetate, catalytic amounts of CoA, and neutral hydroxylamine as described elsewhere? Reagents Potassum phosphate buffer, 1 M, pH 7.0 CoA, 1 mM, pH 6.5 ATP, 0.1 M, pH 7.5 Potassium fluoride, 1 M Magnesium chloride, 0.2 M Reduced glutathione, 0.2 M, pH 4.5 Potassium phenylacetate, 0.2 M, pH 6.0 Hydroxylamine solution, 2 M, pH 6.5, freshly prepared by mixing equal volumes of 4 M hydroxylamine hydrochloride and 4 M potassium hydroxide. Ferric chloride reagent containing 0.37 M ferric chloride, 20 mM trichloroaeetic acid, and 0.66 M hydrochloric acid.
Procedure. To a small centrifuge tube are added 0.1 ml of potassium phosphate buffer, 0.1 ml of CoA, 0.1 ml of ATP, 0.05 ml of potassium fluoride, 0.05 ml of magnesium chloride, 0.05 ml of glutathione, 0.1 ml of potassium phenylacetate, 0.1 ml of hydroxylamine, and enough distilled water to bring the final volume to 1.0 ml after the addition of the enzyme solution. After 5 min of temperature equilibration in a water bath at 37 ° , the enzyme is added and incubation is carried out for periods ' R . Brunner, M. RShr, and M. Zinner, Hoppe-Seyler's Z. Physiol. Chem. 349, 95 (1968). 2 B. Spencer, Biochem. Biophys. Res. Commun. 31, 170 (1968). 3 S. Gatenbeck and U. Brunsberg, Acta Chem. Scc,nd. 22, 1059 (1968). 4 p. Berg, this series, Vol. V [62].
478
ANTIBIOTIC BIOSYNTHESIS
[31]
of 20 or 40 min. The reaction is stopped by the addition of 2 ml of the ferric chloride reagent, and after centrifugation the optical density of the solution is measured at 540 nm in a spectrophotometer. The extinction coefficient of phenylacetylhydroxamate under these conditions is 0.90 X 106 cm 2 mole-1. One unit of enzyme activity is defined as the catalytic activity leading to the formation of 1 t,mole of acylhydroxamate in 1 min. Comment. Omission of CoA in parallel tubes as recommended in the assay of acetyl:coenzyme A ligase 4 frequently does not show any significant difference in absorption. Apparently, phenylacetyl-AMP resulting from the reaction of phenylacetate and ATP can readily react with hydroxylamine, but the possibility cannot be ruled out that catalytic amounts of CoA remain in the enzyme preparation at the low degree of purity achieved. In order to ascertain the participation of CoA in the overall coupled reaction, other procedures such as that of Grunert and Phillips, ~ which gave positive results in the authors' laboratory, may be applied; for assays in the course of enzyme purification the hydroxamate procedure appears to be more advantageous.
Production of Enzyme Enzyme-containing mycelium may be obtained from a penicillin plant (harvested at the third or fourth day of fermentation), or produced in the laboratory as follows: Cultures of a penicillin-producing strain of Penicillium chrysogenum are maintained on Sabouraud-sucrose agar slants. Strains yielding 1000-2000 units of penicillin per milliliter (1 unit = 0.6 t'g sodium benzylpenicillin) are sufficient. Spore suspensions are prepared as follows: barley grains are rinsed with tap water and subsequently steeped in a solution containing: lactose, 30 g/liter; KH2PO~, 3.0 g/liter; K~HP04, 3.0 g/liter; MgS04" 7 H20, 0.1 g/liter; corn steep liquor, equivalent of 0.85 g nitrogen per liter. After 30 min the liquid is decanted and layers of grains about 2 cm deep are placed in Erlenmeyer flasks with cotton plugs and sterilized for 20 min at 120 ° on 3 successive days. One milliliter of a spore suspension obtained from an agar slant is used to inoculate each flask. After 1 week of incubation at 25 °, sterile saline is added aseptically, and the spore suspension obtained by shaking the flasks is transferred aseptically to a sterile flask. It should contain 107-10s spores per milliliter and may be kept in the cold for several months. The nutrient medium contains (in grams per liter): glucose, 10.0; lactose, 30.0; starch, 10.0; dextrine, 5.0; yeast extract, 1.0; citric acid, R. R. Grunert and P. H. Phillips, Arch. Biochem. 30, 217 (1951).
[31]
PHENACYL:COENZYME A LIGASE
479
4.0; lactic acid, 3.0; ammonium acetate, 3.5; ammonia solution (25%), 3.0; KH,,PO,, 1.0; M g S Q " 7 H20, 0.5; FeSO4 • 7 H20, 0.05; Z n S Q . 7 H._,O, 0.01; CUSP4.5 H~O, 0.01; MnSO4" 7 H20, 0.01; Co(NO~)2" 6 H,_,O, 0.005; CaCl.., • 12 H.,O, 0.05; NaC1, 1.0; phenylacetic or phenoxyacetic acid, 2.0. The pH is adjusted to 5.8 with 2 N NaOH and 150 ml of medium are placed in l-liter culture flasks and sterilized at 120 ° for 20 min. After inoculation with 0.5 ml of spore suspension propagation is performed at 25 ° on a rotary shaker (e.g., Model V, New Brunswick Scientific Co.) at 280 rotations per minute. Maximum enzyme production is usually attained after 4 days. The mycelium is filtered with suction and washed three times with cold 0.5% potassium chloride. It may t)e stored in the frozen state; alternatively, freeze-dried mycelium may t)e prepared according to common procedures. Such preparations are very stable, and extraction is less diffficult. Drying with acetone results in considerable losses of enzyme activity. To obtain a crude enzyme extract, the following procedures may be used: Fresh or frozen mycelium is mixed with an equal amount of Celite (Hyflo Super Cel, Johns Manville Co., Baltimore) and ground to a homogeneous paste for 15 min in a cooled mortar. The paste is extracted at 0 ° with 2-3 times the mycelial weight of 10 mM K2HPO~ with stirring, and centrifuged for 30 min at 0 ° at 30,000 g. Freeze-dried mycelimn is ground to a fine powder, mixed with 8 times its weight of glass heads (average diameter, 500 ~m), suspended in 7 times the mycelial weight of 50 mM Tris • HC1 buffer pH 9.0, and treated in a vibrating disintegrator '; for 15 min under cooling at 3-5 °. The suspension is separated from the glass beads by low speed centrifugation for about 5 min, the glass beads are washed with 3 times the mycelial weight of 50 mM Tris . HC1 buffer pH 9.0, and the combined supernatants are centrifuged at 30,000 g for 20 rain at 0% In each case the extract should yield approximately 10-15 mg of protein per milliliter. The use of buffer solutions of higher pH, as indicated in the second procedure, has proved especially efficient in the case of mycelia from industrial fermentations, which frequently give lower protein yields. If not used immediately, the extract should be kept frozen. Partial Purification
Since several of the common procedures of enzyme purification (e.g., adsorption on calcium phosphates, precipitation with organic solvents, 6A "VIBROGEN" cell mill (E. Biihler, Tiibingen, Germany), operated at 4000 rpm, was used by the authors.
480
ANTIBIOTIC BIOSYNTHESIS
v
O
¢9
~0
©
~9 Z
O Z O
y, ~9
¢D
O v
O
L~ tt~
[31]
[31]
PHENACYL:COENZYME A LIGASE
481
absorption by DEAE-cellulose at different pH and ionic strengths) have not been successful, a procedure leading only to a moderate degree of purity has been elaborated. DEAE-cellulose (DE 23, Whatman) is pretreated and equilibrated with 50 mM Tris. HCl-buffer of pH 8.0 according to the instructions given by the manufacturer. The pH of the crude extract is adiusted to 8.0. To each 100 ml of extract about 100 ml of DEAE-cellulose suspension corresponding to 140 g of dry cellulosic material per 100 g of protein are added under stirring. After treatment for at least 15 nfin at 0 ° the suspension is filtered with suction over filter paper and the filtrate eolletted. To each 100 ml of filtrate, 71 g of solid ammonium sulfate are added (95% saturation) at 0 ° under constant stirring; stirring is continued for at least 30 rain. After eentrifugation at 30,000 g for 30 rain at 0 °, the precipitate is dissolved in a sufficient amount of 50 mM Tris • HC1 buffer pH 9.0 containing a few crystals of glutathione. The solution is brought to 40% saturation of ammonium sulfate as described above, centrifuged, and the precipitate discarded. The supernatant solution is brought to 70% saturation of ammonium sulfate as described before, and the precipitate obtained after centrifugation at 30,000 g is dissolved in a minimmn amount of 50 mM Tris • HC1 buffer pH 7.0. An example of the whole procedure is given in the table. Properties
Stabilitg. Whereas crude extracts are rather stable to prolonged storage at --20 ° in a pH range of 7 to 9, partially purified preparations are less stable under these conditions and particularly sensitive to repeated freezing and thawing. Addition of sulfhydryl compounds, e.g., glutathione, gives some protection. Rapid inactivation occurs at temperatures above 40% Specificity. Under conditions of the assay procedure, the enzyme preparation catalyzes the activation of phenylaeetic, phenoxyaeetie, and also acetic acid with similar degrees of activity. No change of the proportions of activity against the respective acids was observed during the purification procedure. Effect of pH. The enzyme preparation is maximally active in a pH range of 6 to 7.
482
ANTIBIOTIC BIOSYNTHESIS
[32]
[32] P h e n y l a c e t y l C o e n z y m e A H y d r o l a s e
By
BBIAN SPENCEa
Phenylaeetyl coenzyme A + H~O--~ phenylaeetie acid + eoenzyme A An enzyme catalyzing this reaction has been recognized in extracts of a member (Wis 51-20 F~) of the "Wisconsin Family" of high penicillinyielding mutants of Penicillium chrysogenum and in a number of related mutants?
Assay M e t h o d
Principle. [1-~4C]Phenylacetyl coenzyme A is incubated at 37 ° with P. chrysogenum extract as well as buffer, EDTA, and a sulfhydryl compound. The reaction is stopped by adding an excess of citrate buffer, pH 5.0, and the mixture is chromatographed on paper. The radioactivity of the spot corresponding to [1-14C]phenylacetic acid is counted by liquid scintillation and quantitated by reference to the radioactivity of standard [ 1-~4C] phenylacetyl CoA.
Reagents [1-14C]Phenylacetyl coenzyme A is prepared by the mixed anhydride method of Stadtman. 2 To 50 ~Ci of [1-14C]phenylacetic acid (39 mCi/mmole, The Radiochemical Centre, Amersham) is added 8.9 mg of phenylacetic acid, and the mixture is dissolved in 400 ~l of ether. After addition of 6,1 ~l of pyridine the mixture is cooled in ice and 7 ~l of ethylchloroformate is added slowly with agitation. During standing for 1 hr in ice, the tube is occasionally twirled by hand, causing the precipitated pyridinium chloride to stick to the sides of the vessel. The clear supernatant is pipetted off and added to a solution of 33.8 mg coenzyme A. (Sigma Chemical Co. Grade 1) in 1 ml of water, which has been adjusted to pH 7.6 with solid potassium bicarbonate. The tube containing the mixture is gassed with nitrogen and shaken for 30 rain. The solution is then adjusted to pH 2.0 with HC1 and extracted 4 times with 1.5 ml ether. The remaining ether is evaporated off by a stream of nitrogen. The solution is adjusted to pH 7.0 with solid lB. Spencer and Chit Maung, Biochem. J. 118, 29P (1970). E.R. Stadtman, this series, Vol. 3, p. 931.
[321
PHENYLACETYL COENZYME A HYDROLASE
483
potassium carbonate and distributed in 100-t~l portions in small tubes for storage at --20 ° . Radioactivity is counted by liquid scintillation and thiol ester content is assayed by the DTNB method of Elhnan 3 scaled down to deal with 40 t~l of diluted (1/9 v/v) sample. The yield is 1 ml of 35 mM [1-14C]phenylacetyl coenzyme A, and 10~1 spotted on paper give 450,000 epm measured as described below. Potassium phosphate buffer, 0.1 M, pH 7.5, containing 1 mM EDTA and 10 mM glutathione or N-acetylcysteine Citrate buffer, pH 5.0, 10% (w/v) Ethanol~n-butanol~28% (w/v) ammonium sulfate (2:1:1 by volume) 4 Whatman No. 1 chromatography paper that has been wetted with potassium phosphate buffer, pH 6.0 (75 g/liter of KH2P04, 25 g/liter of K~HPO~), blotted to remove excess buffer and dried 5 2,5-Diphenyloxazole (PP0), 5 g, and of 2,2-p-phenylenebis(5phenyloxazole) (POPOP), 0.3 g, dissolved in 1 liter of toluene
Assay of the Hydrolase. The enzyme preparation, 20 td, is incubated with l0 t~l of 24 mM [l-14C]phenylacetyl CoA and 10 t~l of 0.1 M potassimn phosphate buffer, pH 7.5 containing 1 mM EDTA and 10 mM glutathione. In controls the enzyme preparation nmst be replaced by the buffer-sulfhydryl mixture in which the enzyme sample is contained and the controls must be incubated along with the enzyme reaction tubes. After incubation for 15 rain, the reaction is stopped by adding 20 ~1 of 10% (v/v) citrate buffer, pH 5.0. Portions (10 ul) of the treated reaction mixtures including controls are chromatographed for 5-10 hr by descending chromatography on treated Whatman No. 1 paper using the ethanol/ n-butanol/28% ammonium sulfate solvent. The spot at RI 0.65, corresponding to ['~CIphenylacetic acid, is cut out, placed in a glass vial with 10-12 ml of the scintillation fluid and counted for sufficient time (usually 2-5 rain) in a Packard Tri-Carb liquid scintillation spectrometer Model 3375 to obtain a count rate with an SE. of less than 1%. The count rate is quantitated by reference to the radioactivity of standard amounts of [1-~C]phenylacetyl CoA which are spotted onto a piece of chromatography paper. The assay is linear with enzyme concentration and with time only over 20 rain. C. Ellman, Arch. Biochem. Biophys. 82, 70 (1959). 4 p. L. Tardrev and M. J. Johnson, J. Bacteriol. 76, 400 (1958). D. L. Pruess and M. J. Johnson, g. Bacteriol. 94, 1502 (1967).
484
ANTIBIOTIC BIOSYNTHESIS
[32]
A unit of phenylacetyl CoA hydrolase is defined in the standard manner as that amount which hydrolyzes 1 ~mole of the substrate per minute.
Production and Purification
Culture on Agar Slopes. Penicillium chrysoqenum Wis 51-20F3 is maintained as a spore suspension in soil and subcultured on tomato juice agar [canned tomato juice adjusted to pH 6 with 1 N NaOH and diluted 1:1 (v/v) with 2.5% (w/v) agar solution (Difco Bacto) ]. Shake Cultures. Inoculations are carried out with fresh spore suspension prepared by washing spores from a tomato-agar slope with 5 ml of sterile deionized water containing 0.1% (v/v) Tween 80. Five milliliters of this spore suspension are used to inoculate 250-ml Erlenmeyer flasks containing 40 ml of the following sterile glucose-lactate salts medium (in g/liter): D-glucose, 40.0; ammonium lactate, 21.0; KH:PO~, 3.0; Na2SO~, 0.74; magnesium acetate, 0.25; ZnCl~, 0.02; CaCO3, 13.0; MnCl~. 4H20, 0.02; FeCl~ • 6H20, 0.02; and CuCl~ 2H~_O,0.005. The glucose/ lactate is sterilized separately from the salts solution. The inoculated flasks are shaken for 2 days at 25 ° and 250 rpm on a gyratory shaker. The preculture (40 ml) is used to inoculate l-liter Erlenmeyer flasks containing 400 ml of sterilized fermentation medium composed of (g/l) : lactose, 50; corn liquor, 50; Na2SO~, 1. Growth is carried out at 28 ° on a gyratory shaker (250 rpm). At 24 hr, 1 ml of lard oil containing 5% v / v Tween 80 is added to prevent foaming. After 3 days growth, the mycelium is harvested by filtering through a double layer of cheesecloth and washed thoroughly with cold water. The mycelium is then pressed between filter papers to remove excess water and is used either immediately or after storage at --20 °. A yield of 15-25 g of "pressed-dry" mycelium per flask is obtained.
Purification
Step 1. Ten grams of press-dried mycelium is suspended in 40 ml of ice-cold 50 mM potassium phosphate buffer, pH 7.6 (containing 5 mM dithiothreitol and 1 mM EDTA), and ground lightly in a cooled mortar for about 2 min. The mixture is then extruded twice in a cooled French press at 5000-7000 psi. The pH of the extract is adjusted to pH 7.6 and mycelial debris is removed by centrifugation. The resulting supernatant, 38-40 ml, has a protein concentration of 15-20 mg/ml.
[32[
PHENYLACETYL COENZYME A HYDROLASE
485
Alternatively, the mycelium can be ground with buffer and sand until a thin paste is given and then centrifuged. The volume of supernatant is less and the protein concentration about half that achieved using the French press. Step 2. Solid ammonium sulfate is added to a supernatant solution to a concentration of 209 g/liter (35% saturation). The precipitate is removed by centrifugation, and further ammonium sulfate is added to a final concentration of 313 g/liter (50% saturation). The precipitate is collected by centrifugation and dissolved in 2 ml of 50 mM phosphate buffer, pH 7.5, containing 1 mM D T T and 1 mM EDTA. The enzyme at this stage is stable for several weeks at --20% Step 3. The 35-50% ammonium sulfate fraction (2.5 ml) is desalted on a Sephadex G-25 (coarse) column which has been equilibrated with 50 mM phosphate buffer pH 7.5 containing 1 mM D T T and 1 mM EDTA, the elution being carried out with the same buffer. A DEAE-cellulose column (0.9 }( 12.5 cm) is equilibrated with 50 mM phosphate buffer pH 7.5 containing 1 mM D T T and 1 mM EDTA. The desalted extract (12 ml) is absorbed onto the colmnn which is then washed with 20 ml of the equilibrating buffer. The enzyme is eluted from the column using the equilibrating buffer containing 0.1 M KC1. After discarding the void volume, the next 6 ml are collected. Step 4. A Sephadex G-100 column (1.2 X 75 cm), Vo 34 ml, flow rate 20-25 ml/hr, is first equilibrated with 50 mM phosphate buffer pH 7.5 containing 1 mM DTT and 1 mM EDTA. The DEAE fraction (6 ml) is applied to the column and eluted with the same buffer. Two-milliliter fractions are collected, and the most active fractions (fraction numbers 27-31) are pooled (10 ml). The pooled fractions from step 4 represent a 130-fold purification from step 1 with recovery of 25% of the activity. The specific activity is 0.15 unit per milligram of protein.
Properties of the Purified Enzyme
Specificity. It has been suggested 1 that phenylacetyl CoA hydrolase is only one of four activities attributable to a single thiol-dependent enzyme whose action involves a Ping-Pong Bi Bi mechanism with an alternate hydrolytic step. The other activities are penicillin acyltransferase, 6-aminopenicillanic acid acyltransferase, and penicillin acylase. The evidence is based on the constant ratio between the activities during various fractionation and purification procedures, inhibition, and pH and temperature inactivation. In the crude extract (step 1) the ratio of phenylacetylCoA hydrolase to the other activities is higher than in the purified ex-
486
ANTIBIOTIC :BIOSYNTHESIS
[32]
tracts, suggesting the presence of nonspecific enzymes at this stage which can hydrolyze phenylacetyl-CoA. The enzyme is approximately 5.0 times as active against phenoxyacetyl-CoA as compared to phenylacetyl-CoA, 6 and this compares to a similar ratio between the acylase activity of the preparation in hydrolyzing penicillins V and G. The purified preparation also hydrolyzes p-nitrophenyl acetate and phenoxyacetyl glycine at about the same rate as the hydrolysis of phenylacety]-CoA. The ratio of these various activities is the same in steps 3 and 4, but there is no other indication to confirm that the same enzyme is responsible. Other Properties. The hydrolase activity shows a pH optimum of 7.6-7.8 and a Km of 3.95 raM. The molecular weight by Sephadex gel filtration 7 is 25,000. The enzyme is sensitive to sulfhydryl inhibitors and is completely inhibited by pretreatment wth 2 mM N-ethylmaleimide, 2 mM dithiobis(2-nitrobenzoic acid), and 2 mM p-mercuribenzoate. The inhibition is reversible by subsequent addition of excess thiol compounds. The enzyme is sensitive to oxidation during the purification procedure, and it is necessary to keep the enzyme in a reduced state by the presence of thiol compounds. The oxidized enzyme fractionates differently on DEAE-cellulose. Free thiol compounds are not involved in the mechanism of action, and when they are removed by passing the enzyme preparation through Sephadex G-25 and thiols are omitted from the assay mixture, some activity is still observed. However, thiol compounds need to be present during the assay for maximum activity. Despite the presence of 1 mM D T T and 1 mM EDTA the purified preparation is not stable, and about 50% of the activity is lost during storage at 0 ° for 24 hr. Comments on the Assay Procedure. The necessity for free thiol during assay introduces complications due to a rapid S -~ S intermolecular acyl migration between phenylacetic acid and the thiol that occurs at pH above 6.5 and which leads to the elimination of free CoA. When the added thiol does not contain NH.. or OH groups proximal to the SH group (e.g., N-acetyl cysteine, thioglycolic acid, glutathione), the acyl group simply equilibrates between CoA and the added thiol, the rate of equilibration increasing with the pH. When proximal NH2 or OH groups are present in the thiol (e.g., DTT, DTE, cysteine, cysteamine, mercaptoethanol), the S-> S acyl migration proceeds to completion owing to subsequent S--> N and S--> O intramolecular acyl transfer. Further reactions can Chit Maung, Ph.D. Thesis, Dublin University, Ireland, 1970. 7p. Andrews, Biochem. J. 91, 22"2(1964).
[331
ERYTHROMYCIN C 0-METHYLTRANSFERASE
487
include elimination of free [14C]phenylacetic acid and the formation of cyclic compounds t h a t contain [1-14C]phenylacetic acid. These reactions not only alter the concentration of substrate during the reaction, but nonenzymieally liberated [1-1~C]phenylacetic acid and other products, which chromatograph at the same R~, can lead to spurious high results. By avoiding those thiols t h a t can carry out intramoleeular S --> 0 and S ~ N aeyl migrations, interference during the assay can be limited, even at its fullest extent, to the equilibrimn position governed by the amounts of phenylacetyl-CoA and thiol used. This interference can be further limited by keeping the ratio, phenylaeetyl CoA:thiol, high and the p H and assay time low. The assay conditions recomn~en(ted take these points into consideration. In the preparation of the enzyme it is convenient to use buffer containing D T T , and the residual amounts of this compound will produce some nonenzymieally liberated [~4C~]phenylacetie acid. I t is therefore necessary for the control to contain the same buffer as t h a t in which the enzyme is dissolved and for the control to be incubated.
[33] S-Adenosylmethionine: Erythromycin C O-Methyltransferase By JOHN W. CORCORAN E r y t h r o m y c i n s A, B, and C, 1 the macrolide antibiotics elaborated by Streptomyces erythreus have the structures shown in Fig. 1. I t has been demonstrated t h a t the methyl groups attached to the C-3"-,3"-0, and N atoms of the sugars of the erythromycins are derived from L-methionine. -°,3 The O-methylation of the L-mycarose moiety of erythromycin C by a partially purified enzyme obtained from extracts of S. erythreus is described here. The reaction catalyzed is shown in Fig. 1Abbreviations : Ea, the lactone of erythromycin A, also called erythronolide A; D, D-desosaminyl group; M, L-mycarosyl group; C, b-cladinosyl group; EaDM, erythromycin C; EaDC, erythromycin A; EDTA, ethylenediaminetetracetic acid, disodium salt; DTT, dithiothreitol; SAM, S-adenosyl-L-methionine; SAH, S-adenosyl-L-homoeysteine; REV, relative elution volume, defined as a ratio of the elution volume (Ve) over the bed volume (Vt). 2 j. W. Corcoran, J. Biol. Chem. 236, PC 27 (1961). 3j. Majer, M. Puza, L. Dole~ilov£, and Z. Vanek, Chem. Ind. (London) 1961, p. 669 (1961).
[331
ERYTHROMYCIN C 0-METHYLTRANSFERASE
487
include elimination of free [14C]phenylacetic acid and the formation of cyclic compounds t h a t contain [1-14C]phenylacetic acid. These reactions not only alter the concentration of substrate during the reaction, but nonenzymieally liberated [1-1~C]phenylacetic acid and other products, which chromatograph at the same R~, can lead to spurious high results. By avoiding those thiols t h a t can carry out intramoleeular S --> 0 and S ~ N aeyl migrations, interference during the assay can be limited, even at its fullest extent, to the equilibrimn position governed by the amounts of phenylacetyl-CoA and thiol used. This interference can be further limited by keeping the ratio, phenylaeetyl CoA:thiol, high and the p H and assay time low. The assay conditions recomn~en(ted take these points into consideration. In the preparation of the enzyme it is convenient to use buffer containing D T T , and the residual amounts of this compound will produce some nonenzymieally liberated [~4C~]phenylacetie acid. I t is therefore necessary for the control to contain the same buffer as t h a t in which the enzyme is dissolved and for the control to be incubated.
[33] S-Adenosylmethionine: Erythromycin C O-Methyltransferase By JOHN W. CORCORAN E r y t h r o m y c i n s A, B, and C, 1 the macrolide antibiotics elaborated by Streptomyces erythreus have the structures shown in Fig. 1. I t has been demonstrated t h a t the methyl groups attached to the C-3"-,3"-0, and N atoms of the sugars of the erythromycins are derived from L-methionine. -°,3 The O-methylation of the L-mycarose moiety of erythromycin C by a partially purified enzyme obtained from extracts of S. erythreus is described here. The reaction catalyzed is shown in Fig. 1Abbreviations : Ea, the lactone of erythromycin A, also called erythronolide A; D, D-desosaminyl group; M, L-mycarosyl group; C, b-cladinosyl group; EaDM, erythromycin C; EaDC, erythromycin A; EDTA, ethylenediaminetetracetic acid, disodium salt; DTT, dithiothreitol; SAM, S-adenosyl-L-methionine; SAH, S-adenosyl-L-homoeysteine; REV, relative elution volume, defined as a ratio of the elution volume (Ve) over the bed volume (Vt). 2 j. W. Corcoran, J. Biol. Chem. 236, PC 27 (1961). 3j. Majer, M. Puza, L. Dole~ilov£, and Z. Vanek, Chem. Ind. (London) 1961, p. 669 (1961).
488
[33]
ANTIBIOTIC BIOSYNTHESIS
R,J, 2
0
~'1
-I,~I g
O
_ HO,-,,,~IV~eN/Me
,,Ho I
~
-'~/-~CH3
~.z/~ORz
~O.~',~OH CH 3
Rz CH5 B H CH3 C OH H Fie. 1. Structure of erythromycins A, B, and C. ERYTHROMYCIN A
o° c . , . . O . o . +
~O-'~CH3
Erythromycin C
R= OH
I -]5 SPECIFIC ~.~-OD CHi3~ 0 OCH~ H TRANSFERASE Eo 3 O SAM
_P-- -Lo
c. '
Erythromycin A
FIG. 2. Conversion of erythromycin C into erythromycin A in the presence of S-adenosyl-L-methionine (SAM). 2 and the enzyme specifically converts erythromyein C into erythromycin A in the presence of S-adenosyl-L-methionine (SAM).45
Assay I n c u b a t i o n Conditions. Characterization of the transmethylase activity is done in a very simple incubation mixture. As employed for studying the kinetic p a r a m e t e r s of the transmethylase and for determiming possible control factors the components of the incubation are as follows (in micromoles per total volume of 1.5 ml) : Buffer (potassium phosphate) 125, p H 7.5 E D T A , 0.1 D T T , 1.0
4 T. S. McAlpine and J. W. Corcoran, Fed. Proc., Fed. Amer. Soc. Exp. Biol. 30, 1168 (1971). 5 T. S. McAlpine, Enzymatic O-methylation of erythromycin C in the biogenesis of erythromycin A. M. S. Thesis, Northwestern University, 1971.
[33]
ERYTHROMYCIN C 0-METHYLTRANSFERASE
489
Erythromycin C, 0.5, except when specified otherwise [~4CH:,]S-adenosyl-L-methionine, 0.5 (50,000 cpm, except when specified otherwise) Enzyme (6-10 mg of protein, microsomal fraction or ammonium sulfate fraction)
Assay Procedures Two different assays of SAM:EaDM transmethylase activity have been used. The first measures the formation of the product erythromycin A, which is detected by partition chromatography as such or by the formation and purification of the crystalline 2-O-benzoyl derivative. The second assay procedure follows the transformation of the L-mycarosyl moiety of erythromycin C into L-cladinose, which is liberated from the product, erythromycin A, by mild acid hydrolysis. Chromatographic separation of the two neutral sugars, L-mycarose coming from the substrate and L-cladinose from the product, is rapid and complete, the only possible radioactive product being L-cladinose.
Assay I. Measurement of the Formation o] Erythomycin A ]ro~t Erythromycin C. The incubation is carried out for 60 min at 33 ° in a water bath without aeration. The reaction is terminated by the addition of acetone (2 volumes). After 30 min, the precipitated protein is removed by centrifugation, and most of the acetone is removed by evaporation. The aqueous residue is then extracted with methylene chloride (2 times, 2 ml each time). The extract is washed with a minimal volume of water, and the organic phase is concentrated to dryness by vaporation under nitrogen. The residue may then be subjected to partition chromatography (see Chromatographic Methods).
Assay II. Measurement o] the Radioactivity Incorporated into the L-Cladinose Moiety o] Erythromycin A. The enzyme assay is the same as that described for Assay I except that the incubation is terminated by the addition of trichloracetic acid (10% to pH 2, approximately 0.25 ml). Nonradioactive erythromycin A is added (2.0 rag), and the mixture is heated at 90 ° for 30 rain. After cooling in an ice bath, the precipitated protein is removed by centrifugation and washed with water (2 times. 1.5 ml each). The combined supernatant fraction and washings are passed through an Amt)erlite MB-3 resin column (1 cm X 14 cm), the material not adsorbed to the resin is collected, and the column is eluted with a small amount of water (8 ml). Nonradioactive L-cladinose (5.0 my, prepared by hydrolysis of erythromyein A) is added to the combined eluate and wash, and water is removed under reduced pressure. The oily residue is dissolved in a minimal amount of ethyl acetate and chromato-
490
ANTIBIOTIC BIOSYNTHESIS
[33]
graphed on a TLC plate to separate L-mycarose from L-cladinose (see Chromatographic Methods).
Erythromycin C (EaDM) and Its Spiroketal (6 --> 9; 12 --->9) Erythromycin C is not commercially available and current production strains of S. erythreus accumulate little, if any. However, a sample of mother liquors from crystallization of erythromycin A supplied by Abbott Laboratories was somewhat enriched in EaDM. Gel filtration using a lipophilic derivative of dextran (Sephadex LH-20, Pharmacia Fine Chemicals) permits very superior fractionation and purification of the EaDM and other erythromycin derivatives as well. A column (2.0 X 120 cm) of Sephadex LH-20 (particle size 25-100 ~m) in a chloroform/hexane mixture (1:1) with a bed volume of 300 ml is prepared, and the mixture containing EaDM dissolved in the same solvent is applied. Fractions (5 ml) are collected at a flow rate of 0.1 ml/cm 2 per minute. The relative elution volumes (Ve/Vt, or REV) of erythromycins B, A, C are 0.8, 0.85, and 0.95 respectively, while the 6-> 9; 12-~ 9 spiroketals of erythromycin A and C eluted at REV 1.0 and 1.10. Thin-layer chromatography is carried out using silica gel plates (F-254, E. Merck, Darmstadt) in a solvent mixture of ethyl acetate, isopropanol, 15% ammonium acetate (pH 9.7), 27:21:24 (upper phase)2 Anisaldehyde and concentrated sulfuric acid in ethanol (1:1:9) are used as a detection reagent (colors develop after heating). RI values relative to erythromycin A (R~ = 1.0) are 1.05 for erythromycin B and 0.87 for erythromycin C. The RI values of the respective 6--> 9; 12--> 9 spiroketals are 1.12 (A) and 0.93 (C).
Erythromycin A Commercial antibiotic (Abbott) is repeatedly recrystallized by dissolving in a minimum amount of ice water and then warming to 37 ° (negative temperature coefficient of solubility) until the crystalline material is homogeneous (TLC, partition paper chromatography, etc., see below). It can also be recrystallized from organic solvents (isopropanol, chloroform, etc.).
S-Adenosyl-L- methionine (SAM) Commercial material, both nonradioactive and radioactive, is used. As usually supplied, it is contaminated with a substantial amount of 6 W. Malczewska-Konecka, Z. Piekarska, and Z. Kowszyk-Gindifer, Chem. Anal. (Warsaw) 14 (5), 1093 (1969).
[33]
ERYTHROMYCIN C 0-METHYLTRANSFERASE
491
S-adenosyl-L-homocysteine. As with many transmethylases, 7-9 the enzyme described here is inhibited by this end product of the enzymic transmethylation reaction. Although the reaction can be monitored with unpurified substrate, the rate is quite dependent on the state of its purity. For studies of the kinetic parameters of the enzyme, the commercial SAM is purified by ion-exchange chromatography. The methods of Schlenk and D e P a l m a TM and Shapiro and Ehringer 11 are suitable.
Purification Gro~vth o] S t r e p t o m y c e s erythreus
A slant culture of S. erythreus (Abbott-CA340) is used to inoculate (dry loop) 50 ml of a sterile soluble vegetative medium (glucose, 5 g; commercial brown sugar, 10 g; tryptone, 5 g; yeast extract, 2.5 g; E D T A , 36 mg; betaine, 1.29 g; sodium propionate, 0.11 g; tap water, 1100 ml; final pH 7.0-7.2 adjusted with K O H ) contained in 500 ml wide-mouth Erlenmeyer flasks. The flasks which are closed with 2 milk filter disks (Filter Fabrics Inc., Goshen, Indiana) secured with rubber bands are shaken at 33 ° for 2.5 to 3 days on a New Brunswick gyratory shaker at 200 rpm. The resultant culture is used (10% v / v ) to inoculate 350 ml of the same medium contained in 2-liter Erlenmeyer flasks, and after growth under the same conditions for 24 hr the second culture is used to inoculate (10%, v / v ) a New Brunswick Microferm fermentor, containing 10 liters of the same medium. The fermentor is stirred at 500 rpm at an aeration rate of 10 liters/min. The growth obtained after 12 hr at 33 ° (75 g net weight) is harvested by continuous flow centrifugation at 27,000 g using a Sorvall RC-2B centrifuge. It is washed twice, each time by suspension in 2 volumes of 10 m M phosphate buffer (pH 7.5) containing 0.1 m M E D T A and 0.1 m M D T T (Buffer A) and stored at --80 ° with 40% glycerol ( v / v ) . Culture conditions for S. erythreus have been described by Kaneda et al. 1'~ and Friedman et al. ~3 A. Y. Akamatsu and J. L. Law, J. Biol. Chem. 245, 709 (1970). 8T. Deguchi and J. Barchas, J. Biol. Chem. 246, 3175 (1971). V. Zappia, C. R. Zydek-Cwick, and F. Schlenk, J. Biol. Chem. 244, 4499 (1969). ~°F. Schlenk and R. E. DePalma, J. Biol. Chem. 229, 1051 (1957). 11S. K. Shapiro and D. J. Ehrimger, Anal. Biochem. 15, 323 (1966). 12T. Kaneda, J. C. Butte, S. B. Taubman, and J. W. Corcoran, J. Biol. Chem. 237, 322 (1962). 1~S. M. Friedman, T. Kaneda, and J. W. Corcoran, J. Biol. Chem. 239, 2387 (1964).
492
ANTIBIOTIC BIOSYNTHESIS
[33]
SAM :EaDM Transmethylase Cell-Free Extract and Microsomal Fraction. Either freshly collected or frozen and thawed cells of S. erythreus suspended in 0.1 M phosphate buffer (pH 7.5) containing 0.1 mM EDTA and 1 mM D T T (Buffer B) are disrupted by a single passage through a French pressure cell at pressures between 8000 and 10,000 psi, and the exudate is centrifuged at 48,000 g for 30 min at 5 °. The resultant supernatant solution may be used as a crude cell-free enzyme preparation. For other experiments this crude extract is centrifuged at 105,000 g for 150 min in a Spinco Model L2-65B preparative ultracentrifuge, and the resultant microsomal pellet was then homogenized gently in a Thomas tissue grinder with one-fourth of the original volume (crude extract) of buffer B. Ammonium Sul]ate Precipitation. All operations are conducted at 5 ° . The crude cell-free extract is gradually brought to 70% of saturation with ammonium sulfate by adding the solid salt (490 mg/ml during 30 min). After 30 rain of equilibration the precipitated protein is collected by centrifugation. The precipitate contains the transmethylase activity, and it can be stored at --15 ° for 1 month without significant loss of activity. For assays, the precipitate is diluted to 25% of the original (crude extract) volume of the crude extract with buffer B and dialyzed for 2 hr with three changes of 100 volumes of phosphate buffer (50 mM, pH 7.5) containing EDTA and D T T (0.1 mM each). The dialyzed enzyme can be kept at 4 ° for several days without significant loss in activity. For studies of enzyme properties and substrate specificity, a further fractionation by ammonium sulfate may be done. In the first step, the crude cell-free extract is brought to 30% of saturation with solid ammonium sulfate (210 mg/ml). After 30 rain, the precipitate is removed by centrifugation and discarded. The supernatant solution is brought to 60% of saturation with ammonium sulfate by adding more solid salt (210 mg/ml). The precipitated protein contains the transmethylase activity.
Chromatographic Methods
Partition Chromatography of Erythromycins A and C. The method is based on the procedure described briefly by Friedman et al. 1" It is a somewhat difficult method, especially when compared to TLC chromatography (above and below), but affords superior resolution of closely related members of the erythromycin family and their derivatives. For analytical work and small-scale preparative separations, the chromatog-
[33]
ERYTHROMYCIN C 0-METHYLTRANSFERASE
493
raphy is done with filter paper as the support for the stationary phase (Whatman 1 or 3 MM, depending on sample amount). The solvent system employed consists of benzene (50 ml), n-heptane (25 ml), acetone (15 ml), isopropanol (10 ml), and potassium phosphate buffer (25 ml, pH 7.0). The paper is impregnated with the stationary (lower) phase of the system and blotted briefly between dry sheets of filter paper. The residue to be analyzed is applied as a solution in acetone, and the chromatogram is then developed by the descending method using the upper phase of the system. Depending on the temperature and relative humidity of the laboratory, the time of development (2-4 hr) and the relative mobilities of the two erythromycins (and other derivatives) vary a great deal. In :::any of the past experiments, erythromycin A had an R r value of 0.6-0.7, that of erythromycin C was much lower, in the range of 0.2-0.3. The product of an enzyme assay is run in a lane separated from the remainder of the chromatogram by a narrow gap cut on either side (scalpel or razor blade), and appropriate standards are put on the same sheet. After removal of the unknown lane(s), the standard samples of erythromycins C and A are detected by spraying with a vanillin (0.5% by weight) perchloric acid (1 M in absolute ethanol) reagent and gentle heating in the absence of air (e.g., in Saran foil) at about 80 ° (grayish blue spots on a light background). The lane to which the residue from the assay system is applied is analyzed with a paper strip counter and the amount of radioactivity with the same migration rate as the reference sample of erythromycin A is a measure of the activity of the SAM:EaDM transmethylase. The chromatographic method described is also adaptable to column separations. In this case the stationary phase is :nixed with silica gel (0.5-1.0 g per gram of gel, giving a friable powder) and packed in a column under the mobile phase of the system. The sample is dissolved either in a m:nimal amount of the mobile phase and added to the top of the column or (if relatively insoluble) it is dissolved in a similar volume of stationary phase. In the latter case the solution is mixed with sufficient silica gel to give a seemingly dry powder, and this is layered on the top of the column. Development with the mobile phase is started and fractions are collected. The relative elution volumes are in the same sequence as the relative migrations of the substances on paper, but the absolute elution volumes are not easily predicted from the R~ values. TLC Chromatography. TLC analysis of the erythromycins is described above. The sugars liberated from the erythromycins by acid hydrolysis also are conveniently separated by TLC chromatography. Plates coated with silica gel are used together with a solvent composed of benzene and acetone (7:3, v/v). When the results of enzyme assays with
494
ANTIBIOTIC BIOSYNTHESIS
[331
radioactive SAM are analyzed, the unknown substances are placed on one side of the plate and reference samples of L-myearose and L-eladinose are placed on the other. After development of the plate, the unknown side is covered with aluminum foil or Saran foil and the standard side is sprayed with the vanillin/perchlorie acid reagent (above). Color development is achieved by gentle drying and heating at 80 °. The sugars afford purplish spots on a light background. The side of the plate containing the radioactive unknowns is divided into zones (ca. 2 X 2 em per lane), and each is scraped into a scintillation vial for measurement of total radioactivity. L-Myearose has an Rf value of 0.25 in this TLC system, and L-eladinose has one of 0.55. Derivatization of Erythromycin A. It is useful, in standardizing the assay for SAM:EaDM transmethylase, to make an independent check of the radiopurity of the erythromyein A produced. It also serves as a control on the functioning of the partition chromatographic separation of the EaDC from possible radioactive contaminants. For this purpose, the erythromyein A produced in an enzyme assay (Assay I) may be diluted with carrier nonradioactive EaDC (17.6 mg, 0.024 mmoles) and a monobenzoyl derivative prepared as described by Kaneda et al. 1~ The combined sample of the carrier erythromyein A and the residue from the assay are dissolved in dry acetone (1 ml) and treated with a solution of benzoic anhydride (8.35 mg, 0.037 mmole) in dry acetone (1 ml). The reaction is conveniently done in a conical centrifuge tube (12 ml) and the solutions are warmed briefly to the boiling point using a fine glass rod to prevent bumping. The mixture is left overnight at 25 ° in the dark, and then the acetone is evaporated under a stream of nitrogen. The oily liquid residue is dissolved in ethanol (95%), and the solution is passed through a small column (1 cm diameter X 5 em in length) of Amberlite IR-45 resin (free-base form) which has been washed with ethanol. The effluent solution plus several washes of the resin (3-5 times the bed volume) contains the etude monobenzoyl derivative of erythromyein A. It is free of excess benzoic anhydride and has been converted to the freebase form of the derivative (product is a benzoate salt prior to the resin treatment). After removal of the ethanol by evaporation under reduced pressure, the 2'-O-benzoyl derivative of erythromyein A is crystallized from minimal amounts of boiling isopropanol (ca. 0.3 ml). Recrystallization is done in the same manner, until a constant specific radioactivity is achieved. The specific radioactivity of the derivative will drop on the first crystallization and rise slightly on successive crystallizations if EaDC is the major radioactive substance isolated from the enzyme assay. Miscellaneous. All reagents not otherwise specified are commercial products used as obtained. Ammonium sulfate is "enzyme grade" as sup-
[33]
E R Y T H R O M Y C ICN 0-METHYLTRANSFERASE
495
plied by Schwarz/Mann. Solvents are analytical reagent grade. The antibiotics tylosin and niddamycin are products of Eli Lilly Co. and Abbott Laboratories, respectively. The sugars, L-mycarose and its anomeric methyl glycosides and L-cladinose, are prepared by the mild acid hydrolysis of tylosin TM and erythromycin A, 15 respectively. The methods used are those described in the literature, and no special purification is done. Mycarosyl erythronolide B, a monoglycoside of L-mycarose and the laetone of erythromyein B (lacking a lactone ring hydroxy group at C12) is a product of Abbott Laboratories. Radioactivity is measured with a liquid scintillation spectrometer (Nuclear Chicago Mark I with use of an external standard and a quench correction) and with a paper strip scanner (Nuclear Chicago Actigraph III). The scintillation system used contains naphthalene (125 g), 2,5diphenyloxazole (7 g), and 1,4-bis-2-(5-phenyloxazolyl)benzene in 1 liter of p-dioxane. Protein is measured by the biuret method; using bovine serum albumin as a standardJ ~
Properties Product o] the Transrnethylase Reaction. The sole radioactive product of the reaction between erythromycin C and S-adenosyl-L-methionine (14CH~) appears to be erythromycin A. This is shown by the apparent homogeniety of the radioactive substance which migrates with the same RI value as EaDC in a partition chromatographic system. This material also cochromatographs with added radioactive EaDC. Further confirmation of the radiopurity of the EaDC is afforded by finding that a monobenzoyl derivative [2'-O-benzoate] retains its radioactivity on repeated crystallization. Reversibility o] the Transmethylase Reaction. Incubation of radioactive erythromycin A, N-[14CH3]EaDC, of very high specific radioactivity obtained from Abbott Laboratories, with the SAM:EaDM transmethylase (mierosomal fraction or ammonium sulfate precipitated protein) yields no detectable erythromycin C (Assay I workup conditions). Thus the transmethylase under these conditions appears to be unable to carry out the transformation of erythromycin A into the C form of the antibiotic. Cellular Location o] the Transmethylase. On comparing the total enzymic activity in a crude cell extract with that in the microsomal frac14R. B. Morin, M. Gorman, R. L. Hamill, and P. V. Demarco, Tetrahedron Lett. 1970, 4737 (1970). ~5F. Wiley, R. Gale, C. W. Pettinga, and K. Gerzon, J. Amer. Chem. Soc. 76, 3121 (1955). '~A. G. Gornall, C. S. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949).
496
ANTIBIOTIC BIOSYNTHESIS
[33]
S-ADENOSYL-L-METHIONINE-DEPENDENT CONVERSION OF ERYTHI~OMYCIN C INTO ERYTHROMYCIN A a
Preparation
Total activity (~moles/hr)
Crude cell-free extract 105,000 g supernatant fraction 105,009 g microsomal fraction
387 67 171
Protein (mg)
275.5 189 75.6
Specific activity (~moles/mg protein/hr) 1.23 0.36 2.97
" Assays conducted as described in Assay Method II. tion, it is apparent that the enzyme is associated with the microsomal material (see the table). The specific activity of this microsomal fraction is approximately 8 times that of the remaining supernatant protein and at least twice that of the original crude extract. Kinetic Properties o] the Transmethylase. The conversion of EaDM to EaDC (Assay II) is dependent on SAM and enzyme. It is linear under the conditions used to about 15 mg of enzyme protein per assay and then falls off slowly up to 25 mg of enzyme protein per incubation. The rate of transmethylation is linear for nearly 60 min and continues at a slightly lower rate for another 60 rain. The effect of pH on the transmethylase activity is not dramatic, and a broad optimum is observed between pH 7.5 and 8.8. The maximum rate is seen at about pH 8.2-8.3. The pK~ of erythromycin C is in the range of pH 8.5-9.0 and the enzyme may use the unprotonated species as its substrate. The temperature dependence of the transmethylase is striking, with a fairly sharp optimum seen at 33-35 °. The strain of S. erythreus used (CA340) as a source of the enzyme grows well between 30 ° and 37 °, but its optimum temperature is also very near 33-34% The amount of EaDM used in the assay is nearly optimal, since the dependency of the enzyme on this substrate is nearly linear to about 0.2 ~mole per assay and falls off rapidly above this value. The amount of EaDM used (0.5 ~mole of EaDM) is seemingly in a nearly saturating range, but changes in conditions may affect this markedly. The products of the enzymic reaction [EaDC and S-adenosyl-L-homocysteine (SAH) ] markedly inhibit the transmethylase activity (43% and 76%, respectively, when 0.5 ~mole of each is added to separate incubation systems). The amount of SAM used to measure enzyme activity is well below the optimal amount, and with a constant amount of EaDM (0.5 ~mole) there is a maximum rate of enzyme activity when 4 ~moles of SAM are present. Increasing the level of SAM above this value produces a reduced rate of transmethylase activity, probably because of end-product inhibition.
[33]
ERYTHROMYCIN C 0-METHYLTRANSFERASE
497
An approximate K,~ for erythromycin C in the SAM:EaDM transmethylase reaction of S. erythreus (CA340) was calculated as 0.30 raM. This value and the total conversion of this substrate to product (ca. 3%) is probably very dependent on the assay conditions, and this K,~ value cannot be more than a rough indication of the enzyme's affinity for erythromycin C. Specificity of the SAM:EaDM Transmethylase. The enzyme shows a very high degree of substrate specificity. Aside from erythrolnycin C, it fails to catalyze the methylation of any L-mycarosyl moiety tested. Substances tested were 3-O-mycarosyloxyerythronolide B, the (6 ~ 9:12--~ 9)-spiroketal of erythromycin C, L-mycarose, the anomeric methyl L-mycarosides, and the antibiotics tylosin and niddamycin) ~ The latter two substances have a mycarosyl moiety present as part of a disaccharide attached to a 16-membered lactone whose structure is not very similar to that in erythromycin. A fourth member of the erythromycin family has been identified in this laboratory. It is called erythromycin D and has the same two sugars as does erythromycin C--namely L-mycarose and n-desosamine. The lactone of erythromyein D is that of erythromycin B and it lacks one tertiary hydroxyl group present in erythromycin C (at C-12). The protein (crude and ammonium sulfate precipitated) used in the study of tho EaDM:SAM transmethylase catalyzes the transformation of erythromycin D (EbDM) into erythromyein B. It remains to be proved that the EaDM:SAM and EbDM:SAM transmethylases are one and the same but it is likely that they are. If so, some variation in lactone structm'e is compatible with enzymic activity (hydrogen vs hydroxy at C-12). Space-filling models of erythromyeins C and A show that the neutral sugar in each is closely aligned (almost parallel) with the basic sugar and that both are roughly perpendicular to the plane of the laetone ring. 's It is somewhat difficult to see how the SAM:EaDM transmethylase can act at all, and it is hard indeed to comprehend the strict substrate specificity that it appears to possess. Significance of the S-Adenosyl-L-methionine :Erythromycin C Transmethylase The best evidence available from studies of physiologically intact S.
erythreus strains indicates that erythromycin C is a precursor of erythro~: G. Huber. K. H. Wallhiiusser, L. Fries, A. Steigler, and H. Weidenmtiller. Arzneim.-Forsch. 12, 1191 (1962). ~ T . J. Perun, in "Drug Action and Drug Resistance in Bacteria" (S. Mitsuhashi, ed.), Vol. I, p. 123. Univ. Park Press, Baltimore, Maryland, 1970.
498
ANTIBIOTIC BIOSYNTHESIS
[34]
mycin A. 10 The properties of the enzyme that produces EaDC from EaDM support this relationship. The potent inhibitory effect of both products, EaDC and SAH, on the methylation reaction also indicates that the activity of the transmethylase may be one control point in the overall biogenesis of erythromycin A. The latter accumulates in very high concentration in the fermentation beer of commercially useful strains of S. erythreus (strain CA340 is a former production strain of Abbott Laboratories), and it may be surmised that some barrier separates the S A M : E a D M transmethylase from this reservoir of inhibitor. The intracellular concentration of SAM is not known, but evidence exists (unpublished) to suggest that methylation steps in general are rate limiting in the biogenesis of the erythromycins. It is possible that the intracellular concentration of SAM and SAH is quite low and that recycling of the SAH to SAM reduces the concentration of this end-product inhibitor. The SAM:SAH ratio could be a major regulatory factor of the final step in erythromycin A formation. Since very little if any erythromycin C accumulates in strains of S. erythreus that are useful for erythromycin A accumulation, the activity of the EaDM:SAM transmethylase clearly is sufficiently great to prevent buildup of this direct precursor of erythromycin A. 19j. R. Martin and A. W. Goldstein, Proc. Int. Congr. Chemother. 6th, 2, 1112 (1970).
[34] S - A d e n o s y l m e t h i o n i n e : I n d o l e p y r u v a t e 3-Methyltransferase
By MARILYN K. SPEEDIE, ULFERT HORNEMANN, and HEINZ G. FLoss O II ~CH2--C--COOH H
+ S-Adenosylmethionine
CHs O F }l ~ C H - C--COOH
~
H
+ 5-S - Adenosylhomocysteine
The enzyme is isolated from an indolmycin-producing strain of Streptomyces griseus and is presumably involved in the biosynthesis of this antibiotic. The reaction catalyzed by this enzyme represents the first spe-
498
ANTIBIOTIC BIOSYNTHESIS
[34]
mycin A. 10 The properties of the enzyme that produces EaDC from EaDM support this relationship. The potent inhibitory effect of both products, EaDC and SAH, on the methylation reaction also indicates that the activity of the transmethylase may be one control point in the overall biogenesis of erythromycin A. The latter accumulates in very high concentration in the fermentation beer of commercially useful strains of S. erythreus (strain CA340 is a former production strain of Abbott Laboratories), and it may be surmised that some barrier separates the S A M : E a D M transmethylase from this reservoir of inhibitor. The intracellular concentration of SAM is not known, but evidence exists (unpublished) to suggest that methylation steps in general are rate limiting in the biogenesis of the erythromycins. It is possible that the intracellular concentration of SAM and SAH is quite low and that recycling of the SAH to SAM reduces the concentration of this end-product inhibitor. The SAM:SAH ratio could be a major regulatory factor of the final step in erythromycin A formation. Since very little if any erythromycin C accumulates in strains of S. erythreus that are useful for erythromycin A accumulation, the activity of the EaDM:SAM transmethylase clearly is sufficiently great to prevent buildup of this direct precursor of erythromycin A. 19j. R. Martin and A. W. Goldstein, Proc. Int. Congr. Chemother. 6th, 2, 1112 (1970).
[34] S - A d e n o s y l m e t h i o n i n e : I n d o l e p y r u v a t e 3-Methyltransferase
By MARILYN K. SPEEDIE, ULFERT HORNEMANN, and HEINZ G. FLoss O II ~CH2--C--COOH H
+ S-Adenosylmethionine
CHs O F }l ~ C H - C--COOH
~
H
+ 5-S - Adenosylhomocysteine
The enzyme is isolated from an indolmycin-producing strain of Streptomyces griseus and is presumably involved in the biosynthesis of this antibiotic. The reaction catalyzed by this enzyme represents the first spe-
[34]
499
INDOLEPYRUVATE 3-METHYLTRANSFERASE
cific step in indohnycin biosynthesis according to the pathway postulated by Hornemann et al2
Assay Method Principle. The enzyme catalyzes the transfer of the methyl group from [methyl-14C]S-adenosylmethionine to the 3-position of the aliphatic side chain of indolepyruvate. The radioactive reaction product can be extracted from the acidified reaction mixture with butyl acetate and then counted in a liquid scintillation spectrometer. Reagents
KH2PO4/Na2HPQ, 10 mM, pH 7.5 Indolepyruvate, 2.5 mM, pH 7.5 [methyl-14C]S-Adenosylmethionine, specific mmole, 2.5 mM
activity
0.20
mCi/
Procedure. Incubations are carried out in 15-ml centrifuge tubes. The reaction mixture contains 0.25 ~mole of indolepyruvate, 0.25 ~mole (0.05 ~Ci) [methyl-14C]S-adenosylmethionine, and enzyme in 10 mM phosphate buffer, pH 7.5, in a total volume of 1.0 ml. The reagents are prepared immediately before addition to the incubation mixture. Incubation is at 30 ° for 1 hr. Boiled enzyme serves as a control. The reaction is terminated by acidification to pH 3 with tartaric acid. One milliliter of distilled water and 4 ml of butyl acetate are added. The mixture is agitated on a Vortex mixer for 30 sec, centrifuged for 5 min, and the top layer (butyl acetate) is withdrawn and placed in a second centrifuge tube. Then 1 ml of water is added to the organic phase, and the mixing and centrifugation are repeated. Two milliliters of the butyl acetate phase are withdrawn and placed in a counting vial. When crude and partially purified enzyme preparations are assayed, S-adenosylmethionine is partially decomposed during the reaction period to form radioactive methanol which interferes with the determination of radioactivity in the product. To overcome this problem, any radioactive methanol is removed by repeated addition of unlabeled methanol and evaporation under nitrogen. With enzyme solutions in the later stages of purification there appears to be no methanol formed, so the butyl acetate phase is simply taken to dryness once under a stream of nitrogen. Ten milliliters of toluene scintillation fluid are added to each vial, and the samples arc counted in a liquid scintillation spectrometer.
1U. Hornemann, L. H. Hurley, M. K. Speedie, and H. G. Floss, J. Amer. Chem. Soc. 93, 3028 (1970).
500
ANTIBIOTIC BIOSYNTHESIS
[34]
Definition o] Activity. One unit of enzyme is defined as that quantity of enzyme which will convert 1 t~mole of substrate in 1 rain as measured by the incorporation of the methyl group of S-adenosylmethionine into 3-methylindolepyruvate under the conditions described. Specific activity is expressed as milliunits per milligram of protein. Protein is estimated by the biuret method 2 for steps 1 and 2 and by absorption at 280 nm for the later steps2 Purification Procedure All steps are performed at 0-4 ° unless otherwise stated. Step 1. Growth o] Cultures and Preparation o] the Crude Cell-Free Extract. A strain of Streptomyces griseus (ATCC 12648) is used as the source of the enzyme. The organism is maintained on slants of Emerson agar at 24 °. For production of cells to be used in enzyme preparations, the culture is first grown on a medium described by Rao 4 which contains dextrose, 1.0 g; K2HPO4, 0.5 g; NaCI, 0.2 g; CaC03, 0.20 g; distiller's solubles, 0.25 g; soybean meal, 0.15 g; and distilled water to 100 ml. A part of the mycelial pad from a slant culture is transferred under sterile conditions to a 500-ml Erlenmeyer flask containing 100 ml of the above medium and is allowed to grow for 4-7 days on a rotary shaker at 180 rpm at 24 °. From this culture 2-ml aliquots are withdrawn and transferred to 500-ml Erlenmeyer flasks containing "Phytone" (Baltimore Biological Laboratories), 2.0 g; yeast extract (Difco), 0.2 g; trace element solution, 5 0.1 ml; FeCI~, 0.1 rag; and distilled water to 100 ml. After 30-42 hr of growth in the latter medium, the mycelium is harvested by vacuum filtration and washed twice with distilled water. The mycelium from each flask is suspended in 15 ml of 10 mM phosphate buffer, pH 7.0. The cells are broken by one passage through a French pressure cell at 15,000-20,000 psi. The resulting suspension is centrifuged at 30,000 g for 20 rain to remove cell debris. Step 2. Ammonium Sul]ate Treatment. Solid ammonium sulfate is added over a period of 15 rain to give 35% saturation. The pH is adjusted to 7.0 with solid Na2HPO4, and the solution is stirred for a further 40 rain. Following 20 min of centrifugation at 30,000 g, ammonium sulfate is added to the supernatant in a similar manner to achieve 55% saturation, keeping the pH at 7.0. The protein that precipitates between 35 A. G. Gornall, C. J. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949); see also this series, Vol. 3 [73]. O. Warburg and W. Christian, Biochem. Z. 310, 384 (1941). K. V. Rao, Antibiot. Chemother. (Washington, D.C.) 10, 312 (1960). E. J, Kirsch and J. D. Korshalla, J. Bacteriol. 87, 247 (1964).
[a41
INDOLEPYRUVATE 3-METHYLTRANSFERASE
501
and 55% saturation contains the enzyme activity and is dissolved in 5-10 ml of 10 mM phosphate buffer, pH 7.0, and dialyzed against two changes of 2 liters of the same buffer for 1.5 hr each. Step 3. Sephadex Chromatography. The dialyzed solution is then applied to a 2.5 X 33 cm column of Sephadex G-150 which has been equilibrated with 10 mM phosphate buffer, pH 7.0, and is eluted with the same phosphate buffer. Fractions of 5 ml each are collected. The C-methyltransferase is recovered in fractions 15 through 22. The total volume of the active fractions is reduced to approximately 4 ml using an Amicon pressure dialysis apparatus. Step 4. DEAE-Sephadex Chromatography. The combined fractions from the previous step are applied to a 1.8 X 23 cm column of DEAESephadex which has been equilibrated with 50 mM phosphate buffer, pH 6.8. A 500-ml gradient of 0-0.4 M NaC1 in 50 mM phosphate buffer, pH 6.8, is used to elute the column. Fractions of 5 ml each are collected and monitored for protein and C-methyltransferase activity. The volume of the combined active fractions is reduced to approximately 5 ml using an Amicon pressure dialysis apparatus. Step 5. Bio-Gel A-5m Chromatography. The enzyme preparation from the preceding step is applied to a 1.0 )< 25 cm column of Bio-Gel A-5m which has been equilibrated with 10 mM phosphate buffer, pH 7.0, and subsequently is eluted with the same buffer. One-milliliter fractions are collected. The enzyme activity is recovered in fractions 9 ~hrough 12. By tim above procedure, the enzyme may be purified approximately ll0-fold with an overall yield of 40-45%. The enzyme can be stored at 2 ° for at least 3 weeks without significant loss of activity. A typical purification is summarized in the table.
Properties
Egect of pH. The enzyme is optimally active between pH 7.5 and 8.5. There appears to be some dependence upon the buffer, since in phosSUMMARY OF ENZYME PURIFICATION
Fraction 1. 2. 3. 4. 5.
Crude extract Ammonium sulfate Sephadex G-150 DEAE-Sephadex Bio-Gel A-5m
Total protein (rag)
Activity (milliunits)
Specific activity (mU/mg)
Yield (%)
358.0 171.0 40.2 2.2 1.5
28.0 22.5 22.0 12.8 12.5
0. 078 0.13 0.55 5.82 8.30
100 80.7 78.3 45.8 44.6
502
ANTIBIOTIC
[35]
BIOSYNTHESIS
phate buffer the enzyme is slightly more active at pH 7.5 than at pH 8.0, whereas in other buffers the optimum pH is slightly higher. The enzyme is irreversibly inactivated at pH 5.5 and below. Specificity. The crude extract is capable of methylating phenylpyruvate and p-hydroxyphenylpyruvate, in addition to indolepyruvate, but this capability is lost upon purification of the indolepyruvate 3-methyltransferase. Kinetic Properties. The Km values for indolepyruvate and S-adenosylmethionine are 4.8 ~M and 13 ~M, respectively. Molecular Weight. The molecular weight of the enzyme is estimated to be 55,000 ± 5000 by Sephadex G-200 gel filtration with reference proteins. Inhibitors. The reaction is strongly inhibited by the thiol reagents p-chloromercuribenzoate (10 ~M) and N-ethylmaleimide (4.0 mM). The Zn ~÷ and Fe 2÷ chelators 1,10-phenanthroline and 2,2P-bipyridine also inhibit the enzyme activity. At 2.0 mM the former compound inhibits 60%, the latter 35%. Ethylcnediaminetetraacetic acid (EDTA) has no effect upon the activity in concentrations up to 5 raM.
[35] N o v o b i o c i c A c i d S y n t h e t a s e By L. A. KOMINEK and H. F. MEYER OH J,~NH2
HOOC. ~
,,CH3 ~CH2CH=C
OH ~-~ ~
0II FCH5 ~NH--C. ~.~ ~CH2CH=C
+ H 0 J'-,~TI"~0 ~ 0 CH3
B ring
r3-Amino-4,7- dihydroxy8-methyl coumorin]
l
CH3
A ring
Novobiocic ocid
~4- Hydroxy-3(3- methy!-2bulenylJbenzoic ocid'l
The antibiotic novobiocin consists of a noviose sugar (C ring), a coumarin moiety (B ring), and a substituted benzoic acid moiety (A ring) linked by a glycosidic and an amide bond as shown in Fig. 1. The enzyme forming an amide bond between the A ring and the B ring to produce novobiocic acid has been demonstrated in cell-free extracts of Streptomyces niveus, z The reaction requires adenosine triphosphate (ATP), which indicates that an activation of the carboxyl group of the A ring is involved. The mode of activation has not been determined. 1L. A. Kominek, Antimicrob Ag. Chemother. 1, 123 (1972).
502
ANTIBIOTIC
[35]
BIOSYNTHESIS
phate buffer the enzyme is slightly more active at pH 7.5 than at pH 8.0, whereas in other buffers the optimum pH is slightly higher. The enzyme is irreversibly inactivated at pH 5.5 and below. Specificity. The crude extract is capable of methylating phenylpyruvate and p-hydroxyphenylpyruvate, in addition to indolepyruvate, but this capability is lost upon purification of the indolepyruvate 3-methyltransferase. Kinetic Properties. The Km values for indolepyruvate and S-adenosylmethionine are 4.8 ~M and 13 ~M, respectively. Molecular Weight. The molecular weight of the enzyme is estimated to be 55,000 ± 5000 by Sephadex G-200 gel filtration with reference proteins. Inhibitors. The reaction is strongly inhibited by the thiol reagents p-chloromercuribenzoate (10 ~M) and N-ethylmaleimide (4.0 mM). The Zn ~÷ and Fe 2÷ chelators 1,10-phenanthroline and 2,2P-bipyridine also inhibit the enzyme activity. At 2.0 mM the former compound inhibits 60%, the latter 35%. Ethylcnediaminetetraacetic acid (EDTA) has no effect upon the activity in concentrations up to 5 raM.
[35] N o v o b i o c i c A c i d S y n t h e t a s e By L. A. KOMINEK and H. F. MEYER OH J,~NH2
HOOC. ~
,,CH3 ~CH2CH=C
OH ~-~ ~
0II FCH5 ~NH--C. ~.~ ~CH2CH=C
+ H 0 J'-,~TI"~0 ~ 0 CH3
B ring
r3-Amino-4,7- dihydroxy8-methyl coumorin]
l
CH3
A ring
Novobiocic ocid
~4- Hydroxy-3(3- methy!-2bulenylJbenzoic ocid'l
The antibiotic novobiocin consists of a noviose sugar (C ring), a coumarin moiety (B ring), and a substituted benzoic acid moiety (A ring) linked by a glycosidic and an amide bond as shown in Fig. 1. The enzyme forming an amide bond between the A ring and the B ring to produce novobiocic acid has been demonstrated in cell-free extracts of Streptomyces niveus, z The reaction requires adenosine triphosphate (ATP), which indicates that an activation of the carboxyl group of the A ring is involved. The mode of activation has not been determined. 1L. A. Kominek, Antimicrob Ag. Chemother. 1, 123 (1972).
[35]
NOVOBIOCIC ACID SYNTHETASE 0 II
O-C -NH2
OH
0
503
CH$ =C~, CH5 o.
CH3
t
CH3
Jl C Ring
Jl B Ring
A Ring
I novobiocic ocid
FIG. 1. Structure of novobiocin.
Assay Method
Principles. Novobiocic acid synthetase can be assayed by measuring the rate of novobiocic acid formation. The quantity of novobiocic acid produced is measurable by spectrophotometric methods after extraction into n-butyl acetate. Reagents Ring A [4-hydroxy-3(3-methyl-2-butenyl)benzoic acid] 10 raM. A solution is prepared by dissolving 2.06 mg/ml of the A ring in 0.1 M Tris buffer, pH 8.0. Ring B (3-amino-4,7-dihydroxy-8-methylcoumarin) 10 mM. A solution is prepared by dissolving 2.44 mg/ml of the B ring hydrochloride in 0.1 M Tris buffer, pH 8.0. The solution is freshly prepared under a nitrogen atmosphere to retard degradation of the B ring. ATP, 50 mM in 0.1 M Tris buffer, pH 8.0 n-Butyl acetate Tris. HC1 buffer, 2.0 M, pH 9.0 Potassium phosphate buffer, 0.5 M, pH 6.5 Enzyme: the cell-free extract in 50 mM Tris buffer is used undiluted. The final reaction mixture should contain 5-20 units per milliliter of enzyme activity.
Procedure. The reaction mixture in both the experimental and blank tubes contains: A ring, 2 ml; B ring, 2 ml; ATP, 1 ml; Tris.HC1 buffer, 1 ml (2.0 M). The reaction is started by the addition of 1-4 ml of enzyme to the experimental tubes. Final volumes of experimental and blank tubes are brought to 10 ml with water. The reaction mixture is preincubated at 30 ° prior to the addition of enzyme. Samples (1 ml) are removed at
504
ANTIBIOTIC BIOSYNTHESIS
[35]
1-min intervals (0-6 min) and placed in a 125-ml glass-stoppered Erlenmeyer flask containing 15 ml of n-butyl acetate and 1 ml of potassium phosphate buffer. The flasks are shaken immediately for 15 rain to stop the reaction and extract novobiocic acid. The butyl acetate is separated from the aqueous phase and its absorbance is determined in a UV spectrophotometer at 360 and 310 nm against an n-butyl acetate blank. The B ring interferes in this assay but is corrected for by the use of a two-component equation: The concentration of novobiocic acid in the presence of B ring can be calculated from the following equation derived from the absorbancies of novobiocie acid (A36o = 42.70; A31o = 25.81) and the B ring (A36o = 3.46; A3,o = 13.05). Novobiocic acid (~g/ml) = (27.89 A36o - - 7.39 A 3 1 o ) X dilution factor
(15)
This assay, based on the novobiocin assay, 2 is sensitive, simple, and rapid but not completely specific. If greater specificity is desired, a quantitative paper chromatographic assay may be used. In this assay 10 ml of the n-butyl acetate extract is taken to dryness and the residue is redissolved in 0.5 ml of ethanol. This sample is spotted on Whatman No. 20 paper in a quantity sufficient to deliver approximately 75 ~g of novobiocic acid to the paper. Prior to application of the sample, the paper is dipped in ethylene glycol containing 2% of 85% lactic acid which is the stationary phase. The mobile phase consists of isopropyl ether saturated with ethylene glycol. The chromatogram is developed by the descending method at 2 8 ° for ca. 16 hr and dried. A standard novobiocic acid solution is also spotted. Novobiocic acid is located with reference to the standard by UV light. These are cut from the paper and eluted with 10 ml of acid methanol (0.002 N H oSO,). The absorbance of the eluate is read at 324 nm, and the concentration of novobiocic acid is calculated from its absorptivity (51.9). Qualitative estimation of novobiocic acid formation can be accomplished with thin-layer chromatography (TLC). The samples and standard are prepared as described previously. They are spotted on silica gel G TLC plates and developed in chloroform:methanol (9:1). Detection is by UV light. U n i t s . One unit of activity is defined as the amount of enzyme catalyzing the formation of 1 ~g of novobiocic acid per minute under the assay conditions described. Specific activity is defined as units per milligram of protein measured. 2R. M. Smith, J. J. Perry, G. C. Prescott, J. L. Johnson, and J. H. Ford, Antibiot. Ann. 1957/1958, 43 (1958).
[35]
NOVOBIOCIC ACID SYNTHETASE 0 II O-C-NH2
OH
0
505
CH
Novobiocin
CH3
CH3
(CH3C0)20/plridine I I
CH3COCl / methanol
0 H O-C-NH 2
0--~i-- CH3
~CH3
+
"%o o
H O O C ~ C H 2 C H = C CH3
x:/ "OAc
CH3
CH3
I
0
tl ~ CH3 NH--C~CH2CH=C ~ CH3 HO'~O~O "¢/~OH CH3
HCl, methanol
Novobiocic
I
acid
NoOC2H5
OH
H O ~
OH
/
/CH3
H2
HOOC.~--~CH2CH =C "~OH ~CH5
CH3 B
ring
A ring
FIG. 2. Hydrolysis scheme.
P r e p a r a t i o n o f S u b s t r a t e s a n d Product
Substrates for the biosynthesis of novobiocin are most conveniently obtained by the hydrolysis of novobioein itself. Two hydrolysis routes devised by J. W. Hinman et al. ~ are summarized in Fig. 2. N o v o b i o c i c Acid. One hundred grams of novobioein are dissolved in 1 liter of methanol; 12.5 ml aeetyl chloride are added, and the solution is refluxed for 80 min (the reaction may be followed by TLC on silica gel with 8:2 benzene:methanol, detection by UV). The solution is then cooled to room temperature and poured into 3 liters of cold water. The yellow precipitate is filtered, washed with 0.5 liter of distilled water, and air dried. The air-dried crude novobiocie acid is reerystallized from a minimum of hot aeetone. The yield is about 51 g (79%); mp 2 1 6 218 ° (d).
Thirty-five grams of this material are purified in a 200 X 50 ml -}- 50 ml eountereurrent distribution machine (CCD). It is dissolved in 500 ml of the upper phase of 1 : 2 : 2:3 water : ethanol : ethyl acetate : eyelohexane, filtered from undissolved material, and filled into the first 10 tubes of the CCD machine. The remaining tubes are filled with 50 ml each of lower phase; upper phase is added automatically. After 500 transfers, the lower phases are assayed by solids determination, the appropriate 3ft. W, Hinman, E. L. Caron, and H. I-Ioeksema, d. Amer. Chem. Sac. 79, 3789 (1957).
506
ANTIBIOTIC BIOSYNTHESIS
[35]
tubes ( ~ N o s . 40-120) are combined and concentrated to 250 ml. The resulting precipitate is recrystallized from a minimum of hot acetone. The yield is 12.9 g (37%), mp 229 ° (d). A second crop of 11.2 g can be obtained by concentrating the mother liquor to 500 ml and adding 100 ml of SkellysoIve B. 4-Hydroxy-3(3-methyl-2-butenyl)benzoic Acid (Ring A). One hundred grams of novobiocin are dissolved in 1 liter of freshly distilled pyridine and refluxed with 216 g acetic anhydride for 4 hr. The reaction solution is cooled to 5 °, 1500 ml of water, and 1030 ml of 12 N hydrochloric acid are added while maintaining the temperature below 20 ° . The resulting precipitate is filtered, washed with 500 ml of water, and dried in a vacuum oven at 50 °. The yield is 114 g of crude mixture. 4-Acetoxy-3(3-methyl-2-butenyl)benzoic acid is extracted from the crude precipitate in a Soxhlet extractor with 2 X 300 ml ether for 1 hr each. The ether extracts are evaporated to form the crude acid; this is recrystallized from 300 ml of ethanol and 600 ml of water. The yield is 74 g crude. A second crystallization from 800 ml of 50% aqueous ethanol yields 31 g of pure acid (76%), mp 120 °. This compound is deacetylated by dissolving 25 g in 1 liter of 1 N sodium hydroxide and 2.5 liters ethanol. After 3 hr the solution is adjusted to pH 2 with 6 N hydrochloric acid and concentrated at reduced pressure to an aqueous concentrate. This solution is extracted with 3 X 1 liter ether; the extracts are combined, dried with sodium sulfate, and evaporated. The residue (20.5 g) is crystallized from 200 ml of hot benzene. The yield is 20 g (80%) ; mp, 104-106 °. The overall yield is 61%. 3-Amino-4,7-dihydroxy-8-methyl Coumarin (Ring B). One hundred grams of novobiocin are hydrolyzed as described under 4-hydroxy-3(3methyl-2-butenyl)benzoic acid. After extracting this compound from the described crude mixture, the remainder (51 g) is recrystallized from 750 ml of ethanol and 200 ml of water to yield 46.5 g (65%) of the sugarcoumarin moiety (rings B-C) ; mp 167-172 °. This compound is hydrolyzed by suspending 20 g in 1 liter of methanol and refluxing with 250 ml 4 N methanolic HC14 (the finely powdered crystals dissolve before the boiling point is reached). After 2-hr refluxing the solution is cooled, concentrated at 35°/vacuum to about 200 ml, and cooled to 5 °. The resulting crystals (8.1 g) are filtered; a second crop (1.3 g) is obtained by concentrating the mother liquor to 60 ml and adding 120 ml of ether. Both crops are combined and recrystallized from 200 ml of hot ethanol and 130 ml of water. The yield is 9.0-9.1 g (90-91%) of the hydrochloride ; mp 90-93 °. 4Dry HCI gas is bubbled into 1 liter of cooled absolute methanol until the weight has increased from 791 g to 1064 g. The acid is titrated with 1 N NaOH; an appropriate amount of absolute methanol is added if the normality is >4.
[35]
NOVOBIOCIC ACID SYNTHETASE
507
The free base may be obtained by recrystallizing 1 g of the hydrochloride from 25 ml 90% aqueous ethanol, yielding about 0.8 g.
Preparation of the Enzyme Cultivation o] the Microorganism. Streptomyces niveus strain BC-345 is maintained on agar slants consisting of glucose, 10 g; brewer's yeast, 10 g; distillers solubles, 5 g; KC1, 4 g; C a CQ , 1 g; agar (Difeo), 20 g; and tap water to 1000 ml. Spores from these slants were suspended in sterile distilled water (10 ml) and used to inoculate a seed medium of the following composition: glucose, 10 g; beef extract, 3 g; NZ Amine B, l0 g; and distilled water to 1000 ml. The inoculated medium was incubated at 28 ° on a reciprocating shaker for 72 hr. The fermentation medium consists of glucose, 30 g; sodium citrate, 6 g; L-proline, 6 g; K2HPO~, 2 g; (NH~)~SO~, 1.5 g; NaC1, 5 g; MgSO4, 1 g; CaCl~, 0.4 g; FeSO4.TH20, 0.2 g; ZnSO4"TH20, 0.1 g; and distilled water to 1000 ml. The pH of this medium is adjusted to 7.2. The glucose is sterilized separately and added aseptically prior to inoculation. The inoculum for the fermentation medium is blended for 3 rain in a sterile, chilled Waring Blendor prior to addition. Blending is essential for rapid growth and enzyme production. The fermentation medium is inoculated with 5% of the blended seed culture and incubated at 28 ° on a rotary shaker. Cell-Free Extract. The cells are harvested from the fermentation medium after 4 days of incubation by centrifugation. The mycelia are washed twice in 50 mM Tris.HC1 buffer, pH 8.0. The mycelia are resuspended in this same buffer to about one-third of the fermentation harvest volume, and broken by sonication with a Bronson sonifier (S-125) run at maximal power output for 5 rain. Cell debris is removed by centrifugation at ~0,000 g for 10 min. All manipulations are carried out at 4 °. Protein concentrations are determined by the method of Lowry et al. 5
Properties Substrate Specificity. In addition to the amide bond formation between the A and B rings the reaction also occurs between the A ring and 3 amino-4-hydroxycoumarin, and between the dihydro A ring [4-hydroxy-3(3 methylbutyl)benzoic acid] and B ring. No reaction occurs between 4-hydroxy benzoate and the B ring or tyrosine and the A ring. 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
508
[36]
ANTIBIOTIC BIOSYNTHESIS
pH Optimum. In Tris.HC1 buffer the optimum pH of the reaction is 9.0 with a rapid decline in activity above this pH. For this reason it is best to run the reaction at pH 8.0 when using crude cell-free extracts. Stability. The stability of the enzyme in crude extract is poor. Storage at 4 ° results in about 50% loss of activity in 24 hr. Freezing or lyophilization results in a complete loss of activity. Relationship to Novobiocin Biosynthesis. The formation of novobiocic acid may not be the initial reaction in the coupling of the ring systems of novobiocin. Other possible coupling mechanisms include the formation of a glycosidic bond between the sugar (C ring) and coumarin (B ring) moieties prior to amide bond linkage with the A ring or a suitable intermediate of ring A.
[36] S - A d e n o s y l m e t h i o n i n e : O - D e m e t h y l p u r o m y c i n O-Methyltransferase By BURTON M. POGELL The antibiotic and antitumor agent, puromycin, is produced by Streptomyces alboniger and is a well characterized specific inhibitor of protein synthesis which terminates peptide bond elongation by competing with AA-tRNA. 1 The presumed last step in its biosynthesis 2 is catalyzed by the following enzymic reaction:
HsC-.N/CHs
HsC\N/CHs
H
O
~
NH
~
OH
I H, / = ~ O=C--CH--C~ - ~ ~ O H NH2 O-Demethylpuromycin
NH
OH
O : C - - CH-- C"~ NH,
~
O--CHs
Puromycin
+ S - Adenosyl- L- methionine
S-Adenosyl-L-homocysteine
: S. Pestka, Annu. Rev. Microbiol. 25, 487 (1971). 2M. M. Rao, P. F. Rebello, and B. M. Pogell, J. Biol. Chem. 244, 112 (1969).
508
[36]
ANTIBIOTIC BIOSYNTHESIS
pH Optimum. In Tris.HC1 buffer the optimum pH of the reaction is 9.0 with a rapid decline in activity above this pH. For this reason it is best to run the reaction at pH 8.0 when using crude cell-free extracts. Stability. The stability of the enzyme in crude extract is poor. Storage at 4 ° results in about 50% loss of activity in 24 hr. Freezing or lyophilization results in a complete loss of activity. Relationship to Novobiocin Biosynthesis. The formation of novobiocic acid may not be the initial reaction in the coupling of the ring systems of novobiocin. Other possible coupling mechanisms include the formation of a glycosidic bond between the sugar (C ring) and coumarin (B ring) moieties prior to amide bond linkage with the A ring or a suitable intermediate of ring A.
[36] S - A d e n o s y l m e t h i o n i n e : O - D e m e t h y l p u r o m y c i n O-Methyltransferase By BURTON M. POGELL The antibiotic and antitumor agent, puromycin, is produced by Streptomyces alboniger and is a well characterized specific inhibitor of protein synthesis which terminates peptide bond elongation by competing with AA-tRNA. 1 The presumed last step in its biosynthesis 2 is catalyzed by the following enzymic reaction:
HsC-.N/CHs
HsC\N/CHs
H
O
~
NH
~
OH
I H, / = ~ O=C--CH--C~ - ~ ~ O H NH2 O-Demethylpuromycin
NH
OH
O : C - - CH-- C"~ NH,
~
O--CHs
Puromycin
+ S - Adenosyl- L- methionine
S-Adenosyl-L-homocysteine
: S. Pestka, Annu. Rev. Microbiol. 25, 487 (1971). 2M. M. Rao, P. F. Rebello, and B. M. Pogell, J. Biol. Chem. 244, 112 (1969).
[36]
PUROMYCIN
509
A s s a y M e t h o d s 2,3
The two procedures described give comparable results and are based on determination of radioactivity incorporated into puromycin after separation from radioactive S-adenosyl-L-methionine (SAM). Method A is a more reproducible, simple one-step assay in which radioactive product is directly extracted into scintillation fluid. Preparation o] O-Demethylpuromycin. 2 The aminonucleoside of puromycin is coupled with O,N-dicarbobenzoxy-L-tyrosine in the presence of Woodward's reagent K, followed by hydrogenolysis of the carbobenzoxy group. Add 0.2 ml of triethylamine (0.34 mmole) in dimethylformamide over a l-rain period to a magnetically stirred suspension of 0.153 g of O,Ndicarbobenzoxy-L-tyrosine (0.34 mmole) and 0.086 g of Woodward's reagent K (0.34 mmole) in 1.6 ml of dimethylformamide. Continue stirring until the Woodward's reagent is completely dissolved (25 min). Then add 3 ml of a solution of 0.10 g of puromycin aminonucleoside (0.34 mmole) and 0.025 mmole of triethylamine in dimethylformamide and stir for an additional 18 hr. Remove the solvent in a vacumn, and triturate the sticky residue with 15 ml of water. Collect the granular precipitate (formed after standing for 3 hr at 4 °) by filtration, wash several times with water, and dry in a vacuum overnight (yield, 0.213 g, 86%). Heat this product to boiling in 5 ml of absolute ethanol, cool, and collect the residue by filtration (rap 184-187°). Dissolve the above residue (0.173 g) in 5 ml of Methyl Cellosolve at 60-70 °, add 0.4 g of 10% palladium charcoal, and place the suspension in a water bath kept at 60-70 °. Pass a slow stream of hydrogen through the solution until the evolution of carbon dioxide, as tested with barium hydroxide solution on the exit gases, is complete (1.5 hr). Remove the catalyst by filtration, the solvent in a vacuum, and dry the glassy residue overnight in a vacuum (yield, 0.105 g). The crude O-demethylpuromyein is further purified by column chromatography on silica gel and paper electrophoresis. Place the sample in 0.1 ml of methanol on a column of silica gel (1 X 10 cm) in chloroform and elute with 3 % methanol in chloroform. The bulk of the product, as measured by absorption at 275 nm, appears between 130 and 410 ml of effluent. Concentrate this material to dryness in a vacuum and repeat the chromatography. Removal of final traces of yellow impurity is achieved by preparative paper electrophoresis in pyridine-aeetic acidwater (2: 1:20) (pH 5.2) and the sample is eluted with ethanol. Absorption maxima and approximate molar extinction coefficients: X~t~~°1 275 nm 3 L. Sankaran and B. M. Pogell, Anal. Biochern. 54, 146 (1973).
510
ANTIBIOTIC BIOSYNTHESIS
(e, 15,000); ~0mla~HCI 245 nm (5, 9,700).
268 nm
[~5]
(5, 14,400); X°m~NNa°~ 277 nm (5, 14,300),
Reagents. All solutions for the enzyme assay are prepared in 0.1 M sodium phosphate-1 mM EDTA (pH 7.5) O-Demethylpuromycin, 4 mM S-Adenosyl-L-methionine (SAM), 0.32 mM (2.5 X l0 s cpm/0.1 ml), either [3H]methyl or [14C]methyl Sodium borate, 0.1 M-NaCI, 5 M, final pH 9.0 Enzyme. Dilute with buffer if necessary.
Procedure A2 Incubate 5 tLl of O-demethylpuromycin, 5 ~l of SAM, and enzyme in a final volume of 20 ~l at 38 ° for suitable time intervals in 1-dram vials with snap-on plastic caps (Rochester Scientific, Cat. No. 7475, 15 X 45 mm). Add all reagents at the same place in the angle between the bottom and side of the vials, and mix the contents by gentle rotation. Stop the reactions by adding 0.7 ml of 0.1 M sodium borate-5 M NaC1 (pH 9) and 3 ml of scintillation fluid (Spectrafiuor, NuclearChicago, 1:25 dilution in toluene) to each vial. Stopper and shake the vials vigorously in a horizontal position for 10 min in a mechanical shaker (Precision Scientific, Cat. No. 65855), centrifuge to clarify the layers, and place in regular-sized glass scintillation vials for radioactive counting. Always include a control incubation minus O-demethylpuromycin, particularly with crude preparations. With 14C-SAM as substrate, transfer 2 ml of the scintillation fluid to a new vial for counting after extraction. This results in much lower background values. Definition o] Unit and Specific Activity. A unit of enzyme is defined as the amount ~hat will form 1 nmole of puromycin per minute at pH 7.5 and 38 ° . Specific activity is expressed as units of enzyme per milligram of soluble protein. Protein is determined by the Lowry procedure 4 with bovine serum albumin as standard. Units of enzyme are calculated as follows: (a) ~H: cpm of sample 0.875 × 1.6 × cpm/nmole of 3H-SAS/I X min of incubation or
(b) 14C: 1.5 X cpm of sample 0.875 X 1.13 × cpm/nmole of [14C]SA5/I × min of incubation 4 E. Layne, this series, Vol. 3 [73].
[36]
PUROMYCIN
511
where 0.875 = fraction of puromycin extracted by scintillation fluid under above conditions; specific radioactivity of SAM is determined by counting in 95% ethanol:scintillation fluid (3:10); 1.6 = ratio of ~H sample counted in scintillation fluid compared to sample counted in ethanol:scintillation fluid; 1.13 = ratio of 1~C sample counted in scintillation fluid compared to sample counted in ethanol:scintillation fluid; 1.5 - correction for 14C because only two-thirds of the scintillation fluid is counted. Procedure B f- Incubate mixtures as above in 10 X 75 mm test tubes. Terminate reactions by adding 20 ul of ethanol-acetic acid (9:1) anti remove protein by centrifugation. Carefully remove one-half of each supernatant with a micropipette and spot on a precoated thin-layer cellulose plastic sheet (Brinkmann). Develop the sheets in saturated ammonium sulfate-1 M sodium acetate-isopropyl alcohol (80:18:2) with puromycin as marker. After drying, repeat the development once or twice. This procedure separates the unreacted SAM and its decomposition products from puromycin, which remains at the origin. Cut out the areas corresponding to puromycin (approximately 1.5-cm squares), place each in a scintillation vial, add 3 ml of ethanol and 10 ml of scintillation fluid, and determine the radioactivity in each sample. Enzyme units are calculated from the specific radioactivity of the added SAM assuming that radioactivity incorporated is directly proportional to product formed.
E n z y m e Purification ~,5,6
Our original studies on this enzyme were carried out in extracts from a mutant strain of S. alboniger, ATCC 12462. Higher specific activities can be obtained with the wild-type organism, ATCC 12461, by harvesting cells during mid- or late logarithmic growth (48-72 hr) and shortening the time of sonication. Inoculate cells of S. alboniger strain ATCC 12461 into 500 ml of 6% corn steep liquor-2% corn starch, pH 6.4, in 2-liter shake flasks with added Dow-Corning Antifoam. Grow at 28 ° with constant agitation on a New Brunswick gyratory shaker. Harvest cells by centrifuging at 4 °, wash twice with cold water, and store frozen at --20 ° . All purification procedures are carried out at 0-4 °. Results of a typical purification procedure with pooled extracts of specific activity >0.5 are shown in the table. 5L. Sankaran and B. M. Pogell, Nature (London) New Biol. 245, 257 (1973). "L. Sankaran, S. V. K. Narasimha Murthy, M. Kariya, and B. M. Pogell, unpublished observations.
512
ANTIBIOTIC BIOSYNTHESIS
[35]
PURIFICATION OF O-I~ETHYLTRANSFERASE
Step 1. Crude extract Protamine sulfate s u p e r n a t a n t 2. A m m o n i u m sulfate precipitate (4,5-70%) 3. DEAE-cellulose column I Fractions 66-84 Amicon 12 concentrate DEAE-cellulose column I I Amicon 12 concentrate
4. Sephadex G-200 Amicon 12 concentrate
Protein (mg)
ReSpecific covery activity (%) (units/mg)
Volume (ml)
Units
15.9 17.1 1.3
43.0 40.6 25.2
76.3 67.5 23.6
100 94 59
38 1.04
17.5 12.9
---
41 30
---
0.82
7.5
1.53
17
4.9
1.4
7.1
0.43
17
16.5
0.56 0.60 1.1
Step 1. Preparation of Cell-Free Extract and Removal of Nucleic Acids. Prepare a cell-free extract by sonic disintegration of a 10% (w/v) suspension of cells in 0.1 M sodium phosphate buffer, pH 7.5, for 3-4 min in an ultrasonic disintegrator (Measuring and Scientific Equipment, Ltd., London, 60 W). Cool the cell suspension by circulating ice water during the sonic disruption. Remove particulate matter by centrifuging for 30 min at 32,000 g. Remove nucleic acids from the above supernatant by adding a 2% solution of protamine sulfate (pH 7) (ca. 0.11 ml per milliliter of enzyme solution). Collect the supernatant by centrifugation. Step 2. Salt Fractionation. Gradually add solid ammonium sulfate to 45% saturation to the protamine supernatant (0.277 g/ml). Remove the precipitate by centrifugation and raise the concentration of ammonium sulfate to 70% saturation by further addition of salt (0.171 g/ml). Collect the precipitate by centrifugation. Upon further purification, the methylase is found to be unstable in dilute solution. Inclusion of 10 ~M SAM in all solutions used for column chromatography prevents this inactivation (see section on Properties). Step 3. DEAE-Cellulose Column Chromatography. Dissolve the ammonium sulfate precipitate in 1.3 ml of 5 mM sodium phosphate-0.1 mM EDTA-10 ~ / / SAM, pH 7.5 (PES), and place on a column of DEAEcellulose (Whatman, DE-52, 5 g, 0.9 X 13 cm) preequilibrated with the same buffer. Wash with PES and elute with a concave salt gradient at a flow rate of 16 ml/hr. A Technicon l-liter capacity Autograd is used to form the gradient. The first 2 chambers contain 70 ml each of 0.1 M NaC1 in PES and the third chamber contains 70 ml of 0.6 M NaC1
[36]
PUROMYCIN
A z ~J
513
z bJ CORN STEEP LIQUOR-CORN STARCH
HICKEY-TRESNER MEDIUM
0 o.
4.0
o
0.I0
f
¢n
GROWTH
FZ
>I-> I--
3.0 0
•
~'~ SPECIFIC ACTIVITY
0.4 E
-g
:~ SPECIFIC
~,
2.0 ~
~_ 0 . 5
_z
0.05
w 0.2
~
0 LO 0,.
._1 I.w a.
I00
200
0
0
_L
L 200
0
TIME IN HOURS
TIME IN HOURS 0
A tO0
0
FIG. 1. Changes in O-methyltransferase activity during growth in (a) HickeyTresner and (b) corn steep liquor-corn starch media. Streptomyces alboniger cells (ATCC 12461) were grown at 28° and cell-free extracts prepared as described in the text. Total protein was estimated in samples of whole-cell sonicates as a measure of growth. Enzyme specific activity was determined in supernatants after centrifugation at 32,000 g for 30 rain. in PES. Collect 2-ml fractions and assay for enzyme activity. The methylase is eluted as one symmetrical p e a k in fractions 66-84. Pool this m a t e rial and concentrate in an Amicon 12 ultrafiltration unit with a P M 30 membrane. Wash and reconcentrate twice with 9-ml volumes of PES. R e p e a t the above DEAE-cellulose c h r o m a t o g r a p h y procedure with concentrated enzyme. Pool fractions 58-76 and reconcentrate as above. Step 4. Sephadex G-200 Gel Filtration. Place the concentrated enzyme on a Sephadex G-200 column (2.5 X 40 cm) equilibrated with P E S and elute at a flow rate of 15 m l / h r with PES. Collect 2-ml fractions and assay for enzyme activity. T h e methylase is eluted as one peak in fractions 46-55. Pool this material, concentrate by ultrafiltration as above, and store at --65 ° . In another preparation, where Sephadex gel filtration was run before DEAE-cellulose chromatography, the final specific activity was 26. Properties o f the E n z y m e 2,5,
The enzyme activity increases with growth, reaches a m a x i m u m during the mid- or late logarithmic growth phase, and then declines rapidly in the stationary phase (Fig. 1). I n a medium designed by H i c k e y and Tresner 7 to enhance sporulation in Streptomyces, the change in enzyme R. J. Hickey and H. D. Tresner, J. Bacleriol. 64, 891 (1952).
514
ANTIBIOTIC BIOSYNTHESIS
[35]
specific activity follows a bell-shaped curve with time, again becoming zero after a high degree of sporulation occurs; also no enzyme activity has been found in spore extracts. Much higher specific activities are obtained in corn steep liquor-corn starch medium. Both glucose (1%) and subinhibitory levels of ethidium bromide (5 ~M) or acriflavine (3 ~g/ml) differentially inhibit enzyme formation; glucose similarly inhibits antibiotic formation on agar. Possibly related to the rapid turnover of the methylase activity is a recent observation that the enzyme is very unstable in extracts at 38 ° and pH 7.5. Addition of assay levels of either SAM or O-demethylpuromycin protects enzyme activity under these conditions. The enzymic methylation is very specific for SAM and O-demethylpuromycin. 5-Methyltetrahydrofolate is completely inactive as methyl donor and no methylation of tyrosine, several tyrosine derivatives, catechol, or L-epinephrine is detectable. The Km for SAM is 0.01 mM and for O-demethylpuromycin, 0.2 mM. A broad pH optimum is found over the range of pH 7-9. Formation of product is linear with respect to time and enzyme concentration.
Other Aspects of Puromycin Biosynthesis Dialyzed supernatants from extracts of S. alboniger catalyze the formation of two aminopentose phosphates, 2-amino-2-deoxy-D-ribose 5-phosphate and 2-amino-2-deoxy-D-lyxose 5-phosphate, from ribose 5phosphate and NH4÷.s,9 However, no evidence has yet been found for the formation of any 3-aminopentose in cell-free extracts. No other enzymic steps in the biosynthesis of puromycin have been characterized. Three possible precursors of the antibiotic have been isolated in small quantities from commercial preparations of puromyein. 1° Advantage was taken of the solubility of puromycin in chloroform at pH 11-12, conditions where O-demethylpuromycin and other derivatives with a free tyrosyl hydroxyl group remain in the aqueous layer. The compounds and structures correspond to the respective demethyl derivatives of puromycin: N6,N6,0-tridemethylpuromycin, N6,0-didemethylpuromycin, and O-demethylpuromycin. Radioactive precursor studies have shown that the methyl group of methionine is incorporated into the 8B. M. Pogell, P. F. Rebello, and P. P. Mukherjee, in "International Symposium on Metabolism, Physiology, and Clinical Use of Pentoses and Pentitols" (B. L. ttorecker, K. Lang, and Y. Takagi, eds.), p. 135. Springer-Verlag, Berlin, 1969. P. F. Rebello, B. M. Pogell, and P. P. Mukherjee, Biochim. Biophys. Acta 177, 468 (1969). 2oT. N. Pattabiraman and B. M. Pogell, Biochim. Biophys. Acta 182, 245 (1969).
[37]
GUANOSINETRIPHOSPHATE-8-FORMYLHYDROLASE
515
N~,N6-dimethyladenine moiety as well as the O-methyl group of puromycin. 6,1~ Adenine is also incorporated into N'%N6-dimcthyladenine during growth. 11 Thus, it appears that these compounds, or derivatives such as 5'-phosphate esters, are probable precursors of puromycin. H a r d l y anything is known about why antibiotics are formed, or what, if any, are the normal functions of these secondary metabolites in normal cell metabolism. Several compounds containing NG,N6-dimethyladenine in glycosidic linkage have been isolated from spore-rich preparations of S. alboniger (2-3 nmoles of base per milligram dry weight)? 1 A major portion is linked to part of the spore coat. In addition, free N~;,N%dimethyladenine is excreted into the growth medium. The absence of methylated adenine in hyphae suggests a role for N-methyla~ion during differentiation in S. alboniger. No O-methyl-L-tyrosinc was found in acidhydrolyzed hyphae or spores.
~ M. G. Sarngadharan, B. M. Pogell, and M. Kariya, Biochim. Biophys. Acta 262, 405 (1972).
[37] G u a n o s i n e
Triphosphate-8-formylhydrolase
B y E. F. ELSTNER and R. J. SUHADOLNIK
GTP ~ 4-N[(5'-triphospho)- l'-ribosylamino]-2, ~diamino-6-hydroxypyrimidine + formic acid
Assay Method Principle. I t is now well established t h a t carbon-8 of guanine, guanosine, or guanosine triphosphate (GTP) is lost as formic acid in the biosynthesis of riboflavin, the pteridines, a pteridine cofactor for phenylalanine hydroxylation, the azapteridine ring, and the benzimidazole ring. ~-7 The enzyme, GTP-8-formylhydrolase, that catalyzes ~he conversion of G T P to formic acid and 4-N-[ (5'-triphospho)-l'-ribosylamino]2,5-diamino-6-hydroxypyrimidine by S. rimosus has been described? This enzyme appears to be involved in the biosynthesis of the pyrrolopyrimi-
1E. F. Elstner and R. J. Suhadolnik, J. Biol. Chem. 246, 6973 (1971). A. W. Burg and G. M. Brown, J. Biol. Chem. 243, 2349 (1968). s B. Levenberg and D. K. Kaczmarek, Biochim. Biophys. Acta 117, 272 (1966). J. Cone and G. Guroff, J. Biol. Chem. 246, 979 (1971). 5W. L. Alworth, S. H. Lu, and M. F. Winkler, Biochemistry 10, 1421 (1971). 6 R. A. Harvey and G. W. E. Plaut, J. Biol. Chem. 241, 2120 (1966). 7A. Bacher and F. Lingens, J. Biol. Chem. 245, 4647 (1970).
[37]
GUANOSINETRIPHOSPHATE-8-FORMYLHYDROLASE
515
N~,N6-dimethyladenine moiety as well as the O-methyl group of puromycin. 6,1~ Adenine is also incorporated into N'%N6-dimcthyladenine during growth. 11 Thus, it appears that these compounds, or derivatives such as 5'-phosphate esters, are probable precursors of puromycin. H a r d l y anything is known about why antibiotics are formed, or what, if any, are the normal functions of these secondary metabolites in normal cell metabolism. Several compounds containing NG,N6-dimethyladenine in glycosidic linkage have been isolated from spore-rich preparations of S. alboniger (2-3 nmoles of base per milligram dry weight)? 1 A major portion is linked to part of the spore coat. In addition, free N~;,N%dimethyladenine is excreted into the growth medium. The absence of methylated adenine in hyphae suggests a role for N-methyla~ion during differentiation in S. alboniger. No O-methyl-L-tyrosinc was found in acidhydrolyzed hyphae or spores.
~ M. G. Sarngadharan, B. M. Pogell, and M. Kariya, Biochim. Biophys. Acta 262, 405 (1972).
[37] G u a n o s i n e
Triphosphate-8-formylhydrolase
B y E. F. ELSTNER and R. J. SUHADOLNIK
GTP ~ 4-N[(5'-triphospho)- l'-ribosylamino]-2, ~diamino-6-hydroxypyrimidine + formic acid
Assay Method Principle. I t is now well established t h a t carbon-8 of guanine, guanosine, or guanosine triphosphate (GTP) is lost as formic acid in the biosynthesis of riboflavin, the pteridines, a pteridine cofactor for phenylalanine hydroxylation, the azapteridine ring, and the benzimidazole ring. ~-7 The enzyme, GTP-8-formylhydrolase, that catalyzes ~he conversion of G T P to formic acid and 4-N-[ (5'-triphospho)-l'-ribosylamino]2,5-diamino-6-hydroxypyrimidine by S. rimosus has been described? This enzyme appears to be involved in the biosynthesis of the pyrrolopyrimi-
1E. F. Elstner and R. J. Suhadolnik, J. Biol. Chem. 246, 6973 (1971). A. W. Burg and G. M. Brown, J. Biol. Chem. 243, 2349 (1968). s B. Levenberg and D. K. Kaczmarek, Biochim. Biophys. Acta 117, 272 (1966). J. Cone and G. Guroff, J. Biol. Chem. 246, 979 (1971). 5W. L. Alworth, S. H. Lu, and M. F. Winkler, Biochemistry 10, 1421 (1971). 6 R. A. Harvey and G. W. E. Plaut, J. Biol. Chem. 241, 2120 (1966). 7A. Bacher and F. Lingens, J. Biol. Chem. 245, 4647 (1970).
516
ANTIBIOTIC BIOSYNTHESIS
[37]
dine nucleoside antibiotics toyocamycin, tubercidin, and sangivamyein. The assay method is based on two procedures. The first method involves the release of 14C-labeled formic acid from carbon-8 of the imidazole ring of [8-~4C]GTP. The formic acid is oxidized to carbon dioxide2 The second method involves the addition of the enzyme reaction mixture, following a 2-hr incubation, to a 1-ml column (Norit A-Celite, 1:1 (w/w). The column is washed with 5 ml of 1 N formic acid to elute the [~4C]HCOOH. The recovery by this method is 75% compared with the first procedure.
Reagents [8-14C]GTP, 12.5 nmoles, 80,000 cpm Tris buffer, 25 mM, pH 8.0 Enzyme, 0.4 unit/ml
Procedure. Mix the above three reagents and incubate for 2 hr at 38°; total volume, 1 ml. The formic acid released is determined by one of the two methods described above. If the oxidation of formic acid is used, the 1~C0~ is removed from the reaction mixture by bubbling with nitrogen gas for 10 min at 100 ° into 1 ml of 1 N Hyamine (NCS solubilizer) ; the 14CO~ is determined by liquid scintillation counting of 0.5 ml of the Hyamine solution using Bray's scintillation fluid. 8 Definition o] Unit and Specific Activity. One unit of enzyme is that amount of protein which causes the formation of 1 nmole of formic acid per hour at 38 ° . Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Murphy and Kies." Purification Procedure
Step 1. Preparation o] Cell-Free Extracts o] S. rimosus. The cells are harvested 46-48 hr after inoculation and centrifuged at 5000 g for 5 min, washed with 50 mM Tris (pH 8.0), centrifuged, and lyophilized. The lyophilized cells are stored at --20 °. The lyophilized cells (5 g, dry weight) are suspended in cold Tris buffer (50 mM, pH 8.0) and treated with a French pressure cell (Aminco, pressure cell No. 4-3396) at 16,000 psi. The slurry is centrifuged at 48,000 g for 30 min. The supernatant is referred to as crude extract. The crude extract is dialyzed overnight against 5 mM Tris buffer, pH 8.0. Ammonium sulfate precipitations are performed with the highest purity ammonium sulfate. All fractionations s G. A. Bray, Anal. Biochem. 1, 279 (1960). PJ. B. Murphy and M. W. Kies, Biochim Biophys. Acta 45, 382 (1960).
[37]
GUANOSINETRIPHOSPHATE-8-FORMYLHYDROLASE
517
are performed at 4 °. GTP-8-formylhydrolase activity is maximal 48 hr after inoculation. Step 2. Purification on DE-52 Cellulose. The dialyzed crude extract (70 ml, no EDTA), 1200 mg of protein, is applied on a column of DE-52 cellulose (2 X 30 cm) after equilibration with 10 mM phosphate buffer, pH 7.4. The linear gradient is from 10 mM to 0.3 M phosphate buffer (pH 7.4), 200 ml each. Fractions of 10 ml are collected at 4 ° and a flow rate of 0.5 ml per minute is maintained. Step 3. Ammonium Sul]ate Fractionation. Solid ammonium sulfate is added with stirring to 30% saturation. After 15 min, the solution is centrifuged at 15,000 g, 10 min. Solid ammonium sulfate is added to the supernatant to bring the solution to 60% saturation, stirred 15 additional min, and centrifuged at 15,000 g, 10 min. The pellet is dissolved in 10 ml Tris buffer (50 mM, pH 8.0) and dialyzed against 2 liters Tris buffer (50 mM, pH 8.0) overnight. Assays of the crude undialyzed extract are not reliable because there are interfering compounds that inhibit the enzyme. These inhibitors are removed by dialysis. Step 4. Purification on Sephadex G-200. Two milliliters (26 mg prorein) of the GTP-8-formylhydrolase are added to a Sephadex column (1.5 cm X 100 era). The column is previously equilibrated with 10 mM phosphate buffer (pH 7.4; EDTA, 1 mM). The protein is eluted with the same phosphate-EDTA solution. Two-milliliter fractions are collected at a flow rate of 0.5 ml/min; the peak tube for elution of the enzyme is usually tube 30. A purification is summarized in the table.
Properties Specificity. The purified enzyme will only eliminate the ureido carbon of GTP as [14C]formic acid. The following [8-1~C]purine ribonucleoside or nucleotides are not substrates: guanosine, GMP, GDP, ITP, and ATP. The addition of guanosine, GMP, or GDP (50 fM) to assays with the purified enzyme does not inhibit the release of [l~C]formic acid from [8-'~C] GTP. Activators and Inhibitors. No cofactors are required for enzymic activity. GTP-8-formylhydrolase is a sulfhydryl enzyme. Sulfhydryl reducing agents such as mercaptoethanol (10 raM) and cysteine (1 mM) stimulate enzyme activity (110 and 140%, respectively). With 0.1 M mercaptoethanol, the GTP-8-formylhydrolase activity decreases to 15%. Ascorbate gives a slight stimulation (120%) and EDTA (1 raM) completely inhibits the enzyme. Sulfhydryl inhibitors such as showdomycin (1 mM) and p-chloromerc-
518
ANTIBIOTIC BIOSYNTHESIS
O
~q
©
©
(9 ©
Z ©
O
0
¢0
[37]
[371
GUANOSINE TRIPHOSPHATE~8-FORMYLHYDROL ASE
519
uribenzoate (0.5 raM) cause a 79% and 95% inhibition of the enzyme; Mg 2÷ (10 mM) and Fe '-'÷ (1 raM) inhibit the enzyme 87% and 41% ; ATP (5 raM) and I T P (5 mM) inhibit the enzyme 60 and 83%. ITP is a mixed type inhibitor (Ki 45 ~M). ATP is a competitive inhibitor (K~ 0.11 raM). The 65% inhibition of the enzyme by showdomycin (1 raM) is reversed by cysteine (3 mM). When incubations are done under anaerobic conditions, about twice as much formic acid is released as under aerobic conditions. Anaerobic conditions are performed in Warburg flasks by flushing for 3 rain with nitrogen. The center well contains 1 g of pyrogallol in 0.5 ml of 1 N NaOH. The reaction vessel contains 1 mM EDTA, 0.025 M Tris buffer, pH 8.0, 50 ~M [8-14C]GTP (90,000 cpm) and 50 mM of glucose. The reaction is started by adding glucose oxidase anti GTP-8-formylhydrolase from the two side arms. A control vessel is run in which pyrogallol, glucose oxidase, and nitrogen are omitted. The reaction is stopped by cooling to 0 °, transferring the reaction contents quantitatively to the microdistillation apparatus, and oxidizing the [14C] formic acid to 1'C0~. The enzyme exhibits maximal activity at pH 8.0, 50 mM Tris buffer. Michaelis Constant and Enzyme Properties. Tile Miehaelis constant for GTP is 20 uM. This was determined by plotting the reciprocal of the velocity of the reaction against the reciprocal of the concentration of GTP (Lineweaver-Burk method). Product formation is linear for 3 hr (125 mnoles of GTP). The rate of reaction is directly proportional to protein concentration up to 10 mg of protein. Molecular Weight. The molecular weight of GTP-8-formylhydrolase has been determined by comparing the elution of the enzyme to that of urease. The molecular weight of the enzyme eluted from a Sephadex G-200 cohlmn is 500,000. Dissociation of GTP-8-formylhydrolase. GTP-8-formylhydrolase c.m be dissociated into two proteins. To do so, it is necessary to dialyze the ammonimn sulfate fraction (without EDTA) prior to chromatograptly on DE-52. Dialysis against EDTA prevents dissociation of the enzyme. The dialyzed crude extract, containing the GTP-8-formylhydrolase, is precipitated between 30 and 60% saturation with ammoniuIn sulfate. The precipitate (380 rag) is dissolved in 2 ml of 50 mM Tris buffer, pH 8.0, and dialyzed overnight against 5 mM Tris buffer, pH 8.0 (no EDTA). The protein is applied to a DE-52 column (1 em X 15 cm) after equilibration with 10 mM phosphate buffer, pH 7.4. The linear gradient is from 10 mM to 0.3 M phosphate buffer (pH 7.4), 150 ml each. Fractions of 8 ml are collected at 4 ° at a flow rate of 0.5 ml per minute. Each tube is assayed for GTP-8-formylhydrolase, phosphatase activity, and protein concentration. Two protein peaks are observed that have GTP-8-formyl-
520
ANTIBIOTIC BIOSYNTHESIS
[38]
hydrolase activity (peak enzyme tubes: 4 and 20). To prove that GTP is the substrate, phosphatase activity is measured in each fraction. Eighty percent of the phosphatase activity is lost in the 30-60% ammonium sulfate fraction. Phosphatase activity is assayed by incubating the enzyme with 25 nmoles [fl-~-32p]GTP (1400 cpm). The reaction mixture is added to 1 ml of a Norit A-celite column (1:1, w/w). The columns are washed with 3 volumes of 0.4 M phosphate buffer, pH 7.4. The inorganic 32p is counted. [fl-y-32p]GTP is prepared from GMP and KH232p04 by the method of Tochikura et al. ~° [fl-y-82P]GTP is purified by DE-52-cellular chromatography (30 g, gradient elution, 500 ml each of water and 0.4 M triethylammonium bicarbonate, pH 7.3). The fl_~/.82p labeled GTP shows a single UV spot on Avicel thin-layer chromatography plates, solvent: 6% ammonium chloride. P r o d u c t F o r m a t i o n . The predicted product following ring opening by GTP-8-formylhydrolase should be 4-N-[(5'-triphospho)-l'-ribosylamino]-2,5-diamino-6-hydroxypteridine. Only 0.16% of this ring-opened product exists following elimination of carbon-8 of GTP. The major product is neopterin. Since riboflavin and folic acid both use the purine ring for their biosynthesis, attempts to isolate these two compounds from the culture medium were made. These compounds are not present in the culture medium. lo T. Tochikura, K. Kawaguchi, T. Kano, and K. Ogata, J. Ferment. Technol. 47, 564 (1969).
[38] 6-Methylsalicylic Acid Synthetase B y G. VOGEL and F. LYNEI~
Under certain culture conditions several P e n i c i l l i u m species produce 6-methylsalicylic acid, the parent compound in the reaction sequence leading to the antibiotic patulin. 1,2 6-Methylsalicylic acid belongs to the class of naturally occurring compounds derived from head-to-tail condensation of acetate units, as postulated in the polyacetate or polyketide hypothesis2 ,4 Malonyl-CoA has been recognized as the building block 1j. D. Bu'Lock and A. J. Ryan, Proc. Chem. Soc. 222 (1958). 2j. D. Bu'Lock, D. Hamilton, M. S. Hulme, A. J. Powell, D. Shepherd, and G. N. Smith, Can. J. Mikrobiol. 11, 765 (1965). 3j. N. Collie, J. Chem. Soc. 91, 1806 (1907). ' A. J. Birch and F. W. Donovan, Austr. J. Chem. 6, 360 (1953).
520
ANTIBIOTIC BIOSYNTHESIS
[38]
hydrolase activity (peak enzyme tubes: 4 and 20). To prove that GTP is the substrate, phosphatase activity is measured in each fraction. Eighty percent of the phosphatase activity is lost in the 30-60% ammonium sulfate fraction. Phosphatase activity is assayed by incubating the enzyme with 25 nmoles [fl-~-32p]GTP (1400 cpm). The reaction mixture is added to 1 ml of a Norit A-celite column (1:1, w/w). The columns are washed with 3 volumes of 0.4 M phosphate buffer, pH 7.4. The inorganic 32p is counted. [fl-y-32p]GTP is prepared from GMP and KH232p04 by the method of Tochikura et al. ~° [fl-y-82P]GTP is purified by DE-52-cellular chromatography (30 g, gradient elution, 500 ml each of water and 0.4 M triethylammonium bicarbonate, pH 7.3). The fl_~/.82p labeled GTP shows a single UV spot on Avicel thin-layer chromatography plates, solvent: 6% ammonium chloride. P r o d u c t F o r m a t i o n . The predicted product following ring opening by GTP-8-formylhydrolase should be 4-N-[(5'-triphospho)-l'-ribosylamino]-2,5-diamino-6-hydroxypteridine. Only 0.16% of this ring-opened product exists following elimination of carbon-8 of GTP. The major product is neopterin. Since riboflavin and folic acid both use the purine ring for their biosynthesis, attempts to isolate these two compounds from the culture medium were made. These compounds are not present in the culture medium. lo T. Tochikura, K. Kawaguchi, T. Kano, and K. Ogata, J. Ferment. Technol. 47, 564 (1969).
[38] 6-Methylsalicylic Acid Synthetase B y G. VOGEL and F. LYNEI~
Under certain culture conditions several P e n i c i l l i u m species produce 6-methylsalicylic acid, the parent compound in the reaction sequence leading to the antibiotic patulin. 1,2 6-Methylsalicylic acid belongs to the class of naturally occurring compounds derived from head-to-tail condensation of acetate units, as postulated in the polyacetate or polyketide hypothesis2 ,4 Malonyl-CoA has been recognized as the building block 1j. D. Bu'Lock and A. J. Ryan, Proc. Chem. Soc. 222 (1958). 2j. D. Bu'Lock, D. Hamilton, M. S. Hulme, A. J. Powell, D. Shepherd, and G. N. Smith, Can. J. Mikrobiol. 11, 765 (1965). 3j. N. Collie, J. Chem. Soc. 91, 1806 (1907). ' A. J. Birch and F. W. Donovan, Austr. J. Chem. 6, 360 (1953).
[381
6 - M E T H Y L S A L I C Y L I C ACID S Y N T H E T A S E
521
for 6-methylsalicylic acid biosynthesis, and a reaction mechanism similar to that for fatty acid synthetase has been proposed2 The 6-methylsalicylic acid synthetase is a stable multienzyme complex which catalyzes 6-methylsalicylic acid formation according to the following equation: CH3CO-SCoA + 3HOOCCH2CO-SCoA + NADPH ÷ H+ CH3 [~COOH+ "OH
3CO2 + 4HSCoA * NADP++ H20
Assay Methods The activity of the enzyme complex can be determined by measuring the incorporation of radioactively labeled acety1-CoA or malonyl-CoA into 6-methylsalicylic acid2 ,7 6-Methylsalicylic acid has to be separated by gaschromatography or thin-layer chromatography prior to determination, because fatty acid synthetase is also present in crude extracts, producing radioactively labeled fatty acids. After complete separation of fatty acid synthetase, the rate of NADPH oxidation can be followed spectrophotometrically at 340 nm. 6 A more sensitive assay, specific for 6-methylsalicylic acid synthetase, is the fluorometric determination of enzymatically formed 6-methylsalicyclic acid. Fluorimetric A s s a y Principle. The fluorescence spectra of 6-methylsalicyclic acid and NADPH are shown in Fig. 1. In 0.1 M potassium phosphate, pH 7.6, containing 1.25 mg/ml bovine serum albumin, the maximal excitation wavelength is 308 nm and the maximal emission wavelength is 410 nm. Using an excitation wavelength of 310 nm and measuring the fluorescence intensity at 390 nm, 6-methylsalicylic acid determination is not disturbed by the concomitant change of NADPH fluorescence. Fluorescence intensity of 6-methylsalicylic acid is increased about 30-fold in the presence of bovine serum albumin. Fluorescence has been measured at 90 ° to the exciting beam using a Hitachi MPF-3 fluorescence spectrophotometer. The reaction may be carried out using any temperature-stabilized fluorometer which incorporates a recorder fitted with a zero suppression de-
F. Lynen and M. Tada, Angew. Chem. 73, 513 (1961). ~'P. Dimroth, H. Walter, and F. Lynen, Eur. J. Biochem. 13, 98 (1970). R. J. Light, J. Biol. Chem. 242, 1880 (1967).
522
ANTIBIOTIC BIOSYNTHESIS
Sl I
o) r-
I
I
I
l | l I I I I l
/
c o o
o I1.
!
/
/
I
\\
\\
I I
/
U
[38]
l I l I l I I l t l
I
! !
\
420
460
/
\
\
\
,
l I
I
280
320
360
380 Wavelength,
480
nrn
FIG. 1. Fluorescence spectra (left set, excitation spectra; right set, emission spectra) of 6-methylsalicylic acid ( ) and NADPH ( - - - ) in 0.1 M potassium phosphate, pH 7.6, containing 1.25 mg of bovine serum albumin per milliliter. The excitation spectrum of 6-methylsalicylic acid (c = 0.5 #M) was measured by setting the emission wavelength at 410 nm, and the emission spectrum was measured by setting the excitation wavelength at 310 nm. The excitation spectrum of NADPH (c = 20 #M) was measured by setting the emission wavelength at 455 nm, and the emission spectrum was measured by setting the excitation wavelength at 334 nm. The excitation and emission wavelengths used in the assay of 6-methylsalicyclic acid synthetase are indicated by the arrows. vice, n e c e s s a r y for zeroing o u t e x t r a n e o u s fluorescence i n t e n s i t i e s due to the high p r o t e i n c o n t e n t in the cuvette. Reagents P o t a s s i u m p h o s p h a t e buffer, 1 M , p H 7.6 B o v i n e s e r u m a l b u m i n , 25 m g / m l N A D P H , p H 8, 20 m M A c e t y l - C o A , p H 5-7, 10 m M 8 M a l o n y l - C o A , p H 5-7, 20 m M 9 sPrepared from the corresponding anhydrides by the method of E. J. Simon and D. Shemin, J. Amer. Chem. Soc. 75, 2520 (1953). 9For preparation, see F. Lynen, this series, Vol. 5 [60].
[]8]
6-METHYLSALICYLIC ACID SYNTHETASE
523
Procedure. The reaction mixture (in a 3.0-ml cuvette, d = 1 cm) contains in a total volume of 2 ml, 0.2 ml of 1 M potassium phosphate, pH 7.6, 2.5 mg of bovine serum albumin, 0.2 mM NADPH, 0.1 mM acetyl-CoA, and 0.2-1.0 mU of enzyme. The assay is performed at 25 °. The background fluorescence is compensated for by an electrical offset circuit. After establishment of a stable baseline, 0.02 ml of malonyl-CoA (0.2 mM) are added, and the increase in fluorescence associated with 6methylsalicylic acid formation is followed. The extent of fluorescence change is related to that obtained with a standard solution of 6-methylsalicylic acid. TM Sensitivity of the fluorometer is adjusted so that addition of 2 nmoles of 6-methylsalicylic acid (10 ~l of a 0.2 mM solution) gives a full-scale deflection on the recorder under the usual assay conditions. This assay also works with crude extracts of enzyme provided that the solution is clear. Units. One unit of enzyme is defined as that amount of enzyme catalyzing the formation of 1 ~mole of 6-methylsalicylie acid per minute at 25 ° under the previously described assay conditions. Protein is determined by the biuret method 11 or in solutions of low protein content by the spectrophotometric method at 260 and 280 nm. 11 Organism and Growth Conditions Agar slants of Penieillium patulum N R R L 679 (Central bureau voor Schimmelcultures, Baarn, The Netherlands) are maintained at room temperature for a minimum of 10 days with transfers at 4-6-week intervals. To prepare the medium, 15 g of bacto agar (Difco) are dissolved with heating in 1 liter of H~O, and 20 ml of wort (LSwenbrauerei, Munich) arc added. Stock cultures are kept at 4 ° to prevent strain variation. A separate slant is used to inoculate each 2-liter Fernbach flask containing 650 ml of a modified Czapek-Dox medium, ( N a N Q , 3 g; KH2P04, 2.5 g; K2HPO~, 2.5 g; MgSO~'7H20, 0.5 g; KC1, 0.1 g; yeast extract (Difco), 3 g; glucose, 30 g; and distilled water to 1 liter). Flasks are incubated at 30 ° for 36 hr on a rotary shaker using a 5-cm stroke and 100 rotations per minute. The contents of two of these flasks are then added to 10 liters of the modified Czapek-Dox medium in a 14-liter fermentor vessel (New Brunswick, F-14). Cultures are grown in a Fermentor (New Brunswick, FS314) for 24 hr at 25 ° with an aeration rate of 6 liters per minute and an agitation rate of 200 rpm. Foaming is prevented by addition of 0.5 ml of Niax Polyol (Brentag GmbH, Mfilheim, Germany). Yield from lo For preparation, see this volume [39]. 1~E. Layne, this series, Vol. 3 [73].
524
ANTIBIOTIC BIOSYNTHESIS
[38]
a typical 10-1iter fermentation is 150-200 g of wet cells. The mycelia are collected on a cheesecloth lining a wire basket and washed with 0.9% of sodium chloride solution. The cheesecloth is then wrung out forcibly to remove residual liquid. This is essentially the method used by Dimroth et al2 Fresh cells are used immediately or are lyophilized and stored at --20 ° without loss of enzymic activity over several months, if kept in tightly sealed containers. A shake culture technique, which is of advantage to laboratories lacking a fermentor, is described in the article on 6-methylsalicylic acid decarboxylase?°
Purification Procedure All operations are carried out at 0-4 °. Step 1. Cell Breakage and Extraction of the Enzyme. About 1000 g of fresh cells or 250 g of lyophilized mycelia are suspended in 6-8 liters of 0.2 M potassium phosphate, pH 7.6, containing 6% (w/v) polyethylene glycol 6000 (Serva, pract, grade), 5 mM 2-mercaptoethanol, and 5 mM EDTA. The suspension is cooled to 3-5 °, and then the cells are disrupted by passage once through a Gaulin press (Manton-Gaulin, Everett, Massachusetts) at 600 arm. Unbroken cells and cell fragments are removed by centrifugation at 5000 g for 40 min, and the supernatant fraction is saved. Small-scale extraction is carried out by agitating a suspension of 7.5 g wet weight cells in a cell homogenizer12 for 1 min with 30 g of glass beads (size 31/10, Dragonwerk Wild, Bayreuth) in 0.2 M potassium phosphate, pH 7.6, containing 5 mM 2-mercaptoethanol, and 5 mM EDTA. Step 2. Polyethylene Glycol 6000 Precipitation. The desired concentration of polyethylene glycol is achieved by using a stock solution prepared by dissolving 50% (w/w) of polyethylene glycol 6000 in 50 mM potassium phosphate, pH 7.6. To 1 liter of the supernatant fraction 360 ml of this polyethylene glycol solution are added slowly while the mixture is stirred mechanically. The solution is stirred for a further 20 min and centrifuged for 40 rain at 5000 g. Step 3. Polyethylene Glycol 1500 Fractionation. The supernatant fraction is discarded, and the precipitate is well homogenized using a plastic homogenizer with a Teflon plunger in about 300 ml of 0.2 M potassium phosphate, pH 7.6, containing 8% (w/v) polyethylene glycol 1500 1~M. Merkenschlager, K. Schlossmann, and W. Kurz, Biochem. Z. 329, 332 (1957).
[381
6-METHYLSALICYLIC ACID SYNTHETASE
525
(Serva, pratt, grade), 0.5 M sodium chloride, 5 mM 2-mercaptoethanol, and 5 mM EDTA. The resulting precipitate is removed by centrifugation at 10,000 g for 20 min. To each 100 ml of the green supernatant liquid a 70-ml portion of a solution containing 50% (w/w) polyethylene glycol 1500 dissolved in 50 mM potassium phosphate, pH 7.6, is then added dropwise, while the mixture is stirred gently. Stirring is continued for a further 20 min, and the suspension is centrifuged at 15,000 g for 15 min. Step 4. Ammonium Sul]ate Fractionation. The green precipitate resulting from step 3 is homogenized in about 300 ml of 0.2 M potassium phosphate, pH 7.6, containing 38.4 g ammonium sulfate (22% saturation), 5 mM 2-mercaptoethanol, and 5 mM EDTA. The cloudy liquid is centrifuged at 10,000 g for 20 rain, and the supernatant fraction is taken to 38% saturation by the addition of solid ammonium sulfate. The supernatant fraction, containing a green protein component which shows peroxidase activity, is discarded. The precipitated protein is dissolved in a minimal volume (about 50 ml) of 50 mM potassium phosphate, pH 7.6, containing 5 mM 2-mercaptoethanol (EDTA omitted), and desalted by filtration on a 5 X 25 cm column of Sephadex G-25 previously equilibrated against the same buffer. Step 5. Hydroxyapatiie Chromatography. The desalted, slightly yellow solution is then placed on a 5 X 20 cm column of hydroxyapatite (Bio-Gel HT, Bio-Rad) preequilibrated with the desalting buffer. A layer of about 1 cm of the adsorbant is whirled up carefully so as to mix with the protein solution and is then allowed to settle again. By this procedure a sharp starting zone is obtained. This starting zone is readily observable by examining the fluorescence of flavin components of the solution using a UV-lamp. Up to this stage of the purification procedure, the 6-methylsalieylic acid synthetase and the fatty acid synthetase of this organism are purified together, with similar yields and a comparable purification factor. As can be seen from Fig. 2, they are completely separated by hydroxyapatite chromatography. 6-Methylsalicylic acid synthetase is eluted from the column witll a linear gradient established between 800 ml of 50 mM and 800 ml of 0.22 M potassium phosphate, pH 7.6, containing 5 mM 2-mereaptoethanol. An uniform flow rate of 100 ml per hour is maintained by means of a pump. The eluent is collected, and active fractions arc pooled. If desired, fatty acid synthetase can be isolated by a further stepwise elution with potassium phosphate, pH 7.6, of increasing ionic strength (see Fig. 2). Elution, readily observable by the bright yellow fluorescence of the enzyme, usually occurs during the application of a 0.25 M phosphate buffer to the column.
526
[38]
ANTIBIOTIC BIOSYNTHESIS
.....
.,~t100'
OD280n m
"//~
"/t' 10.05M
mm~
eoo~
:. ".
t ~,
"
t
i
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:" :"'"
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80 Fractions
I
I
D 0.22M I Potassium phosphate rnolarity
"~
~"."
I I i
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I
120 0.25M
I 0.5M
I
FIG. 2. Hydroxyapatite column. Desalted protein from step 4 (1.6 g in a volume of 72 ml, 60 units) was placed on a 5 × 20 cm column as described in the text. 6-Methylsalicylic acid synthetase was eluted in 14 hr (overnight) with a linear gradient of 0.05 M and 0.22 M potassium phosphate (pH 7.6, 5 mM 2-mercaptoethanoD, 1600 ml total volume. The flow rate was maintained at about 110 ml/hr, and 15-ml fractions were collected. Fatty acid synthetase was eluted by a stepwise increase of phosphate concentration to 0.25 M. Peak I and peak II contained green fluorescent proteins, visualized with a UV lamp, whereas fatty acid synthetase is bright yellow fluorescent.
Step 6. Gel Filtration on Sepharose 6B. The solution, containing 6methylsalicylic acid synthetase activity, is t a k e n to 45% saturation with solid a m m o n i u m sulfate. The precipitated protein is brought down by centrifugation at 10,000 g for 30 min and dissolved in a minimal volume (about 25 ml) of 50 m M potassium phosphate, p H 7.6, containing 5 m M 2-mercaptoethanol and 5 m M E D T A . The concentrated enzyme solution is placed on a 5 X 90 cm column of Sepharose 6B (Pharmacia) previously equilibrated with the above buffer. The protein is then eluted from the column at a flow rate of approximately 100 ml per hour, 12-ml fractions being collected. E n z y m e activity appears after an elution volume of about 800 ml in the second of three m a j o r protein peaks. Tubes containing activity are pooled for subsequent fractionation. The leading and trailing edges of the enzyme p e a k are discarded. Step 7. DEAE-CeUulose Chromatography. The pooled fractions from the Sepharose-6B column are loaded onto a 5 X 16 cm DEAE-cellulose
[38]
6-METHYLSALICYLIC ACID SYNTHETASE
527
column (Whatman DE-52) preequilibrated with 50 mM potassium phosphate, pH 7.6, containing 5 mM 2-mercaptoethanol, and 5 mM EDTA. The enzyme is eluted by a linear gradient established between 500 ml of the starting buffer and 500 ml of the same buffer containing 0.2 M sodium chloride, 10-ml fractions being collected. The enzyme appears at the end of the gradient as a broad peak. Fractions containing the major portion of the enzyme are pooled and concentrated by ammonium su~.fate precipitation as described in step 6. The precipitated protein is dissolved in a minimal volume of an appropriate storage buffer (see below). Better recovery of enzymic activity is sometimes observed using an alternative procedure. The DEAE-cellulose column is equilibrated with the same equilibration buffer described above, but containing 5% (w/v) polyethylene glycol 1500. In this case the enzyme is eluted by a linear gradient of 0.1 M to 0.4 M sodium chloride in the starting buffer, 1000 ml total volume. The enzyme again appears as a broad peak at the end of the gradient. Fractions containing the bulk of the activity are pooled and the enzyme is concentrated by centrifuging this solution at 100,000 g for 14 hr. The resulting pellet is homogenized in an appropriate storage buffer (see below). The results of a typical purification procedure starting with 950 g of wet weight cells are summarized in the table and show that there is approximately a ll0-fold increase in specific activity with a 20% recovery of enzymic activity.
PURIFICATION OF 6-METHYLSALICYLIC ACID SYNTHETASE
Step and fraction 1. Centrifuged extract 2. Polyethylene glycol 6000 precipitation 3. Polyethylene glycol 1500 fractionation 4. 22-38% Ammonium sulfate fraetionation 5. Hydroxyapatite eluate 6. Sepharose-6B eluate 7. DEAE-cellulose eluate a
Volume (ml)
Protein (mg)
Total Specific activity activity Re(milli(milli- covery units) units/rag) (%)
8200 320
48,000 10,800
93,000 82,000
280
4,700
75,000
16
80
48
1,600
60,000
37.5
64
320 290 270
410 180 92
38,000 29,000 19,000
92.5 161 206
41 31 20
1.9 7.5
100 88
DEAE-eellulose chromatography without polyethylene glycol 1500 in the equilibrating buffer.
528
ANTIBIOTIC BIOSYNTHESIS
[38]
Properties
Stability and Storage. The 6-methylsalicylic acid synthetase is unstable in solutions of low ionic strength or when exposed to acid or alkaline pH. The purified enzyme can be stored for several weeks at --15 ° in 0.2 M potassium phosphate, pH 7.6, containing 50% glycerol, and 1 mM dithiothreitol, or at 2 ° in 0.2 M potassium phosphate, pH 7.6, containing 5% (w/v) polyethylene glycol 1500, and 1 mM dithiothreitol. Pure or partially purified enzyme preparations dissolved in 0.2 M potassium phosphate, pH 7.6, can be lyophilized and stored at --25 ° for at least one year with only slight loss of activity, when protected from moisture. Homogeneity. The purified enzyme forms a single peak in the ultracentrifuge and migrates as a single protein band upon electrophoresis on cellogel strips (Serva) at various pH values. It forms a single precipitation band in Ouchterlony double-diffusion plates and immunoelectrophoresis against rabbit antisera prepared against purified enzyme preparations. With acrylamide disk electrophoresis, two or sometimes three diffuse bands are observed. Possibly dissociation of the enzyme takes place under these conditions. The enzyme is completely free of fatty acid synthetase. Kinetic Properties. The reaction has an optimal pH of about 7.6. 6 The Km values for acetyl-CoA and malonyl-CoA are both 20 ~M. 6 Molecular Weight. A molecular weight of about 1.2 X 106 has been determined by Dimroth et al., 6 using sucrose density gradient centrifugation with catalase and yeast fatty acid synthetase as references. A molecular weight of about 3.7 X 10~ is reported by Light and Hager, 1~ using gel filtration on a standardized Sephadex G-200 column and sucrose density gradient centrifugation. This great discrepancy has not been explained to date. The preparations used in the latter case have been unstable crude extracts. Perhaps the enzyme complex is dissociated under the conditions employed. Inhibition. The enzyme is inhibited by sulfhydryl reagents such as iodoacetamide and N-ethylmaleimide. 6 The inactivation rate with iodoacetamide is pH independent whereas that with N-ethylmaleimide increases with increasing pH. This indicates the participation of two types of SH-groups in the catalytic process, analogous to the yeast fatty acid synthetase. 14 One of these SH groups is identified as part of a 4'-phosphopantetheine residue of the "acyl carrier protein. ''1~ 4'-Phosphopantetheine is released by alkaline hydrolysis from the enzyme complex, and ~' R. J. Light and L. P. ttager, Arch. Biochem. Biophys. 125, 326 (1968). 14F. Lynen, this series Vol. 14 [3]. is G. GreulI, Doctorate Thesis, University of Munich, Munich, Germany, 1973.
[38]
6-METHYLSALICYLIC ACID SYNTHETASE
529
E-SH 1. CH3CO-SCoA ÷ 2HOOCCH2CO-SCoA ~ " CH3COCH2COCH2CO-SE÷2CO2 OH l /,~ (%J..
Triacetic acid lactone
/ H3C~XO/"~O 2. CH3COCH2COCH2CO~SE+ NADPH + H*-<---~ CH3COCH2CH(OH)CH2CO-SE+NADP+
3. CH3COCH2CH(OH)CH2CO-SE -
~ CH3CO-CH=CH-CH2CO-SE * H20 cis
4. CH3CO-CH=CH-CH2CO-SE-"HOOCCH2CO-SCoA~ CH3CO-CH=CH-CH2COCH2CO-SE cis H3C'~c//O HC/ CH,)-CO-SE 5.
II I ~ HC~c/C=O
Atdol condensation Hydrolysis
~
/J,,~CH3 F(,--~T COOH ----~-T~'OH
H2
FIG. 3. Hypothetical reaction scheme for 6-methylsalicylicacid synthesis. pantothenate is determined microbiologically with LactobacilIus plantarum. Inhibition of the enzyme by the hex-3-ynoyl-N-acetylcysteamine (CH~CH2C~CCH2COSCH2CH2NHCOCH~) at concentrations around 110 ~M is reported. 1~ Enzyme activity is inhibited completely by rabbit antisera prepared against highly purified enzyme preparations. Substrate Specificity. Replacing NADPH by NADH as reducing agent, the reaction rate is reduced to 10-20%. The specificity of the "priming" reaction is studied by measuring the rate of acyl transfer from acyl-CoA to pantetheine. Propionyl-CoA is found to be transferred 13 times more slowly to pantetheine than acetyl-CoA.17 Incubating the enzyme with propionyl-CoA as a "primer" in the presence of malonyl-CoA and NADPH, 6-ethylsalicylic acid is formed although with a reduced rate? 7 Butyryl-CoA is ineffective in this respect. Incubation of 6-methylsalicylic acid synthetase with acetyl-CoA and malonyl-CoA in the absence of NADPH leads to the formation of triacetic acid lactone as the only product, at a rate of one-tenth that of 6-methylsalicylic acid production G (see also Fig. 3). The specificity of the reductase in 6-methylsalicylic acid synthetase is different from that of the fatty acid synthetase. Triacetic acid ethyl ester as a model substrate is readily reduced by 6-methylsalicylic acid synthetase, whereas S-acetoacetyl-N-acetylcysteamine is inactive in this respect. TM The opposite is the case in fatty acid 16A. I. Scott, IUPAC Meeting, Sec. 0-13, Boston, Massachusetts,July 1971. 1~p. Dimroth, unpublished results. ~ R. Seyffert, unpublished results.
530
[391
ANTIBIOTIC BIOSYNTHESIS
synthetase. S-Acetoacetyl-N-acetylcysteamine is actively reduced, whereas triacetic acid ethyl ester is only a poor substrate. Enzyme Mechanism. In analogy to fatty acid synthesis, and supported by the experimental evidence described above, a hypothetical reaction scheme for 6-methylsalicylic acid synthesis has been proposed (Fig. 3).5,6 According to this scheme, all the intermediates in 6-methylsalicylic acid synthesis are covalently bound as thioesters to a multienzyme complex. The reaction sequence is initiated by the binding of an acetyl and a malonyl residue to the enzyme. Condensation between the acetyl and malonyl group with concomitant loss of carbon dioxide gives rise to the formation of ucetoacetyl-enzyme. In a second condensation step triacetoacetyl-enzyme is formed. The 3,5-diketoacyl-enzyme is reduced then to the fl-hydroxy-5-ketohexanoyl-enzyme and is then dehydrated with the formation of the cis fl,7-unsaturated ester. The stereochemistry in this step is opposite to that in fatty acid synthesis, where a trans a,fl-unsaturated ester is formed. By a final condensation step with malonyl-CoA, the carbon skeleton of 6-methylsalicylic acid is completed. Finally, enzyme-bond 6-methylsalicylic acid is formed by aldol condensation and is then liberated by hydrolysis from the thioester bond.
[39] 6-Methylsalicylic Acid (2,5-Cresotic Acid) Decarboxylase By
ROBLEY J. LIGHT and GUNTER VOGEL COOH
H Q ~ cH3
~
H~
cH3 +
6-MethylsaUcyllc Acid (2,6- Cresotlc Acid)
C02
m- Cresol
The decarboxylation of 6-methylsalicylic acid (6-MSA) represents the first step in the conversion of this aromatic polyketide into patulin.
Assay Methods
Principle. Four assay methods have been used for the measurement of decarboxylase activity: (1) liberation of 14C02 from [carboxyl-14C] 6MSA1; (2) disappearance of 6-MSA by decrease in absorption at 300 1R. J. Light, Biochim. Biophys. Acta 191, 430 (1969).
530
[391
ANTIBIOTIC BIOSYNTHESIS
synthetase. S-Acetoacetyl-N-acetylcysteamine is actively reduced, whereas triacetic acid ethyl ester is only a poor substrate. Enzyme Mechanism. In analogy to fatty acid synthesis, and supported by the experimental evidence described above, a hypothetical reaction scheme for 6-methylsalicylic acid synthesis has been proposed (Fig. 3).5,6 According to this scheme, all the intermediates in 6-methylsalicylic acid synthesis are covalently bound as thioesters to a multienzyme complex. The reaction sequence is initiated by the binding of an acetyl and a malonyl residue to the enzyme. Condensation between the acetyl and malonyl group with concomitant loss of carbon dioxide gives rise to the formation of ucetoacetyl-enzyme. In a second condensation step triacetoacetyl-enzyme is formed. The 3,5-diketoacyl-enzyme is reduced then to the fl-hydroxy-5-ketohexanoyl-enzyme and is then dehydrated with the formation of the cis fl,7-unsaturated ester. The stereochemistry in this step is opposite to that in fatty acid synthesis, where a trans a,fl-unsaturated ester is formed. By a final condensation step with malonyl-CoA, the carbon skeleton of 6-methylsalicylic acid is completed. Finally, enzyme-bond 6-methylsalicylic acid is formed by aldol condensation and is then liberated by hydrolysis from the thioester bond.
[39] 6-Methylsalicylic Acid (2,5-Cresotic Acid) Decarboxylase By
ROBLEY J. LIGHT and GUNTER VOGEL COOH
H Q ~ cH3
~
H~
cH3 +
6-MethylsaUcyllc Acid (2,6- Cresotlc Acid)
C02
m- Cresol
The decarboxylation of 6-methylsalicylic acid (6-MSA) represents the first step in the conversion of this aromatic polyketide into patulin.
Assay Methods
Principle. Four assay methods have been used for the measurement of decarboxylase activity: (1) liberation of 14C02 from [carboxyl-14C] 6MSA1; (2) disappearance of 6-MSA by decrease in absorption at 300 1R. J. Light, Biochim. Biophys. Acta 191, 430 (1969).
[39]
6-METHYLSALICYLIC ACID DECARBOXYLASE
531
nm2; (3) disappearance of 6-MSA by decrease in fluorescenceS; and (4) appearance of COs by manometric techniques, s Only two methods will be described. Method 1. Fluorescence Assay The method is based upon a decrease in fluorescence intensity as 6MSA is decarboxylated. Measurement is made in a solution containing 1.25 mg/ml of bovine serum albumin, which enhances the 6-MSA fluorescence intensity about 30-fold. The method is described for use with a Hitachi MPF-3 spectrofluorometer with an excitation wavelength of 310 nm and fluorescence intensity measurement at 420 rim. It can be adapted to a filter fluorometer with excitation in the 310-360 nm range and fluorescence intensity measurement in the 380-2000 nm range. Reagents 6-MSA, 2 mM in water Potassium phosphate buffer, 1 M, pH 7.5 Bovine serum albumin, 25 mg/ml Procedure. The reaction mixture (in 3.0 ml cells, d = 1 cm) contains in a volume of 2 ml: 200 ~moles of potassium phosphate, pH 7.5 (0.2 ml); 2.5 mg of bovine serum albumin (0.1 ml); and 200 nmoles of 6-MSA (0.1 ml). The reaction is carried out at 25 °. By the associated zero suppression device, fluorescence intensity is set on a recorder deflection of 100. After establishment of a stable baseline of fluorescence, the reaction is started with 0.5-1 mU of enzyme, and the decrease in fluorescence is followed. Fluorescence change is related to that obtained with a standard solution of 6-MSA. The sensitivity of the fiuorometer is adjusted so that addition of 10 nmoles of 6-MSA (5 ~1 of a 2 mM solution) gives a full-scale deflection on the recorder under the chosen assay conditions. The rate of reaction is linear for at least 15 min. The assay is applicable to crude extracts, provided the solution is clear. Method 2. Radioactive Assay 1 Reagents [Carboxyl-14C]6-MSA, specific activity about 0.03 ~Ci/~mole, 0.5 mM in water. Potassium phosphate buffer, 20 mM, pH 7.5 2G. Vogel, Doctorate Thesis, University of Munich, Munich, Germany, 1971.
532
ANTIBIOTIC BIOSYNTHESIS
[39]
Hyamine hydroxide, 1 M i~ methanol H2S04, 10 N 6-MSA decarboxylase preparation in 20 mM phosphate buffer, pH 7.5. Scintillation counting fluid, 4 g of 2,5-diphenyloxazole, 0.05 g of 1,4bis [2- (5-phenyloxazolyl) ] benzene in 1 liter of toluene.
Procedure. A folded paper (Whatman No. 1, 2 X 4 cm), soaked in 0.2 ml of a 1 M Hyamine hydroxide solution is placed in the center well of a Warburg flask, and 0.3 ml of 10 N H_.S04 is added in the side arm. About 10-~ unit of 6-MSA decarboxylase is placed in the main compartment of the flask together with sufficient phosphate buffer to give a final volume of 2.0 ml. [Carboxyl-14C]6-MSA (0.08 ml, 40 nmoles) is then added to the main compartment. The flask is stoppered and allowed to incubate at room temperature (25 ° ) for 1 hr. The incubation is terminated by mixing the side arm and main compartment contents, and the flask is kept closed at room temperature for an additional 2 hr to allow complete adsorption of the C02 by the Hyamine-soaked paper. The Hyamine-soaked paper is then transferred to a scintillator counting bottle, the center well is rinsed twice with 0.2 ml of methanol into the bottle, 15 ml of scintillation fluid is added, and the sample is counted in a scintillation spectrometer. Other scintillation counting fluids can be used. The amount of quenching from the Hyamine hydroxide and the efficiency of counting can be determined by adding an internal standard. The quantity of 14C0~ released can then be calculated from the known specific activity of the starting [carboxyl-i4C]6-MSA and the counting efficiency. The 14C02 released is linear with added protein up to about 5 X 10-4 unit of decarboxylase activity. It is possible to adapt this assay to smaller volumes in order to conserve radioactive substrate. In this case the enzyme and buffer are added to the sidearm of the Warburg flask in a total volume of 0.5-0.8 ml, together with 10-15 nmoles of [carboxyl-14C]6-MSA, while the main compartment contains 3 ml of 3 N H~S04. Units. One unit of activity (in either assay) is defined as the amount of enzyme that catalyzes the decarboxylation of 1 ~mole of 6-MSA per minute at 25 ° and pH 7.5. Preparation of 6-MSA
Chemical Synthesis. Two methods for chemical synthesis of 6-MSA have been reported. One 3 involves sulfonylation of m-cresol in fuming 8 E. L. Eliel, D. E. Rivard, and A. W. Burgstahler, J. Org. Chem. 18, 1679 (1953).
[~9]
6-METHYLSALICYLIC ACID DECARBOXYLASE
533
sulfuric acid to block the reactive 4 and 6 positions, bromination in the 2 position, and removal of the sulfonic acid groups by steam distillation with superheated steam. The resulting 2-bromo-m-cresol is converted to the lithium derivative with butyl lithium and carboxylated with COs, a step easily adapted to synthesis of [carboxyl-14C]6-MSA. 2 A simpler alternative synthesis, but not so easily adapted to isotopic labeling, involves heating the cupric salt of o-toluic acid in mineral oil at 215%~ Since these procedures are adequately described elsewhere, 3,4 they will not be given here. Isolation of Biosynthetic 6-MSA. 6-MSA is produced as an extracellular metabolite during the "idiophase" of growth of submerged Penicillium patulum cultures. 5,6 Yields of 6-MSA can vary considerably, sometimes unpredictably, depending upon the precise timing of appearance and the relative levels of activity of 6-MSA synthetase and 6-MSA decarboxylase. Conditions of idiophase growth which lead to appearance of 6-MSA can be triggered artificially by using a combination of step-down culture from rich to poor medium together with a partial inhibition of protein synthesis. 8,9 This procedure has some advantages in timing and can be used with shake cultures, and will therefore be described here. Alternatively the fermentor culture technique described in the article on 6-MSA synthetase 5 could be used by isolating 6-MSA when it is near its maximum level, usually at some time between 24 and 72 hr of growth2 ,7 Levels of 6-MSA in the medium can sometimes exceed 0.5 mg/ml, and for the best yields one should harvest cultures when levels approach this value. For simple estimation of 6-MSA, 1 ml of culture medium is diluted to 4 ml with water, and I ml of FeC13 reagent (0.2 M FeCI~ in 0.1 N HC1) is added. The optical density of the dark red complex formed is determined in a spectrophotometer at 550 nm or in a Klett-Summerson colorimeter with a 540 nm filter. A solution of 100 mg of 6-MSA per milliliter gives an optical density of 0.165 or a Klett reading of 94. While other phenols also give a color with the FeCl.~ reagent, 6-MSA is the first major phenolic substance to appear in the medium, and hence isolation should be carried out soon after the initial rise in phenolic content, before appreciable degradation to m-cresol and other products has occurred. A more specific estimation of 6-MSA content 4W. W. Kaeding and G. R. Collins, J. Org. Chem. 30, 3750 (1965). 5This volume [38]. 6j. D. Bu'lock, D. Hamilton, M. S. Hulme, A. J. Powell, H. M. Smalley, D. Shepherd, and G. N. Smith, Can. J. Microbiol. 11, 765 (1965). P. Dimroth, H. Walter, and F. Lynen, Eur. J. Biochem. 13, 98 (1970). s R. J. Light, Arch. Biochem. Biophys. 122, 494 (1967). R. J. Light, J. Biol. Chem. 232, 1880 (1967).
534
ANTIBIOTIC BIOSYNTHESIS
[39]
can be carried out by gas-liquid chromatography of a diazomethanetreated ether extract of the growth medium,2 or by thin-layer chromatography (see below) of an aliquot of growth medium. Cultures of Penicillium patulum N R R L A-14806, which was an isolate from NRRL 2159A (early MSA isolateS,9), are maintained on malt agar slants (20 g of malt extract, 20 g of glucose, 1 g of peptone, 20 g of agar, 1 liter of distilled water) at 25 ° with transfer every few weeks. Spores from a slant at least 2 weeks old are washed into 150 ml of germinating medium contained in a 500 ml Erlenmeyer flask. Germinating medium is prepared by adding the following chemicals to hot (90-95°), stirred distilled water: 20 g of peptone, 10 g of malt extract, 40 g of glucose, 20 g of soluble starch (suspended first in cold water, with lumps broken up), 3 g of NaNO~, 1 g of KH.~P04, 0.5 g of 65% MgS04, 0.5 g of KC1, and 0.02 g of FeSO~. 7H~O per liter of distilled water. Heating and stirring are continued until solution is achieved, and the medium is then sterilized by autoclaving. The germinating medium culture is incubated on a rotary shaker (1 inch stroke, about 150 rpm) at room temperature for 16-24 hr. The culture is blended for 10 sec in a Waring blender and mycelium is harvested by filtration on a Biichner funnel and washed with Czapek-Dox medium (50 g of glucose, 2 g of NaNOs, 1 g of KH2PO~, 0.5 g of KC1, 0.37 g of 65% MgS04, 0.01 g of FeSO4.7H20, and 0.029 mg of ZnSO4.7H20 per liter of distilled water). Yields of 3-4 g of mycelium, wet weight, are usually obtained. The mycelium is then resuspended in Czapek-Dox medium containing 0.3 ~g/ml of cycloheximide (Actidione, Up John Co., Kalamazoo, Michigan) which is added after autoclaving. Suspensions of about 0.5-1.5 g wet weight of mycelium per 100 ml of medium are then incubated on the rotary shaker in Erlenmeyer flasks. 6-MSA is detectable after about an hour and reaches high levels about 4-10 hr after transfer, s,9 Because of the short incubation time, sterile technique is not necessary during the harvest and transfer of mycelium. Mycelium is removed from the cultures by filtration through filter paper on a Biichner funnel, and the medium is acidified with dilute mineral acid and extracted four times with ether. The ether extracts are combined, and the solvent is removed in vacuo with a rotary evaporator. The residue is dried by adding 100 ml of benzene and evaporating in vacuo. The residue is triturated with warm benzene (1-5 ml), which is applied to a silicic acid chromatographic column (Mallinckrodt SilicAR CC-4, 100-200 mesh) packed in benzene. It is recommended that 10 g of silicic acid be used for every 100 ml of culture medium extracted, but in no case should the column loading exceed 20 mg of residue per gram of silicic acid. The residue is triturated with three additional portions of
[39]
6-METHYLSALICYLIC ACID DECARBOXYLASE
535
warm benzene, which are also applied to the column. The column is then eluted with benzene (20 ml per gram of silicic acid) followed by 10% ether in hexane (20 ml per gram of silicic acid). Each solvent is collected in five or more fractions, and 100-~l aliquots of each fraction are spotted on a silica gel coated thin-layer plate (Adsorbosil, Applied Sciences Laboratories). The plate is developed in ether-hexane-acetic acid, 15:80:2, v/v/v, and 6-MSA is visualized as a blue fluorescent spot under ultraviolet light or as a brown spot upon exposure to iodine vapor. The Rr may vary from about 0.5 to about 0.8, depending upon the activity of the thin-layer plate and the batch of silica gel used. The 6-MSA-containing fractions, usually the late benzene and early ether/hexane fractions, are pooled, and the solvent is removed in vacuo. The residue can be further purified by recrystallization from chloroform. Purified 6-MSA has a melting point of 174-175 °. It has the following ultraviolet absorption maxima (molar extinction coefficient in parentheses): water sh 240 nm (3460), 300 nm (1730) ; 0.01 M phosphate buffer, pH 7, sh 238 nm (4150), 298 nm (1840). Its fluorescence spectrum is given in the 6-MSA synthetase article. ~ Preparation o] Biosynthetic [I~C]6-MSA. The biosynthetic preparation of [14C]6-MSA is conveniently carried out using the cycloheximidestepdown shake culture described above. Mycelium harvested from germinating medium (0.8 g, wet weight) is dispersed into 50 ml of Czapek-Dox cycloheximide medium contained in a 125 ml Erlenmeyer flask, and shaken at room temperature on a rotary shaker. Sodium [1-14C]acetate (125 ~Ci) is added after about 1 hr, and the [I~C]6-MSA is isolated after an additional 3-6 hr of shaking. The extraction, silicic acid chromatography, and recrystallization are carried out as described above. Use of a 10-g column of silicic acid, elution by 200 ml each of benzene and 10% ether in hexane, and collection of 25-ml fractions are recommended. Yields and final specific activity might vary, but one should expect at least 10% of the added radioactivity to be incorporated into the 6-MSA. Carrier 6-MSA may be added at any step during the isolation if desired and may improve the yield of ~4C isolated as 6-MSA at the cost of reducing the specific activity of the product. Chemical degradation of [14C]6-MSA produced from [1-~C]acetate has shown that one-fourth of the radioactivity is found in the carboxyl group, ~°,~1 and hence the specific activity as [carboxyl-14C]6-MSA, calculated for the decarboxylase assay described above, should be one-fourth the total specific activity of the [14C]6-MSA sample. 1~R. J. Light, Arch. Biochem. Biophys. 112, 163 (1965). 11A. J. Birch, R. A. Massey-Westropp, and C. J. Moye, Aust. J. Chem. 8, 539 (1955).
536
ANTIBIOTIC BIOSYNTHESIS
[39]
Purification Procedure Penicillium patulum N R R L 679 is grown in fermentor culture as described for the isolation of 6-MSA synthetase. ~ Mycelium is harvested after 70 hr of growth. Cells are used fresh or may be lyophilized and stored at --20 ° without loss of activity for several months. All operations are carried out at 0-4 ° unless otherwise stated. The procedure is described for 2.2 kg wet weight of mycelium. Step 1. Cell Breakage and Extraction o] the Enzyme. The mycelium is suspended in about 10 liters of 0.2 M Tris.HC1, pH 7.5, containing 100 g per liter of ammonium sulfate. Cells are disrupted by passing them twice through a Gaulin press (Manton-Gaulin, Everett, Massachusetts) at 600 arm. Temperature rises in this procedure to about 25 °. Small-scale extraction may be carried out by suspending 7.5 g wet weight of mycelium, 30 g of Ballotini glass beads (31/10, Dragonwerk Wild, Bayreuth, Germany), and 24 ml of the above buffer. The suspension is agitated for 90 sec in the cell homogenizer of Merkenschlager et al. 12Release of activity by sonication has also been reported. 1 Step 2. First Ammonium Sul]ate Fractionation. The resulting suspension is brought to about 40% saturation by the addition of 133 g of solid ammonium sulfate per liter. The mixture is stirred slowly and mechanically for 30 min and centrifuged for 2 hr at 3000 g. The slightly turbid supernatant is taken to 65% saturation by addition of 165.6 g of ammonium sulfate per liter, stirred mechanically for 20 min, and centrifuged at 10,000 g for 1 hr. The supernatant liquid is discarded. The precipitate, lyophilized after dissolving in a minimal volume of 0.2 M Tris.HC1, pH 7.5, can be stored as a dry powder for several months without loss of activity. It may be more convenient to extract only small portions of fresh mycelium, carry out the first ammonium sulfate fractionation, and store the lyophilized active fraction. Difficulties involved in centrifuging large volumes are thereby avoided. Step 3. Ultracentri]ugation. The fresh precipitate or the dried powder from the first ammonium sulfate step is dissolved in about 1 liter of 0.2 M Tris-HC1, pH 7.5, containing 100 g of ammonium sulfate per liter. This turbid solution is then centrifuged for about 5 hr at 100,000 g, and the resulting supernatant liquid, containing the activity, is saved. Step 4. Second Ammonium Sul]ate Fractionation. The dark brownish but clear supernatant fraction is taken to 60% saturation by addition of solid ammonium sulfate. The precipitate, which contains all the decarboxylase activity, is collected by centrifugation at 15,000 g for 30 min, and the supernatant liquid is discarded. ~ M. Merkenschlager, K. Schlossmann, and W. Kurz, Biochem. Z. 329, 332 (1957).
[39]
6-METHYLSALICYLIC ACID DECARBOXYLASE
537
The precipitate is transferred to a 250-ml polycarbonate centrifuge tube and extracted successively with a series of solutions of decreasing ammonium sulfate concentrations prepared at 4 ° by dissolving solid ammonium sulfate in a 0.2 M Tris.HC1 buffer, pH 7.5. The solutions, used in the order given and expressed as percent saturation, are 200 ml of 55%, 200 ml of 52%, 150 ml of 50%, 100 ml of 48%, and 100 ml of 46%. The precipitate is stirred for 10 rain and removed by centrifugation at 15,000 g for 20 rain. The supernatant liquid is decanted, and the precipitate is suspended in the next extracting solution. 6-MSA decarboxylase is found primarily in the 48-52% fractions. Active fractions are combined and taken to 70% saturation with solid ammonium sulfate. The resulting precipitate is collected by centrifugation at 15,000 g for 20 min. The pellet is dissolved in about 100 ml of 0.2 M Tris.HC1, pH 7.5, containing 100 g per liter of ammonium sulfate. Step 5. Sephadex G-IO0 Gel Filtration. The enzyme solution is applied to a 10 X 90 cm column (Pharmacia chromatography tube K100/100) of Sephadex G-100 equilibrated with the same buffer. The enzyme is then eluted from the column at a flow rate of approximately 120 ml per hour, 30-ml fractions being collected. 6-MSA decarboxylase appears after an elution volume of about 3.5-4 liters. Active fractions are pooled and taken to 70% saturation with solid ammonium sulfate; the precipitate is collected by centrifugation at 15,000 g for 30 rain. Step 6. DEAE-Sephadex A-50 Column Chromatography. The pellet of step 5 is dissolved in approximately 20 ml of 50 mM Tris-HC1, pH 7.5, containing 20% glycerol, and desalted by passage through a 3 X 25 cm column of Sephadex G-25 equilibrated against the same buffer. The enzyme solution is then placed on a 5 X 35 cm DEAE-Sephadex A-50 column previously equilibrated with the above buffer. The column is washed once with starting buffer to remove unadsorbed protein. The enzyme is subsequently eluted with a linear gradient established between 1.5 liters of starting buffer and 1.5 liters of the same buffer containing 0.2 M sodium chloride. Enzyme appears at about 0.1-0.15 M sodium chloride. Active fractions are pooled and concentrated by ammonium sulfate as described previously. Step 7. Hydroxyapatite Column Chromatography. The precipitated prote~n, which is recovered from the preceding step by centrifugation, is dissolved in about 5 ml of 50 mM Tris.HC1, pH 7.5, containing 20% glycerol, and desalted by filtration on a 2 }{ 6 cm column of Sephadex G-25. The desalted enzyme solution is applied to a 3 }( 7 cm colmnn of hydroxyapatite, prepared according to Tiselius et al.,'3 and previously equilibrated with the above buffer. (The elution pattern of Bio-Rad HT, ~ A. Tiselius, S. Hjert~n, and ~. Levin, Arch. Biochem. Biophys. 65, 132 (1966).
ANTIBIOTIC BIOSYNTHESIS
538
PURIFICATION OF
Step 1. Centrifuged extract 2. 40-65% Ammonium sulfate fractionation 3. Supernatant of ultracentrifugation 4. 48-52% Ammonium sulfate extraction 5. Sephadex G-100 eluate 6. DEAE-Sephadex A-50 eluate 7. Hydroxyapatite eluate
[39]
6-MSA DECARBOXYLASE
Volume (ml)
Total Specific ReProtein a activity activity covery (mg) (units) (units/mg) (%)
11,200 1,000
67,000 25,000
500 450
0.0075 0.018
100 90
920
11,900
420
0. 035
84
90
3,300
380
0. 115
76
420 490 32
600 32 12
300 200 145
0.5 6.25 11.6
60 40 28
a Protein is determined by the biuret method, or, when the protein content of the enzyme solution is below 2 mg/ml, by the optical method at 280-260 nm. Both methods are described by E. Layne, Vol. 3 [$7]. a commercial preparation of h y d r o x y a p a t i t e from Bio-Rad, was not tested in this procedure.) Stepwise elution is carried out with 50 ml of starting buffer, 80 ml of buffer containing 15 m M phosphate, and 80 ml of buffer containing 20 m M phosphate. Elution of enzyme usually occurs during the application of the buffer containing 15 m M phosphate. Active fractions are pooled and precipitated by 70% a m m o n i u m sulfate saturation. The highly purified enzyme is dissolved in an appropriate storage buffer (see below). The results of a typical purification procedure are s u m m a rized in the table and show a 1500-fold increase in specific activity with a 28% recovery of enzymic activity.
Properties 2
Stability and Storage. 6-MSA decarboxylase is very unstable in solutions of low ionic strength. Dilute solutions of the enzyme lose most of their activity within 15 hr at 0 °. The enzyme is s t a b l e / o r several months at - - 1 5 ° in a 0.2 M T r i s . H C l buffer, p H 7.5, containing 50% of glycerol and 5% (w/v) of a m m o n i u m sulfate. I n the same buffer, glycerol omitted, it can be lyophilized and stored at - - 2 5 ° for at least one year without loss of activity if moisture is excluded. Homogeneity. The purified enzyme forms a single, symmetrical p e a k in the ultracentrifuge. I t migrates as a single protein band upon electrophoresis on cellulose acetate strips (Cellogel, Serva) and acrylamide disc gel electrophoresis. T h e purified enzyme contains no chromophore and is also free of nucleotides and nucleic acids.
[39]
6-METHYLSALICYLIC ACID DECARBOXYLASE
539
Physical Properties. The approximate molecular weight is determined by two methods. The sedimentation profile of purified enzyme in a sucrose gradient yields a size of 56,000-60,000 when compared to the sedimentation of alkaline phosphatase (calf intestine) and peroxidase (horseradish). This value is in agreement with that (58,000) obtained upon Sephadex G-100 gel chromatography on a column standardized with cytochrome c, chymotrypsinogen, ovalbulmin, and bovine serum albumin. The isoelectric point, determined by isoelectric focusing, is pH 5.6. Kinetic Properties. The decarboxylase is active over a broad pH range with maximal activity between pH 5.5 and 7.5. The Michaelis constant (Kin) measured under the conditions of the fluorometric assay is 10 ~M, and Lineweaver-Burk plots are linear over the concentration range studied (3-30 ~M). When pK,,1 (--log Kin) is plotted against pH, a break is observed at pH 6, while Vm is 100% at this pH. Therefore only the coefficient of the substrate dependent term in the rate equation contains a rate constant dependent on some group (histidine?) in the enzyme having a pK of about 6. By measuring the influence of temperature on reaction rate, an Arrhenius activation energy of 13.5 kcal/mole has been found. Isotope Ef]ects. Cleavage of the C--C bond is not rate limiting in the decarboxylation step. No decrease in reaction rate is observed when the normal carboxyl-carbon in 6-MSA is substituted by 1~C. Enzymic decarboxylation performed in D~O is slower than in H,_,O, showing an isotope effect (vH~o/V,2o) of 2.7. The isotope effect does not vary between pH 5 and 9, an indication that the effect does not reflect changes in the ionization behavior of the enzyme or the substrate. The rate effect is seen predominantly in V...... the intercept terra of the Lineweaver-Burk plot, while the slope term is increased only about 10-15%. To exclude an effect of D20 on alteration of enzyme conformation, the tritium isotope effect in HOT was determined by comparing the specific activity of the product m-cresol with that of the water. The percent of HOT in H20 is so small as to have no effect on the protein conformation. An isotope effect (VH20/VHoT) of 3.7 was found, in good agreement with the value 3.6 calculated from the Swain equation. ~4 From these observations it is proposed that a protonation step is rate limiting in the decarboxylation process. Substrate Specificity. The enzyme also decarboxylates 6-ethylsalicylic acid (Kin = 10 tiM, relative V..... = 100%); homoorsellinic acid (K,I = 40 t~M, relative V...... = 100%); orsellinie acid (Kin = 100 ~M, ~ C. G. Swain, E. C. Stivers, J. F. Reuwer, and L. J. Schaad, J. Amer. Chem. Soc. 80, 5885 (1958).
540
ANTmmTrC BIOSYNTHESIS
[40]
relative V ~ x = 80%); and 3-bromoorsellinic acid (Kin--150 ~M, relative Vm~x = 6%). Inhibitors. Salicylic acid, 2-methylbenzoic acid, 3-methylsalicylic acid, and 4-methylsalicylic acid show noncompetitive inhibition with K~'s in the range between 1 and 2 mM. Competitive inhibitors are a-hydroxy6-methylsalicylic acid (Ki = 10 ~M), 6-methylanthranilic acid (K~ = 40 ~M) and 2-hydroxy-l-naphthoic acid (K~ = 40 t~M). The enzyme is not inhibited by iodoacetamide (10 mM), p-chloromecuribenzoate (1 mM), N-ethylmaleimide (1 mM), KCN (10 raM), NaN~ (10 mM), acetylacetone (1 mM), EDTA (10 raM), o-phenanthroline (10 raM), or a,a-dipyridyl (10 mM). It is inhibited only 20% by NaNO.~ (10 raM), and is completely inhibited by diethylpyrocarbonate (10 mM). 6-MSA decarboxylase is photooxidized in the presence of RoseBengal. When the enzyme is exposed to light at neutral pH in the presence of an excess of this dye, inactivation follows the course of a firstorder reaction. The susceptibility to photoinactivation decreases in the range of pH 8.5-5 and is found to follow closely the curve for the photooxidation of imidazol. 15 Photoinactivation is prevented in the presence of substrate or of the competitive inhibitor a-hydroxy-6-methylsalicylic acid, whereas the noncompetitive inhibitor salicylic acid shows no effect. 1~E. W. Westhead, Biochemistry 4, 2139 (1965).
[40] m - H y d r o x y b e n z y l - a l c o h o l
Dehydrogenase
By G. M. GAUCHER The antibiotic patulin is a labile, structurally simple (C7H~04) secondary metabolite 1 produced by the fungus Penicillium urticae. Extensive studies have shown that patulin is synthesized via an essentially oxidative pathway from the classic aromatic polyketide 6-methylsalicylic acid2 This phenolic acid is in turn synthesized from acetyl-CoA and malonyl-CoA in an enzymic process closely related to fatty acid biosynthesis. While of little pharmaceutical interest because of its toxicity, patulin has aroused recent interest as a fungal toxin in food 2 and as a general plant toxin. 3 However, the persistent interest in patulin biosyn1W. B. Turner, "Fungal Metabolites." Academic Press, New York, 1971. 2p. M. Scott and B. P. C. Kennedy, J. Ass. Offic. Agr. Chem. 56, 813 (1973). 3j. R. Ellis and T. M. McCalla, Appl. Microbiol. 25, 562 (1973).
540
ANTmmTrC BIOSYNTHESIS
[40]
relative V ~ x = 80%); and 3-bromoorsellinic acid (Kin--150 ~M, relative Vm~x = 6%). Inhibitors. Salicylic acid, 2-methylbenzoic acid, 3-methylsalicylic acid, and 4-methylsalicylic acid show noncompetitive inhibition with K~'s in the range between 1 and 2 mM. Competitive inhibitors are a-hydroxy6-methylsalicylic acid (Ki = 10 ~M), 6-methylanthranilic acid (K~ = 40 ~M) and 2-hydroxy-l-naphthoic acid (K~ = 40 t~M). The enzyme is not inhibited by iodoacetamide (10 mM), p-chloromecuribenzoate (1 mM), N-ethylmaleimide (1 mM), KCN (10 raM), NaN~ (10 mM), acetylacetone (1 mM), EDTA (10 raM), o-phenanthroline (10 raM), or a,a-dipyridyl (10 mM). It is inhibited only 20% by NaNO.~ (10 raM), and is completely inhibited by diethylpyrocarbonate (10 mM). 6-MSA decarboxylase is photooxidized in the presence of RoseBengal. When the enzyme is exposed to light at neutral pH in the presence of an excess of this dye, inactivation follows the course of a firstorder reaction. The susceptibility to photoinactivation decreases in the range of pH 8.5-5 and is found to follow closely the curve for the photooxidation of imidazol. 15 Photoinactivation is prevented in the presence of substrate or of the competitive inhibitor a-hydroxy-6-methylsalicylic acid, whereas the noncompetitive inhibitor salicylic acid shows no effect. 1~E. W. Westhead, Biochemistry 4, 2139 (1965).
[40] m - H y d r o x y b e n z y l - a l c o h o l
Dehydrogenase
By G. M. GAUCHER The antibiotic patulin is a labile, structurally simple (C7H~04) secondary metabolite 1 produced by the fungus Penicillium urticae. Extensive studies have shown that patulin is synthesized via an essentially oxidative pathway from the classic aromatic polyketide 6-methylsalicylic acid2 This phenolic acid is in turn synthesized from acetyl-CoA and malonyl-CoA in an enzymic process closely related to fatty acid biosynthesis. While of little pharmaceutical interest because of its toxicity, patulin has aroused recent interest as a fungal toxin in food 2 and as a general plant toxin. 3 However, the persistent interest in patulin biosyn1W. B. Turner, "Fungal Metabolites." Academic Press, New York, 1971. 2p. M. Scott and B. P. C. Kennedy, J. Ass. Offic. Agr. Chem. 56, 813 (1973). 3j. R. Ellis and T. M. McCalla, Appl. Microbiol. 25, 562 (1973).
[40]
m-HYDROXYBENZYL-ALCOHOL DEHYDROGENASE
541
CH3 f ~ COOH NADP~NADPH+ H~ ~,(,Acetyl- CoA+ 3 Melonyl - CoA OH 6-Methylsalicylic acid 5 CO2 + 4 CoA- SH C02"~ CH3
CH2OH
NADP~ NADPH -,.
OH m-Cresol
CHO
COOH
~
--->
OH m-HO-Benzyl olcohol
OH m - HO- Benzoldehyde
HOO:
CHO
OH m - H O - Benzoic acid
I
HOO: CH3
OH Toluquinol
CH20H NADP~ NADPH OH
COOH OH
Gentisyl olcohol
OH
Genlisaldehyde
Gentisic acid
NADPH
02
~NADP~
r CHO l" L
~. COOH -CH20H
OH coo.] CHO CHzOH
~o0 ~0 H
J
P r e - palulin
Potulin
FIG. 1. The major pathway for patulin biosynthesis in Penicillium urticae (NRIRL 2159A) is indicated by heavy arrows with branch reactions indicated by light arrows and probable additional reactions by dashed arrows. Metabolites which often accumulate to significant levels in the culture medium are drawn with heavy lines. Square brackets indicate proposed intermediates.
thesis steins from its use as a simple model system in examining the enzymology, regulation and raison d'gtre of an important group of secondary metabolites, the polyketides. 1 These secondary metabolites are produced principally b y the fungi, and to a lesser extent by bacteria and higher plants. Among the fungi, polyketides are produced primarily by the fungi imperfeeti and the aseomyeetes and rarely by the basidiomyeetes. The few polyketides of major commercial interest are the antibioties, griseofulvin and the tetraeyelines, and the animal toxins, the aflatoxins. Only the biosyntheses of patulin and the tetraeyelines have been extensively studied at the enzymologieal level. The major in vivo pathway for patulin biosynthesis has been determined ~ by kinetic pulse labeling techniques, and an up-to-date scheme is given in Fig. 1. With reference to this .figure, it is noteworthy that the mono-oxygenases catalyzing steps 3 and 5 of the pathway are the only enzymes not yet detected in cell-free extracts of P. urticae. The most recent prog4 p. I. Forrester and G. M. Gaucher, Biochemistry 11, 1102 (1972).
542
ANTIBIOTIC BIOSYNTHESIS
[40]
ress has been a demonstration of the dioxygenase mediated ring cleavage of gentisaldehyde2 Only 6-methylsalicyclic acid synthetase,6 6-methylsalicylic acid decarboxylase7 and m-hydroxybenzyl-aleohol dehydrogenases have been purified to any extent. The latter enzyme (3-hydroxybenzylalcohol:NADP ÷ oxidoreductase, EC 1.1.1.97) which catalyzes step 4 of the pathway is the subject of this article. Source of E n z y m e
Vegetative cells of a white variant (NRRL 2159A) of the relatively common soil fungus,~ P. urticae, 9 are the source of this enzyme. On the basis of a preliminary survey of seven fungis this dehydrogenase may be restricted to closely related fungi possessing all or part of the patulin pathway. Five milliliters of a detergent spore suspension are prepared from an agar slant IN 15 cm2; 49 g of Czapek solution agar (Difco) plus 5 g of Bacto-Agar (Difco) per liter], incubated for 7 days at 28 °, and stored at 4 ° for not more than 1 month. Three such spore suspensions are used to inoculate 3 liters of medium (Difco yeast extract, 5.0 g; glucose, 40.0 g; and distilled water to 1 liter) in a 4-liter New Brunswick Microferm fermentor operating at 28 ° and with aeration and agitation rates initially at 0.5 liters/min and 200 rpm, and then increased to 3 liters/min and 500 rpm at 5 hr and to 5 liters/min and 700 rpm at 18 hr. Cells (,~ 5-6 g dry/liter) harvested at 28-34 hr yield approximately 180 mU of enzyme per milligram of dry cells. Similar growth conditionss using medium containing 0.3% yeast extract and salts, yield cells (,-, 4 g dry/ liter) at 20-24 hr containing approximately 58 mU of enzyme per milligram of dry cells. After harvesting by suction filtration, cells are washed twice with distilled water, lyophilized, and stored dry at --15 °. Before storage these lyophilized cells contain the same dehydrogenase activity as fresh cells, whereas after 2 months of storage they retain approximately 75% of their dehydrogenase activity. Shake cultures s in 250-ml conical flasks (26-28°; 180 rpm on rotary shaker) containing 100 ml of an essentially identical medium except for the omission of yeast extract yield cells at 8 days containing an e s t i m a t e d 1.6 mU of 5A. I. Scott and L. C. Beadling,Bio-organ. Chem. 3, 281 (1974). eThis volume [38]. This volume [39]. 8p. I. Forrester and G. M. Gaucher,Biochemistry 11, 1108 (1972). ' Penicillium urtlcae Bainier, Penicillium patulum Bainier, and Penicillium flexuosum Dale are synonyms; Penicillium griseo]ulvum Dierckx is closely related.
[40]
?~t-HYDROXYBENZYL-ALCOHOL DEHYDROGENASE
543
enzyme per milligram of dry cells. These latter authors 5 used fresh cells. Preparation of Cell-Free Extracts Large quantities of cells are best broken using the liquid shear techniques of sonication or liquid pressing. 1° Lyophilized cells (2 g) are suspended in 60 ml of 10 mM TES [N-Tris (hydroxymethyl)methyl-2aminoethanesulfonic acid] buffer (pH 7.6) containing 1 mM (DTT) dithiothreitol and 1 mM MgClz. 11 The cell slurry is sonicated in a 100-ml rosette cell immersed in an ice bath for 7 min at the maximum setting of a Bronson sonicator (model 5125). Alternatively 10 g of wet cells per 60 ml of sonicating buffer can be treated as above. The supernatant from a 30-rain centrifugation at 30,000 g yields the crude cell-free extract. In an alternative procedure ~ a slurry of 30 g of wet cells per 100 ml of 50 mM phosphate buffer (pH 7.6) containing 1 mM DTT is passed through a French press at 10,000-20,000 psi. For small samples more reproducible cell breakage is obtained by the liquid shear technique of ballistic disruption. Lyophilized cells (0.1 g), glass beads (20 g; 0.45-0.50 mm; Braun) and 8 ml TES buffer (identical to sonicating buffer above) are added to a 50-ml Braun flask. The flask is then shaken for 30 sec in a Braun shaker (model MSK) at 4000 rpm. The resulting suspension may be clarified by suction filtration through Whatman No. 4 filter paper and by centrifugation. This latter method generally releases more enzyme than sonication. Despite the addition of various stabilizing additives, s such crude extracts retain only 11% dehydrogenase activity after 24 hr at 3% However, treatment with Polyclar AT (insoluble polyvinylpyrrolidone; GAF Corp., New York, New York) has been shown to stabilize and activate (1.5fold) such extracts. 5 After boiling for 10 min in 10% HC1, Polyclar AT was washed free of chloride with distilled water, equilibrated with buffer, and then separated from excess buffer by decantation. A wet weight of this Polyclar slurry equal to the original wet weight of cells broken is added to the cell extract. The suspension is stirred for 10 min and then clarified by centrifugation. Extracts from cells grown for only 28-34 hr as described above are, however, not activated by Polyclar. lOVarious grinding and homogenization techniques generally liberate less dehydrogenase. Since two freeze-thaw treatments yield cells devoid of dehydrogenase activity, the solid shear technique of freeze pressing with a Hughes press is also unsatisfactory. 11In all these procedures the use of freshly prepared buffer containing dithiothreitol is imperative.
544
ANTIBIOTIC BIOSYNTHESIS
[40]
Dehydrogenase Assay Principle. This kinetic, spectroscopic assay is typical of dehydrogenases and depends upon determining the rate of loss of NADPH during the reduction of m-hydroxybenzaldehyde. Reagents Buffer: 10 mM TES (pH 7.6) containing 1 mM MgC12 Substrate: 2 mM m-Hydroxybenzaldehyde (Sigma; recrystallized from water; mp 106 °) in water Cofactor: NADPH.Na4 (Sigma), 5 mM in a 1% NaHC03 solution
Procedure. An appropriate amount (usually 0.02-0.40 ml) of enzyme solution is added to 0.5 ~mole of NADPH and TES buffer previously equilibrated at 30 °. After a stable baseline is achieved, the reaction is started by adding 0.4 ~mole of substrate to yield a total volume of 3.0 ml in a quartz cuvette in the 30 ° thermostated cell compartment of a spectrophotometer. The loss of NADPH is monitored by the decrease in absorbance at 340 nm. The assay is linear with increasing enzyme concentration up to a A absorbance of 0.25. Units and Calculations. One unit of activity is equal to that amount of enzyme necessary to oxidize 1 ~mole NADPH per minute. Assay results are routinely expressed in milliunits (mU), that is nanomoles of substrate converted per minute. Since both NADPH QpH \ 340 7.~ 6.22 × 103) and substrate \(~pH 340 7.6 1.6 × 103) absorb at 340 nm, the sum of their extinction coefficients is used in converting AOD/min into enzyme units. ~ Other Assays. A similar gentisylalcohol dehydrogenase assay may be carried out using gentisaldehyde ( ~ m 2.7 X 103) as substrate. 5 A more time consumingfixed-time radiochemical assay using [1-~4C]m-hydroxy benzaldehyde as substrate has also been reported2 Purification Despite this enzyme's instability some purification has been possible. The crude cell-free extract is brought ~o 2% (w/v) in streptomycin sulfate (Sigma) and after 10 rain is centrifuged at 10,000 g for 10 min. Solid ammonium sulfate (Mann Ultra Pure) is added to the resulting supernatant until 40% saturation is reached. After centrifugation at 10,000 g for 10 min the resulting precipitate is discarded and the supernatant is brought to 65% saturation in ammonium sulfate. The precipitate obtained by centrifugation at 10,000 g for 10 min is dissolved in 12Given that substrate (S) and cofactor (C) both absorb at 340 nm, AOD3,0= esA[S] + e¢A[C], and since MS] = A[C] then A[S] = AOD34o/¢~+ e¢.
[40]
m-HYDROXYBENZYL-ALCOHOL DEHYDROGENASE
545
TABLE I PARTIAL PURIFICATIONOFTn-HYDROXYBENZYL-ALCOHOL])EHYDROGENASE ~
Purification step Crude supernatant Steptomycin sulfate precipitation Ammonimn sulfate fractionation Sephadex G-200
Volume Total (ml) (milliunits) 275 275
115,500 115,500
12
70,000
42
17,000
Protein concentraiion b (mg/ml) 4.2 13 0.55
Specific activity (milliunits/rag of Yield proleiT~) (%)
Purifi alion (-fold)
100 -
100 100
--
455
6l
4.55
655
15
6.55
Reproduced by permission of the American Chemical Society from Biochemislrg 11, 1108 (1972). b Both the Folin-Lowry method and the 280:260 nm ratio method were used ~o determine protein concentration using bovine serum albumin (Sigma) as ~t standard. a minimum volume of 10 m M T E S buffer (pH 7.6) which is 1 m M in both dithiothreitol and MgCl~. This a m m o n i u m sulfate fraction is applied to a P h a r m a e i a K25/100 column packed with Sephadex G-200 (fine) which has been previously equilibrated with the above T E S buffer which also is 0.02 m M in N A D P +. Elution is carried out using the above equilibration buffer and 5 ml fractions are collected. Fractions containing dehydrogenase activity are pooled. The 6.6-fold purification yields a preparation with a specific activity of 655 mitliunits per milligram of protein as detailed in Table I. As recently reported '~ improved purification of what is probably the same dehydrogenase results in a 46-fold purification and a final specific activity of approximately 1100 milliunits per milligram of protein. Tile enhanced purification results from a Polyclar AT treatment of the crude extract and a diethylaminoethyl (DEAE)-cellulose column chromatography step using a 0.05-0.50 M phosphate huffer (pH 7.6) gradient. This purified preparation is 50% active after 48 hr. Finally, this preparation exhibits m-hydroxybenzyl alcohol and some gentisyl "tlcohol dehydrogenase activity. Hence the existence of one or two dehydrogenases is uncertain.
Properties Despite the fact t h a t a stable, highly purified preparation of this dehydrogenase is not available, some characterization has been possible and
546
ANTIBIOTIC BIOSYNTHESIS
[40]
TABLE II INHIBITION STUDY OF UNPURIFIED m-HYDROXYBENZYLALCOHOL DEHYDROGENASE a
Preassay treatment b Control + Iodoacetic acid + NADPH, then iodoacetic acid + NADP +, then iodoacetic acid + m-Hydroxybenzaldehyde, then iodoacetic acid + Diethylpyrocarbonate + NADP +, then diethylpyrocarbonate + m-Hydroxybenzaldehyde, then diethylpyrocarbonate
Relative activity (%) 100 46 97 89 45 19 65 19
a Reproduced by permission of the American Chemical Society from Biochemistry 11, 1108 (1972). b Ten-milliliter portions of crude supernatant were incubated at 0° with either 1 ~mole of m-hydroxybenzaldehyde, 1 ~mole of NADP +, or 1 ~mole of NADPH. After 5 min equilibration, 10 ~moles of iodoacetic acid (BDH, recrystallized before use) were added to each sample. After a further incubation at 0° of 2 hr the activity of each sample was determined by assaying 0.1-ml aliquots. Similarly, 10-ml portions of crude supernatant, adjusted to pH 6.0 with 1 N HC1, were incubated at 0° with 1 ~mole of NADP + or with 1 ~mole of m-hydroxybenzaldehyde. After 5 min of equilibration, 10 ~moles of diethylpyrocarbonate (Eastman) were added. After a further 3 min at 0° the activity of each sample was determined by assaying 0.1-ml aliquots.
has been carried out using crude enzyme preparations, s This intracellular, soluble dehydrogenase (EC 1.1.1.97) has an approximate molecular weight of 120,000 (gel filtration method) and can be readily detected in crude extracts as a single enzymically active band in polyacrylamide disc gel electrophoresis. As indicated in Table I I the inactivation of the dehydrogenase by the sulfhydryl reagent, iodoacetate, and the histidine reagent, diethylpyrocarbonate, and the protection provided by N A D P H agree with what has been found for other dehydrogenases. The enzyme has a fairly sharp pH optimum at 7.6 and exhibits classic MichaelisMenten plots of initial rate versus substrate concentration (approximate K~ values = 4 to 5 X 10-5 M). The equilibrium constant and standard free-energy change for the reaction have been calculated to be: K'app -- [aldehyde] [ N A D P H ] / [ a l c o h o l ] [ N A D P +] = 0.18 (pH 7.6, TES, 30 °) and AG°1 ---- + 1.04 kcal/mole (pH 7.6, TES, 30°). The reduction of m-hydroxybenzaldehyde, which is opposite to the preferred pathway direction, is clearly favored (i.e., the reverse rate relative to the
[40]
m-HYDROXYBENZYL-ALCOHOL DEHYDROGENASE
547
TABLE I I I SUBSTRATI~ SPECIFICITY OF UNPURIFIED m-HYDROXYBENZYLALCOHOL DEHYDROGENASE a
Relative substrate Relative inhibitor activity activity
m-Hydroxybenzaldehydeg m-Methoxybenzaldehyde~ AcetaldehydeS Benzaldehyde p- Hydroxybenzaldehyde o-Hydroxybenzaldehyde Acetophenone m-Hydroxyacetophenone p-Hydroxyacetophenone p-Hydroxyoctaphenone Gentisaldehydeh
(~o)b
(G~)C
100 100 29 20 10 0 0 0 0 0 0
-0 16 0 37g 0 0 0 5()g 70~ 36
a Reproduced by permission of the American Chemical Society from Biochemistry 11, 1108 (1972). b In each case the cofactor NADPH and standard assay conditions of pH 7.6 and 30 ° were used. c Percent decrease in m-hydroxybenzyl-alcohol dehydrogenase activity upon addition of an equimolar amount of various substrate analogs. d Substitution of NADH for NADPH yielded a relative activity of ~12%, while substitution of m-hydroxybenzyl alcohol and NADP O for substrate and cofactor, respectively, yielded a relative activity of ~18%. Furthermore substitution of NADP® or NAD@ for NADPH yielded no activity. e Substitution of m-methoxybenzyl alcohol and NADP O for substrate and cofactor, respectively, yielded a relative activity of ~ 9 %. ] Substitution of NADH for NADPH yielded a relative activity of 35%. g Concentration of added compound equal to only yl~ the substrate concentration since these compounds absorb strongly at 340 nm. h Substitution of NADH for NADPH yielded a relative activity of ~12 %.
forward rate is favored 6: 1). T h e r e a c t i o n ' s r e v e r s i b i l i t y clearly indicates t h a t the e n z y m e is a dehydrogenase, n o t a reductase. F i n a l l y , a l t h o u g h a crude e n z y m e p r e p a r a t i o n was used, the d e h y d r o g e n a s e ' s s u b s t r a t e specificity is d e l i n e a t e d in T a b l e I I I . T h e e n z y m e is clearly a n alcohol r a t h e r t h a n a n a l d e h y d e d e h y d r o g e n a s e a n d is N A D P + specific. A specificity for a r o m a t i c aldehydes with a meta(3)-hydroxy or m e t h o x y s u b s t i t u e n t is a p p a r e n t a n d a r o m a t i c k e t o n e s are good i n h i b i t o r s r a t h e r t h a n substrates. T h e presence of some of both N A D a n d N A D P e t h a n o l d e h y d r o genase a c t i v i t y is p r o b a b l y due to other enzymes. A l t h o u g h no gentisyl alcohol ( N A D P ) d e h y d r o g e n a s e a c t i v i t y was found in this p r e p a r a t i o n ,
548
ANTIBIOTIC BIOSYNTHESIS
[41]
such activity has recently been reported in a different preparation from P. urticaeP As has been discussed, both kinetic pulse-labeling experiments 4 and the characteristics of this enzyme 8 suggest that this dehydrogenase probably catalyzes the rate-determining step of the patulin biosynthetic pathway. Acknowledgment The development of unpublished procedures in this manuscript by Mr. Jan Groot Wassink is gratefully acknowledged.
[41] Bacitracin Synthetase B y HANSPETER RIEDER, GERHARD HEINRICH, EBERtIARD BREUKER,
MAHAVIR M. SIMLOT, a n d PETER PFAENDER
The bacitracins (Fig. 1) are a group of potent peptide antibiotics that are active against a variety of gram-positive, but only a few gram-negative, microorganisms. 1 The bacitracins and licheniformins ~ are produced by Bacillus licheni]ormis. Bacitracins A and B, whose structures are known, are the most common members of the group3,4; bacitracins D and E, whose structures are only partially known, contain valine, bacitracin C also yields glycineP In aqueous solution bacitracin A is transformed into the antibiotically inactive, yet equally nephrotoxic bacitracin F. 1 (B)
VAL
I I ~ (A)
~oRF-' .PHE
C~H~--C--C~-'# x'/---CO-LEU-D-GLU-I LE-*LYS ~ I I 's-# ,~SN CH 3 NH 2 --C ~N
)
HI S
"~-D.ASI~
Fro. 1. Bacitracins A, B, and F. B and F are similar to A except for the variations shown. 1R. J. Hickey, Progr. Ind. Microbiol. 8, 95 (1964). R. K. Callow and T. S. Work, Biochem. J. 51, 558 (1953). a L. C. Craig, J. R. Weisiger, W. Hausmann, and E. J. Harfenist, J. Biol. Chem. 199, 259 (1952). 4C. Ressler and D. V. Kashelikar, J. Amer. Chem. Soc. 88, 2025 (1966). G. G. F. Newton and E. 19. Abraham, Biochem. J. 53, 597 (1953).
548
ANTIBIOTIC BIOSYNTHESIS
[41]
such activity has recently been reported in a different preparation from P. urticaeP As has been discussed, both kinetic pulse-labeling experiments 4 and the characteristics of this enzyme 8 suggest that this dehydrogenase probably catalyzes the rate-determining step of the patulin biosynthetic pathway. Acknowledgment The development of unpublished procedures in this manuscript by Mr. Jan Groot Wassink is gratefully acknowledged.
[41] Bacitracin Synthetase B y HANSPETER RIEDER, GERHARD HEINRICH, EBERtIARD BREUKER,
MAHAVIR M. SIMLOT, a n d PETER PFAENDER
The bacitracins (Fig. 1) are a group of potent peptide antibiotics that are active against a variety of gram-positive, but only a few gram-negative, microorganisms. 1 The bacitracins and licheniformins ~ are produced by Bacillus licheni]ormis. Bacitracins A and B, whose structures are known, are the most common members of the group3,4; bacitracins D and E, whose structures are only partially known, contain valine, bacitracin C also yields glycineP In aqueous solution bacitracin A is transformed into the antibiotically inactive, yet equally nephrotoxic bacitracin F. 1 (B)
VAL
I I ~ (A)
~oRF-' .PHE
C~H~--C--C~-'# x'/---CO-LEU-D-GLU-I LE-*LYS ~ I I 's-# ,~SN CH 3 NH 2 --C ~N
)
HI S
"~-D.ASI~
Fro. 1. Bacitracins A, B, and F. B and F are similar to A except for the variations shown. 1R. J. Hickey, Progr. Ind. Microbiol. 8, 95 (1964). R. K. Callow and T. S. Work, Biochem. J. 51, 558 (1953). a L. C. Craig, J. R. Weisiger, W. Hausmann, and E. J. Harfenist, J. Biol. Chem. 199, 259 (1952). 4C. Ressler and D. V. Kashelikar, J. Amer. Chem. Soc. 88, 2025 (1966). G. G. F. Newton and E. 19. Abraham, Biochem. J. 53, 597 (1953).
[411
BACITRACIN SYNTHETASE
549
The nonribosomal synthesis of cyclic peptide antibiotics, such as gramicidin S and the tyrocidines, 6 on enzyme templates suggested the present study of baeitracin synthetase. 7-9 The study is concerned with the purification and partial characterization of bacitracin synthetase from a m u t a n t of B. licheniformis, L o h m a n n cells, exhibiting a high rate of bacitracin synthesis. Principle. Bacitracin is synthesized by the enzyme via Eq. (1): 3 lie + Cys + Leu + Glu + Lys + Orn + Phe + l i i s + Asp + Asn
n A T P , M g ~+ p H 7.5
, bacitracin + n PPI
(1I
The exact amount of A T P needed is not known. For bacitracin-synthesizing particles it has been determined to be about 2 A T P for 1 peptide bond. The amount of bacitracin formed can be measured by incorporation of one or more labeled amino acids into bacitraein, by radio thin-layer chromatography, 9 or selectron filters, 9 or by growth inhibition test after incubation, lyophilization of the mixture and application to culture plates planted with Micrococcus flavus according to Hoff. TM The activity of baeitracin synthetase can also be evaluated by ATP-32PPi exchange hie a su re menU. 9,~1 One unit of enzyme is defined as t h a t amount of bacitracin synthetase that incorporates 1 ~mole of one of the amino acids (1 t~mole of isoleucine) into baeitracin per minute at 25 °. Materials
Buffers M E , 2-Mercaptoethanol, Merck, D a r m s t a d t , G e r m a n y Buffer A: 25 m M sodium phosphate, p H 7.5 Buffer B: buffer A + 0.25 m M E D T A , 10 m M MgC1._,, 10 m M M E Buffer C: buffer B + 0.5 m M ATP, 0.4 m M bacitracin amino acids except cysteine 1 m M 6F. Lipmann, W. Gevers, H. Kleinkauf, and R. Roskoski, Jr., Advan. Enzymol. 35, 1 (1971). 7p. Pfaender, Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg, I. Abt. Orig. A 230, 319 (1972). 8 p. Pfaender, D. Specht, G. Heinrich, E. Schwarz, E. Kuhnle, and M. M. Simlot, FEBS Lett. 32, 100 (1973). M. M. Simlot, P. Pfaender, and D. Specht, FEBS Lett. 35, 231 (1973). 4oD. A. Hoff, R. E. Bennett, and A. R. Stanley, Science 106, 551 (1947). 11M. M. Simlot and P. Pfaender, FEBS Lett. 35, 201 (1973).
550
ANTIBIOTIC BIOSYNTHESIS
[41]
Enzymes Alcohol dehydrogenase (ADH), EC 1. 1.1.1 Catalase, EC 1.11.1.6 Deoxyribonuclease (DNase), EC 3.1.4.5 Gtyceraldehydephosphate dehydrogenase (GA-3P-DH), EC 1.2.1.12 Lactate dehydrogenase (LDH), EC 1.1.1.27 Leucine aminopeptidase (LAP), EC 3.4.1.1 3-Phosphoglycerate kinase, EC 2. 7.2.3 Ribonuclease A (RNase), EC 2. 7.7.16 Urease, EC 3. 5.1.5 (All from Boehringer, Mannheim, Germany) Lysozyme, EC 3.2.1.17, (from Serva, Heidelberg, Germany) Other proteins Crystalline bovine serum albumin (BSA) Crystalline soybean trypsin inhibitor (SBTI) (Both from Serva, Heidelberg, Germany) Amino acids Asparagine, aspartic acid, glutamic acid, histidine, isoleucine, leucine, lysine, ornithine, phenylalanine; all from Merck, Darmstadt, Germany; cysteine from Schuchardt, Munich, Germany Labeled compounds Algae protein total hydrolysate, 250 ~Ci/5 ml [U-14C]Lysine, 50 ~Ci/0.023 mg Tetrasodium pyrophosphate, 2.48 ~Ci/~mole (All from Buchler, Braunschweig, Germany.) Fermentation media Bolus alba (white clay), Haiger, mine Niederdresseldorf, Germany Corn steep liquor, Kellogg, Osterholz, Scharmbeck, Germany Pharmamedia, Traders Protein Division, Fort Worth, Texas Someel, Holtz & Willemsen,Krefeld, Germany Column chromatography steps DEAE-cellulose, Merck, Darmstadt, Germany Sephadex G-50, Sephadex G-200, Pharmacia, Uppsala, Sweden Concentration step XM-50 Amicon membrane filters, 25 ram, Amicon, Oosterhout, The Netherlands Electrophoresis Sodium dodecyl sulfate (SDS), Merck, Darmstadt, Germany Growth inhibition test Triphenyltetrazolium chloride (TTC) Merck, Darmstadt, Germany Bacitracin, 68 IE per mg, Serva, Heidelberg, Germany
[41]
BACITRACIN SYNTHETASE
551
Liquid scintillation counting PPO, diphenyloxazole, Merck, Darmstadt, Germany
Assay Methods The following methods are described: (1) Micrococcus flavus growth-inhibition test (2) ATP-3'-'PPi exchange measurement (3) Millipore filter test (4) radio thin-layer chromatography of incorporated [14C]lysine 1. Micrococcus flavus Growth Inhibition Test. Fifteen milliliters of media I containing 4.0 g/liter of nutrient broth (Difco), 3.0 g/liter of yeast extract (Difco), 3.0 g/liter of Peptone (Difco), and 1.0 g/liter of glucose (Merck) are inoculated with a loop of M. flavus (ATCC 10240) and incubated at 37 ° for 18 hr. Culture plates are prepared with base agar containing 15 g of agar in 1 liter of media I without glucose, and seed agar, containing 15 g of agar, 4.0 g of Casamino acids (Difco), and 10 ml of M. flavus culture in 1 liter of media I. A 3 mm deep layer of base agar at 50 ° is poured onto sterile polyethylene culture plates with a diameter of 8.2 cm. After 20 rain a 1 mm-deep layer of seed agar at 45 ° is poured onto the base agar. Poured plates can be kept for 1 week at 4 °. Just before the assay, 5 sterile steel cylinders (0.5 X 0.7 cm) are gently placed onto each culture plate, 0.1 ml of bacitracin containing solution are filled into each cylinder and the plates are incubated at 37 ° for 18 hr. After incubation the cylinders are removed and the plates are reincubated at 37 ° with 0.1% TTC for 15 rain. The circular zones of inhibition are then measured against the red background. A diameter of 1.5 cm corresponds to 0.1 unit or 1.74 ~g of bacitracin. 2. ATP-3'2PP~ Exchange Measurement. The composition of the incubation mixture is ATP, 4.5 mM; MgCI~, 3.75 mM; KF, 22.5 mM; 2-mercaptoethanol, 7.5 raM; Na432P~O,, 4.5 mM (specific activity 2.48 mCi/mmole) ; BSA, 0.23 mg/ml; cysteine, 15 mM; the nine amino acids of baeitracin, each 4.0 mM; Tris.HC1 buffer, 109 mM; final pH 7.5. The enzyme solution in buffer B (20 ~l) is mixed in the cold with 40 ~l of incubation mixture and incubated at 0 °, 4 °, or 25 ° for the stated periods (Table I). A control is also incubated which contains water in place of amino acids. After incubation, the mixture is immediately frozen to stop the reaction, thawed, and 10 ~l of a solution of 0.4 M Na,P~O; in 15% perchloric acid is added. After shaking the solution it is centrifuged and 50 ~l of
552
ANTIBIOTIC
E~
o~
[41]
BIOSYNTHESIS
=
'°
l
g ©v
@,1 E~ Z
v
©
~°I, i ~ ~_ ~
i®
o
r~
©
0
[41]
BACITRACIN SYNTHETASE
553
the clear solution is then applied to the charcoal filter disk. Disks are held either on pins or laid flat on a bed of pins and numbered. The filter paper disks are allowed to dry at room temperature. For washing the disks, any of the techniques used in filter paper disk methods can be used. However, we have found the following procedure to be satisfactory: filter paper disks are placed in a large petri dish separated from each other and a cold solution of 40 mM sodium pyrophosphate in 1.5% perchloric acid is added. Disks are submerged in the solution and, with the help of forceps, kept from settling to the bottom and adhering to each other. After about 5 rain, the wash solution is withdrawn and replaced with a fresh cold solution. This is followed by a third wash. Disks are now washed in the same manner with cold distilled water. Finally individual disks are held with forceps, washed in a stream of distilled water and placed on a bed of pins for drying. After partial air drying (30-60 rain), the disks are completely dried for 10 rain at 110% After cooling, the disks are placed under 5 ml of 5% PPO in toluene in scintillation counter vials, and the radioactivity is measured in a liquid scintillation counter tLS 150, Beckman Inc., Fullerton, California) using the channel range of 14C + 32p. 3. Millipore Filter Test. The synthesis of bacitracin is measured by incorporation of radioactive amino acids into baeitracin and its retention hy selectron filters (0.45 t~m, 24 mm diameter, Sehleicher & Sehuell, Dassel, Germany). The incubation mixture of 0.1 ml, pH 7.3, contains 8.0 mM ATP; 10 mM cysteine; 3 mM each of the other 9 amino acids (all L-form) ; 0.16 M Tris.HC1 buffer and 50 nCi [U-~C]protein hydrolyzate. Enzyme solution in buffer B (0.] ml) is added in the cold and incubated 10 rain at 4 ° and 25 ° for the stated periods. Blanks are prepared as above, but are frozen immediately. Blanks and the incubated mixtures are filtered through Selectron filters in the cold, washed with huffer B (2 }( 1 ml}, and once with water (1 ml). The filter disks are dried at 50 ° for 20 rain and counted in 5 ml of 57; PPO in toluene in a Beckman scintillation counter.
4. Radio Thin-Layer Chromatographg of Incorporated [HC ]Lgsi~w. The incubated mixture from the Millipore filter test whieh contains 550,000 cpm of [~4C]lysine instead of 1~C label protein hydrotysate, is ehromatographed on TLC-plates silica gel F~.~ (Merck) using r~-butanol:acetie acid:water (4:1:2, v/v) as the developer. After development, the areas corresponding to haeitraeins A and F (Rr 0.43 and 0.46) are measured and integrated by a windowless thin-layer scanner (Duennsehicht-scanner II, Berthold, Wildbad, Germany) (Table I). The areas corresponding to baeitraeins are scraped off, extracted with methanol (2 X 1 ml) and then with water (2)4 0.5 ml). The pooled ex-
554
ANTIBIOTIC BIOSYNTHESIS
[41]
tract is dried under vacuum and lyophilized with a small amount of water to remove any trace of methanol. The residue is taken up in water, and the antibiotic activity is determined. As the antibiotic activity in these samples is usually low, the above incubation is also directly tested for growth inhibition after extraction with methanol. Other Methods
SDS Disc Gel Electrophoresis. Protein, 0.04 ml or 83 ~g, of fractions 18-21 (DEAE-cellulose run) and 0.04 ml or 160 ~g of protein of fractions 10-11 (Sephadex G-200 run) are each diluted with 0.02 ml of an electrode buffer solution from gel-system No. 612 (20% sucrose, 1% SDS, 0.1% ME). Lucite tubes of 0.5 cm diameter containing 7.5 cm of separation gel and 0.5 cm of spacer gel serve as carriers. Electrophoresis is allowed to proceed over a period of 4 hr with a current of 4 mA. The exposed gels are then developed with Amido black and finally decolorized with 7% CH3COOH (20% ethanol). Marker enzymes are LAP, catalase, and ADH. Disc Gel Electrophoresis. Portions, 0.02 ml of fractions 18-21 (DEAEcellulose run) and fractions 10-11 and 14 (Sephadex G-200 run) are diluted as described except that 1% SDS and 0.1% ME are omitted (Fig. 4). Protein Determination. Protein is determined according to the method of Folin-Ciocalteu.is Sucrose Density Gradient Centri]ugation. Protein, 0.1 ml or 0.4 mg, of fractions 10-11 (Sephadex G-200 run) are carefully layered on top of a sucrose density gradient (5-20% sucrose in buffer C), and centrifuged at 204,000 g and 3 ° for 3 hr. Marker enzymes are ADH, LAP, and urease. Two-drop fractions are collected by puncturing the bottom of the tube. All fractions are assayed for bacitracin synthesizing activity utilizing the ATP-PPi exchange method. Three active bands are found (Table II). Bands of marker enzymes are located by measuring the optical density of all fractions in a Zeiss M4QIIId manual UV spectrophotometer (Zeiss, Oberkochen, Germany) at 280 nm. Preparation o] Inoculum/or Fermentation. (A) Mutant 4 rough cells of Bacillus licheni]ormis are obtained from 3% tryptic soy broth slant cultures no older than 24-28 hr. (B) Tryptic soy broth (30 ml) in a 300-ml Erlenmeyer flask is inoculated with a large loop from a slant culture. The flask is then incubated at 36-37 ° for 12 hr and shaken at a rate of 180 strokes per minute with an amplitude of 3 cm. (C) Two 12H. R. Maurer, in "Disc Electrophoresis," pp. 44-45. de Gruyter, Berlin, 1971. l~j. R. Spies, this series, Vol. 3, pp. 467--468.
[41]
B&CITRACIN SYNTHETASE
555
TABLE II MOLECULAR WEIGHT DETERMINATION
Fraction
Molecular weight
DEAE-cellulose fraction 18-21
148,000
Sephadex G-200 fraction 10-I 1
148,000 SDS disc gel electro74,000 phoresis 431,000 Sucrose density gradient 327,000 centrifugation 64,700
Method SDS disc gel electrophoresis
ATP-a'PP~ exchange (cpm) --5;5 92 1092
2000-ml Erlenmeyer flasks containing 500 ml of 3% tryptic soy broth each, pH 6.0-6.2 after sterilization, are inoculated with 5 ml of culture from part B, incubated and shaken for 10 hr at 36-37 °. Fermentation. Fermentation takes place in a K 7 fermenter with 1000liter capacity. The fermentation media (1000 liters) contains 10 kg of Pharmamedia, 30 kg of Someel, 20 kg of starch, 5 kg of corn steep liquor, 5 kg of Bolus alba, 5 kg of CaCO~, 5 g of FeSO~.7 H.20, and 5 g of M n S Q . H 2 0 . The media is sterilized at 121 ° for 40 min. The pH values of the media before and after sterilization are 6.2 and 6.5-7.0, respectively. During fermentation, the media pH is determined every 2 hr (Fig. 2), and adjusted with NaOH or H2S04 when necessary. Fermentation is started by adding to the 1000 liters of sterile media 80-
7.5
T 70
6.51
0
I0
Hours
20
30
FIG. 2. Fermentation, 1000 liters, of Bacillu~ licheni]ormis, mutant 4, rough; pH profile.
556
ANTIBIOTIC BIOSYNTHESIS
[41]
the two 500-ml portions of inoculum prepared (see C, above) and is allowed to proceed at 34 ° for 33-34 hr. The fermentation mixture is stirred with a "Turbo-stirrer" at 180 rpm and aerated with an air flow of 0.25 liter/liter per minute for the first 4 hr, 0.5 liter/liter per minute for the next 4 hr, and 1.0 liter/liter per minute until fermentation is terminated. An antifoaming mixture of soy-oil/lard is added as necessary. Harvesting and Storage of Cells. Figure 2 shows the pH changes of the fermentation media from which the organism utilized in the present study was harvested. The arrow indicates harvesting time. Eight hours before harvesting the fermenter was slowly cooled and the air flow was reduced gradually to allow proper removal of 100 liters of cell suspension at 26 hr of fermentation. The cell suspension was centrifuged to yield 5 kg of sediment (wet weight, 70% H20). Cells were frozen in 100-g portions. Purification of Bacitracin Synthetase All procedures were carried out at 4 ° unless noted otherwise. Step 1. Cell Lysis. Rough mutant 4 cells (RC4) of B. licheniformis, 100 g wet weight, are thawed in 150 ml of 25 mM phosphate buffer, pH 7.5. After 2 hr of gentle stirring the cell suspension is centrifuged for 40 min at 27,000 g. T h e pellets are resuspended in 200 ml of 25 mM phosphate buffer, pH 7.5, containing 0.75 mM EDTA, 1 mM MgCI_~, and 10 mM mercaptoethanol. To the clay-colored suspension are then added 160 mg of lysozyme, 400 ~g of DNase, and 100 ~g of RNase. The gently stirred mixture is incubated at 30 ° for 20 min. After centrifugation at 27,000 g for 40 rain, the pellets are resuspended and treated once more with lysozyme, DNase, and RNase as described above. The turbid light yellow supernatant from both lysis cycles is combined and, under slow stirring, is brought to 45% saturation with crystalline ammonium sulfate. After standing for 2 hr, a minimal amount of precipitate is removed from the suspension by centrifugation at 27,000 g for 40 min. The supernatant is brought to 80% ammonium sulfate saturation in a similar fashion after removal of a thin surface layer of lipid material. Step 2. Sephadex G-50 Chromatography. The suspension from step 1 (saturated to 80% with ammonium sulfate) is centrifuged at 27,000 g for 40 rain. The sediment, containing approximately 120 mg of protein, is dissolved in 5 ml of buffer B and the resulting 8.5 ml of solution are eluted from a Sephadex G-50 column (2.2 X 32 cm) with buffer C. Fractions of 8 ml are collected using a Uvicord with UltroRac (LKB Produkter AB, Bromma, Sweden). Fractions 5, 6, and 7 show bacitracin synthesizing activity (Fig. 1, Table I).
[41]
BACITRACIN SYNTHETASE
557
Step 3. DEAE-Cellulose Chromatography. Fractions 5, 6, and 7 from step 2 are combined to give 18 ml of solution. This solution is then eluted from a DEAE-cellulose column (2 X 24 cm) with an exponential NaC1 gradient in buffer C. The gradient is achieved by running 300 ml of 0.5 M NaCt in buffer C into a mixing chamber containing a constant volume of buffer C (100 ml). Fractions of 8 ml are collected as in step 2. Fractions 14-]7 show bacitracin-synthesizing activity (Fig. 2, Table I). Step 4. Diafiltration and Concentration. Fractions 14-17 from step 3 (23 ml) are concentrated to 6 ml in a Multi-Micro Ultra,filtration System (Amicon, Oosterhout, The Netherlands) with 30 psi of N._,. Step 5. Sephadex G-200 Chromatography. The concentrate from step 4 (6 ml) is eluted with buffer C from a Sephadex G-200 column (2 X 61 cm) in fractions of 8 ml each. Fractions 8-11, 14 show bacitracin synthesizing activity (Fig. 3, Table I). •
-
,
.
,
A[3° 2.O ~.
c
2 ~ 50 o
i.o
I i
-':
"'" .......
'..''""
iO
B L04
:
S 7
~oI00 "'"I,
"k
,~. , 1001
:, '~
~1008 c
~
-
.02 "6
, ." , . , 10 20 30 8 ml F r a c t i o n s
Fro. 3. Column chromatography of bacitraein synthetase. % Transmittance ( ), milligrams of protein per milliliter (---). (A) Sephadex G-50. (B) DEAE-cellulose; 0-0.5 M NaC1 gradient ( . . . ) . (C) Sephadex G-200.
558
ANTIBIOTIC BIOSYNTHESIS
[41]
0-
5.0
cm I 2 3 FIG. 4. Disc gel electrophoresis of enzyme fractions. 1, DEAE-cellulose chromatography, fractions 18-21; 2, Sephadex G-200 chromatography, fractions 10W 11; 3, fraction 14, treated similarly to fraction 10 T 11 in 2.
The Millipore filter test, which measures the sum of specifically, at low temperature, acid stable (thioesters?) TM, and nonspecifically bound intermediates 'of the biosynthesis of bacitracin also shows that the rate of formation of bacitracin from these intermediates rises at 25 °. The 0 ° and 4 ° values of the growth inhibition test reflect only roughly the rise of specific activity of the enzyme containing material over the course of the three column chromatography steps. The growth inhibition test is necessary, however, because during radio thin-layer chromatography, which measures the sum of bacitracin A and F (Rf bacitracin A: 0.43; R~ bacitracin F: 0.46) at least 90% of bacitracin A is transformed into bacitracin F. Lysis and Ammonium Sul]ate Precipitation. Approximately 120 mg of bacitracin synthetase are recovered from a bulk of 15 g of bacterial protein (0.8%). Properties
ATP-82PPI Exchange, Millipore Filter Test, Radio Thin-Layer Chromatography, and Growth Inhibition Test (Table I). The results of ATP-82PPi exchange measurements of the three column chromatography steps clearly show two facts: (a) the purification factor for the very fast reaction. T M E + ATP + AA ~- AMP-AA-E + PPi
(2)
is of the order of 100 at 4 °. (b) While the rate of formation of s2P-labeled A T P rises throughout the three column chromatography steps at 4 °, this rate reaches its peak in step 2 of the assays at 25% 1, W. Gevers, H. Kleinkauf, and F. Lipmann, Proc. Nat. Acad. Sci. U.S: 60, 269 (1968).
[42]
EDEINE SYNTHETASE
559
A comparison with the amounts of bacitracin found in the radio thinlayer chromatography analyses suggests that the rate of formation of bacitracin from aminoacyl adenylates on the highly organized and purified multienzyme complex of column chromatography, step 3, has increased at 25 ° and has reached the same order of magnitude as the back reaction of reaction (2). SDS Disc Electrophoresis, Disc Electrophoresis, and Sucrose Density Gradier.t CentriJugation. The molecular weights as determined by SDS disc gel electrophoresis with a single sharp band of MW 148,000 for the DEAE-cellulose run (Fig. 3) and of MW 148,000 and 74,000 for the Sephadex G-200 run show clearly that bacitracin synthetase has a molecular weight of 431,000 (Table II, sucrose gradient centrifugation) and is composed of 7 subunits. The Sephadex G-200 fractions produce a more differentiated electrophoretic pattern because of constant association and dissociation of enzyme subunits and because of the comparatively low protein concentration. Accordingly, in Fig. 4, bar 2 (Sephadex G-200, fractions 10-11) and Fig. 4, bar 3 (Sephadex G-200, fraction 14) identical bands at 1.8 and 2.4 cm may be seen. The same bands are visible in Fig. 4, bar 1, in the still impure DEAE-cellulose fractions (see bands below 4.1 cm). In contrast, the band of highest molecular weight is seen only in bars 1 and 2 of Fig. 4. Molecular weight determination with a number of marker proteins (LAP, LDH, ADH, BSA, and SBTI) on the Sephadex G-200 column yielded a molecular weight of ~300,000 for fractions 8-9 (elution within exclusion volume).
Acknowledgments Thanks are due to Deutsche Forschungsgemeinschaftand to Fonds der Chemischen Industrie for grants, and to Lohmann & Co. AG for RC4 cells. The skillful assistance of Miss Annerose Bahnmfiller and of Miss Dorothee Specht is greatly appreciated.
[42] Edeine Synthetase By ZOFIA KURYLO-BoRoWSKA Edeines A and B are linear oligopeptides. 1 They are mixtures of biologically active compounds (edeine A1 and B1) (Fig. 1) and inactive isomers (edeine A~. and B2) in which isoserine is linked to the ~- rather T. P. Hettinger, Z. Kurylo-Borowska,and L. C. Craig, Ann. N.Y. Acad. Sci. 170, 1002 (1970).
[42]
EDEINE SYNTHETASE
559
A comparison with the amounts of bacitracin found in the radio thinlayer chromatography analyses suggests that the rate of formation of bacitracin from aminoacyl adenylates on the highly organized and purified multienzyme complex of column chromatography, step 3, has increased at 25 ° and has reached the same order of magnitude as the back reaction of reaction (2). SDS Disc Electrophoresis, Disc Electrophoresis, and Sucrose Density Gradier.t CentriJugation. The molecular weights as determined by SDS disc gel electrophoresis with a single sharp band of MW 148,000 for the DEAE-cellulose run (Fig. 3) and of MW 148,000 and 74,000 for the Sephadex G-200 run show clearly that bacitracin synthetase has a molecular weight of 431,000 (Table II, sucrose gradient centrifugation) and is composed of 7 subunits. The Sephadex G-200 fractions produce a more differentiated electrophoretic pattern because of constant association and dissociation of enzyme subunits and because of the comparatively low protein concentration. Accordingly, in Fig. 4, bar 2 (Sephadex G-200, fractions 10-11) and Fig. 4, bar 3 (Sephadex G-200, fraction 14) identical bands at 1.8 and 2.4 cm may be seen. The same bands are visible in Fig. 4, bar 1, in the still impure DEAE-cellulose fractions (see bands below 4.1 cm). In contrast, the band of highest molecular weight is seen only in bars 1 and 2 of Fig. 4. Molecular weight determination with a number of marker proteins (LAP, LDH, ADH, BSA, and SBTI) on the Sephadex G-200 column yielded a molecular weight of ~300,000 for fractions 8-9 (elution within exclusion volume).
Acknowledgments Thanks are due to Deutsche Forschungsgemeinschaftand to Fonds der Chemischen Industrie for grants, and to Lohmann & Co. AG for RC4 cells. The skillful assistance of Miss Annerose Bahnmfiller and of Miss Dorothee Specht is greatly appreciated.
[42] Edeine Synthetase By ZOFIA KURYLO-BoRoWSKA Edeines A and B are linear oligopeptides. 1 They are mixtures of biologically active compounds (edeine A1 and B1) (Fig. 1) and inactive isomers (edeine A~. and B2) in which isoserine is linked to the ~- rather T. P. Hettinger, Z. Kurylo-Borowska,and L. C. Craig, Ann. N.Y. Acad. Sci. 170, 1002 (1970).
560
ANTIBIOTIC BIOSYNTHESIS . . . . . . . . . . .
r- . . . .
p GUANYL) SPERMIDINE I I
/ r "/
r / N H ~ N H
OH
~
I
J
I r HO/~/NH2 ,____L--__~__J I I
J
I
I NH2 0
i
1
I I0
r~ I~
~ N H R TI L--T ....... 0
~ . . . . . .
GLY
r~,/~NH-
/ /
[42l
I
I
I r i
J
~
,8- TYR / ISER ~ . . . . . . . . . . . . . . . . . . .
EDEINE
or DAPA
AA
/ I L .........
A,
R=H
B,
R = C ( = N H ) NH2
FIG. 1. Structure of edeine A1 and B~. GLY, glycine; DAPA, ~,fl-diaminopropionic acid; DAHAA, 2,6-diamino-7-hydroxyazelaic acid ; ISER, isoserine ; #-TYR, #-tyrosine. than to the a-amino group of a, fl-diaminopropionic acid. 2 The biosynthesis of edeines resembles that of tyrocidines2 It is accomplished by two complexes of soluble enzymes of Bacillus brevis Vm4. Properties of the enzymes are reminiscent of those participating in fatty acid synthesis2 The formation of aminoacyl-AMP of constituent amino acids of edeines is catalyzed by two polyenzymes: I, M W 210,000; II, M W 180,000. Prior to the polymerization the activated amino acids are bound to the sulfhydryl group of 4'-phosphopantetheine, which is covalently bound to protein of both polyenzymes. Activation of fl-tyrosine is catalyzed by polyenzyme I, whereas activation of isoserine, a,fl-diaminopropionic acid, 2,6-diamino-7-hydroxyazelaic acid, and glycine involves polyenzyme II. The presence of both polyenzymes and spermidine are required for the formation of edeines. The mechanism of addition of spermidine to the peptide chain is not known; however, its presence is sufficient for the formation of edeine A as well as edeine B2
Assay
Methods
Activation reaction R. CH. COOH 4- ATP [
NH2
Mg2 + polyenzyme
, R. CH. CO. AMP 4- PPI
I or II
I
(1)
NH2
2 T. P. Hettinger and L. C. Craig, Biochemistry 9, 1224 (1970). 3S. G. Lee, R. Roskowski, Jr., K. Bauer, and F. Lipmann, Biochemistry 12, 398 (1973). 4F. Lynen, Biochem. J. 102, 381 (1967). 5Z. Kurylo-Borowska and J. Sedkowska, Biochim. Biophys. Acta 351, 42 (1974).
[42l
EDEINE SYNTHETASE
561
Binding reaction polyenzymeI R. CH. CO. AMP + , R- CH. CO--S--polyenzyme + AMP t or polyenzymeII [ NH~ NH~
(2)
Polymerization reaction R. CH. CO--S--polyenzyme I -I- 2?(R. CH. CO)--S--polyenzyme II r I NH2 NH~ -I- spermidine--~ edeine A +edeine B + HS-enzymes (3) Reaction (1), conducted with fl-tyrosine and polyenzyme I, or with isoserine, a,fl-diaminopropionic acid, 2,6-diamino-7-hydroxyazelaic acid, glycine, and polyenzyme II is determined by the ATP-3zPPi exchange. If experiments are conducted with polyenzyme II purified by DEAEcellulose chromatography only, it is advisable to measure the ATP-32PPi exchange dependent on isoserine, DAP, and DAHAA rather than glycine, since the contamination of this fraction with t R N A g'y and a corresponding ligase can occur. Reaction (2): binding of the amino acyl to the enzyme complex can be measured only if radioactive edeine constituent amino acids are available. Reaction (3): formation of edeine A and B is measured by incorporation of radioactive glycine and spermidine into these two compounds or microbiological assay. Reagents
Amino acids a,fl-Diaminopropionic acid, 10 m M (Calbiochem) Isoserine, 10 m M (Nutritional Biochemicals Corp.) 2,6-Diamino-7-hydroxyazelaic acid, 10 m M (obtained by hydrolysis of edeines ~) Glycine, 10 m M fl-tyrosine, 10 m M (obtained by hydrolysis of edeines ~ or synthetically 7) Reagents for the measurement of the activation reaction Tris.HC1 buffer, 1 M, pH 8.0 Mg acetate, 1 M EDTA, 0.1 M DTT, 0.1 M NaF, 0.1 M G. Roncari, Z. Kurylo-Borowska, and L. C. Craig, Biochemistry 5, 2153 (1966). 'V. M. Rodinov, A. A. Dudinskaya, V. G. Avramenko, and N. N. Suvorov, J. Ge,. Chem. USSR 28, 2279 (1958).
562
ANTIBIOTIC BIOSYNTHESIS
[42]
Na4P207, 0.1 M Na4~2P207 Bovine serum albumin (10 mg/ml) Polyenzyme I or polyenzyme II, 0.2-1.0 mg protein/ml in a solution of 0.1 M Tris, pH 7.6, 2 mM D T T Amino acid solutions: 10 mM Reagents for the biosynthesis of edeine A and B Tris buffer, 1 M, pH 7.9 Mg acetate, 1 M KC1, 1 M DTT, 0.1 M ATP, 0.1 M Phosphoenolpyruvate, 0.2 M Phosphoenolpyruvate kinase, 10 mg/ml Spermidine, 10 mM Amino acids, 10 mM [U-14C] Glycine [U-I'C] Spermidine Solution of polyenzyme I and II or of fraction A, fraction B, and fraction C. Procedure Activation o] Amino Acids. Standard reaction mixture consists of 20 ~l of 1 M Tris.HC1 buffer, pH 8.0, 1 ~l of 1 M Mg acetate, 2.5 ~l of 0.1 M DTT, 7 /~1 of 10 mM EDTA, 5 ~l of 0.1 M ATP, and 2 ~l of 1% bovine serum albumin. To this mixture add 50-150 ~l of enzymes solution (fraction I or II), 2.5 ~l of 0.1 M NaF, 5 ~l of 0.1 M Na~P207, 100,000 cpm of Na43~P20~, 25 ~1 of 10 mM amino acid and water to a total volume of 0.25 ml. The reaction is carried at 35 ° for 30 min, then terminated by addition of 100 ~l of charcoal suspension in a solution of Na4P20T and perchloric acid. s Charcoal is collected on glass fiber disks (Whatman GFA, 2.4 cm) and washed with 30 ml of water. Filters are dried, and the radioactivity of adsorbed [3-~p]ATP is measured in a liquid scintillation counter. Biosynthesis o] Edeine A and Edeine B. The reaction mixture consists of: 80 ~l of 1 M Tris buffer, pH 7.9, 20 ~l of 0.1 M DTT, 10 ~l of 1 M KC1, 10 ~l of 1 M Mg acetate, 2.5 ~l of 0.1 M ATP, 5 ~l of 0.2 M phosphoenolpyruvate, 5 ~l of (10 mg/ml) phosphoenolpyruvate kinase, 25 ~l of 0.1 M ATP, 10 ~l of each 0.1 M edeine constituent amino acid, 8 R. Calendar and p. Berg, "Procedures in Nucleic Acid Research" (G. L. Cantoni and D. R. Davies, ed.), p. 384. Harper, New York, 1966.
[42]
EDEINE SYNTHETASE
563
SUMMARY OF THE PURIFICATION OF EDEINE BIOSYNTHETIC ENZYMES a
Stage of purification
Protein (mg)
Specific activity Purifica(ATP nmoles/mg tion protein) (fold)
A. ¢~-TyrosineActivating Enzymes 1. 30,000 g supernatant fraction 2. Ammonium sulfate precipitate (30-55 % saturation) 3. DEAE-cellulose column Fraction I Fraction II 4. Sephadex G-200 of fraction I: fraction A, MW 210,000 Sephadex G-200 of fraction II: fraction A, MW 210,000
920 300
4.0 10.0
2.5
25 18 2.5
75.0 26.0 280.0
18.7 6.5 70.0
2.0
85.0
21.2
B. Diaminopropionic Acid Activating Enzymes 1. 30,000 g supernatant fraction 2. Ammonium sulfate precipitate (30-55% saturation) 3. DEAE-cellulose column Fraction I Fraction II 4. Sephadex G-200 of fraction I Fraction B, MW 180,000 Fraction C, MW 100,000 Sephadex G-200 of fraction II Fraction B, MW 180,000 Fraction C, MW 100,000
920 300
6.2 18.0
2.8
25 18
80.0 160.0
13.2 25.8
2.0 2.0
250.0 360.0
40.0 60.0
3.0 3.0
360.0 150.0
60.0 24.2
Specific activity of the enzymes was calculated from the ATP-~2PPI exchange dependent on a,f~-diaminopropionic acid or ¢~-tyrosine. One nanomole of ATP corresponded to 200 cpm. including [U-14C]glycine (25,000 cpm/tLmole), and 10 tL1 of 0.1 M [U-14C]spermidine (25,000 cpm/~mole). Equal amounts of polyenzymes I and I I are added (50-100 ~g of each), and the volume is adjusted with water to 1 ml. If polyenzymes I and I I are purified further by Sephadcx G-200 filtration (see the table) equal amounts of fractions A, B, and C are used. After incubation at 35 ° for 30-min, samples are acidified to p H 5.5 with 1 M acetic acid. The precipitate is removed by centrifugation. The resulting supernatant solution is neutralized with 1 N N a O H and adsorbed to Dowex 50-X4 H +, on a column of 2 cm X 1 cm. The resin is washed successively with 20 ml of 0.5 N ammonium formate buffer pH 7, 20 ml of water, and 5 ml of 1 N NH4OH. The fraction
564
ANTIBIOTIC BIOSYNTHESIS
[42]
eluted by the NH40H is concentrated under vacuum to 0.4 ml. Aliquots of 100 ~l are chromatographed on Whatman 3 MM paper With isopropanol-NH~OH-H20 (4: l : l , v/v) in the presence and in the absence of standards of edeine A and edeine B. Chromatograms are dried, and the parts which were eochromatographed with standards of edeines are stained with ninhydrin. These spots are excised and counted. The parts of chromatograms developed without standards are cut into strips and placed on agar plates, inoculated with B. subtilis 168. After 3 hr at 4 ° strips are removed, and plates are incubated for 10 hr at 37 °. The zones of inhibition corresponding to the position of edeine A and B are measured and compared with those of standard edeine solutions chromatographed under the same conditions.
Purification
Preparation of Crude enzymes. The enzymes are obtained from Bacillus brevis Vm4. The organism is grown in Bactopeptone-yeast extract medium at 30 ° with shaking2 The cells are collected by centrifugation from 10 liters of a 10-12-hr-old culture. After washing 3 times with 600 ml (each time) of cold 0.1 M Tris.HC1 buffer pH 7.6 in 2 mM dithiothreitol (buffer A) the pellet (about 120 g wet weight) is suspended in 60 ml of buffer pH 7.2 consisting of morpholinopropanesulfonic acid and 2.5 mM EDTA. The cells are lysed by incubating the suspension with 36 mg of lysozyme 'and 1.2 mg of DNase at 30 ° for 20 rain and then at 0 ° for 20 min. The lysate of cells is centrifuged 90 min at 30,000 rpm at 4 °. All further operations are conducted at 4 °, unless stated otherwise. To the supernatant fraction solid ammonium sulfate is added to 30% saturation. After 1 hr the precipitate is removed by centrifugation and the concentration of ammonium sulfate of the supernatant fraction is adjusted to 55%. This is kept 1 hr at 0 ° and afterward the precipitate is collected by centrifugation. The precipitate is dissolved in a small volume of 1 M Tris.HCl buffer, pH 7.6, and dialyzed 5 hr against 2 liters of buffer A, changing the buffer twice. DEAE-Cellulose Chromatography. The dialyzed crude enzymes (about 300 rag) are applied to the column of DEAE-cellulose (2.2 X 25 cm) equilibrated with buffer A. The column is washed successively with 500 ml of buffer A and 250 ml (each): 0.1 M, 0.2 M, and 0.3 M KC1 in buffer A. Fractions of 6 ml (flow rate 40 ml/hr) are collected. Proteins and the ATP-3-~PPi exchange dependent on edeine constituent amino acids are measured in aliquots of fractions. Polyenzyme I elutes with 9Z. Kurylo-Borowskaand E. L. Tatum, Biochim. Biophys. Acta 113, 206 (1966).
[42]
EDEINE SYNTHETASE
565
0.3M K(
O.IM KCI
50
I I
1.5 30 ~
~6
L a E
1.0
<
.E
oJ
E
2 o g¢'- o_ ~:
B
ge ~
0._
cl
0.5
I0C3
"
60
90
120
150
F r a c t i o n No.
FIG. 2. Resolution of edeine biosynthetic enzymes into two fractions by DEAEcellulose column chromatography. Protein precipitated with 30-55% saturation of ammonium sulfate (300 mg) are dialyzed against buffer A and then applied to '~ column of DEAE-cellulose (2.2 cm X 25 cm). Enzymes are eluted stepwise with 0.1 M, 0.2 M, and 0.3 M KC1 in buffer A. Fractions of 6 ml are collected and assayed for protein (O O) and diaminopropionic acid-dependent ATP-3~'PP~ exchange ( 0 0),
0.2 M KC1 (fractions: 100-120) and polyenzyme II with 0.3 M KC1 (fractions: 130-140) (Fig. 2). To the concentrate of eluted enzymes solid ammonium sulfate is added to obtain 60% saturation. Samples are kept at 0 ° for 2 hr, and the resulting precipitate is collected by centrifugation. The precipitate is dissolved in 2.5 ml of 1 M Tris.HC1 buffer pH 7.6 and dialyzed against 1 liter of Buffer A. Sephadex G-200 Filtration. Further purification of polyenzymes (see the table) is obtained by filtration through a column (1.8 X 70 cm) of Sephadex G-200 in buffer A. Polyenzyme I or polyenzyme II (about 30 rag) is applied to the column and eluted with buffer A. Fractions of 2.8 ml with a flow rate 5 ml/hr are collected. Proteins and ATP-a2PP~
566
ANTIBIOTIC BIOSYNTHESIS
[42]
exchange dependent on edeines constituent amino acids are measured. This procedure resolves polyenzyme I and polyenzyme II into fractions: A (tubes 15-20), B (tubes 18-22), and C (tubes 28--32) (Figs. 3 and 4). Properties of the Enzymes
Stability. The enzymes are quite stable at --70 ° . At 4 ° about 30% of activity is lost within 2 days. Incubation at 60 ° for 20 min destroys most of the activity. pH Optimum. The enzymes operate in Tris buffer in the pH range 7.2-8.4 with the optimum at pH 7.9. Veronal and glycine-NaOH buffers significantly lower the enzymic activity. Activators and Inhibitors. Mg 2÷ is required. Its optimal concentration for biosynthesis of edeines is 10 raM. Higher concentrations are inhibittory. ATP is required at concentrations of 2.5 mM.
6O 0.4-
-6
I
E
"t2_
-- O.5E
o
g
13-
~
I
0.2-
50 c. E o 4O
X
C:
~g so gg ~~, g
I
-0.4
~
r
~
x
o
0,~" 0 -0.2 I
i
0
5
I~0
15
20
25
3'0
40
Fraction No. FIG. 3. Sephadex G-200 filtration of fraction I. Protein (30 rag) in 2.5 ml of buffer A is applied to a column of Sephadex G-200 (1.8 cm X 70 cm). Fractions of 2,8 ml are eluted with buffer A and assayed for protein (O O), fl-tyrosine-dependent (X X) and diaminopropionic acid-dependent ATP2~PP1 exchange (0 " 0 ) and pantothenic acid ( A - - A ) .
[43]
GRAMICIDIN S SYNTHETASE
567
O-
o_
c
x2_ u
,30 ~-
o
c
20 ~ E
(3_
0.2.
~"o
T ,Q E ._c
o
o
Jc
Q; c Q;
Q; ~
, o ' ~ o_
....
._~'n
-0.4
(3-
O.D
t
"~
~', T?0 (2. x
(3-
7
-0.2
o
,'o
,5
2'0
2's
3'0
35
Froction No.
FIG. 4. Sephadex G-200 filtration of fraction Ii. The experimental conditions are as described in Fig. 3.
Molecular Weight. Polyenzyme fractions A, B, and C have molecular weights of 210,000, 180,000, and 100,000, respectively.
[43] Gramicidin S Synthetase By
TRINE-LISE ZIMMER a n d SgREN G. LALAND
The antibiotic gramicidin S, which belongs to a group of peptides synthesized by different strains of Bacillus brevis, was first isolated by Gause and Brazhnikova I and has been shown to have the following structure: D-Phe-L-Pro-L- Val-L-Orn-b-Leu
I
I
L-Leu-L-Orn-L-Val-L-Pro-D-Phe i G. F. Gause and M. G. Brazhnikova,
Lancet, 715
(1944).
[43]
GRAMICIDIN S SYNTHETASE
567
O-
o_
c
x2_ u
,30 ~-
o
c
20 ~ E
(3_
0.2.
~"o
T ,Q E ._c
o
o
Jc
Q; c Q;
Q; ~
, o ' ~ o_
....
._~'n
-0.4
(3-
O.D
t
"~
~', T?0 (2. x
(3-
7
-0.2
o
,'o
,5
2'0
2's
3'0
35
Froction No.
FIG. 4. Sephadex G-200 filtration of fraction Ii. The experimental conditions are as described in Fig. 3.
Molecular Weight. Polyenzyme fractions A, B, and C have molecular weights of 210,000, 180,000, and 100,000, respectively.
[43] Gramicidin S Synthetase By
TRINE-LISE ZIMMER a n d SgREN G. LALAND
The antibiotic gramicidin S, which belongs to a group of peptides synthesized by different strains of Bacillus brevis, was first isolated by Gause and Brazhnikova I and has been shown to have the following structure: D-Phe-L-Pro-L- Val-L-Orn-b-Leu
I
I
L-Leu-L-Orn-L-Val-L-Pro-D-Phe i G. F. Gause and M. G. Brazhnikova,
Lancet, 715
(1944).
568
ANTIBIOTIC BIOSYNTHESIS
[43]
The synthesis of gramicidin S is catalyzed by two enzymes (light and heavy) designated gramicidin S synthetase; the net reaction is as follows: 10 amino acids T 10 ATP -~ gramicidin S -~- 10 AMP ~ 10 PPi A detailed description of the mechanism of synthesis, for which we have suggested the name "thiotemplate" mechanism, has recently been published. 2
Assay of Gramicidin S Synthetase Principle. In the biosynthesis of gramicidin S, the individual amino acids are activated by ATP through the formation of the aminoacyl adenylates. The aminoacyl moieties are then transferred to specific thiol groups on the enzymes (see below). The resulting thioester-bound amino acids are the immediate precursors in the stepwise polymerization process leading to the formation of the thioester-bound pentapetide n-Phe-LPro-L-Val-L-0rn-L-Leu. Gramicidin S is then formed by head-to-tail condensation of two such pentapeptides on the same enzyme molecule (intramolecular reaction). The light enzyme of gramicidin S synthetase activates and racemizes L- and D-phenylalanine, and the heavy enzyme activates L-proline, L-valine, L-ornithine, and L-leucine. Formation of the thio ester-bound precursor peptides and the cyclization reaction are also catalyzed by the latter enzyme.
ATP PPi R.CH.COOH:t % J-- ~R" NH~
AMP
H.CO.AMP+R.CH.CO.SE-*-*--)
NH2
gramicidin S
~
E SH
NH~
According to the reaction scheme outlined above, the following enzymic activities may be used for assaying gramicidin S synthetase: (1) synthesis of gramicidin S; (2) amino acid-dependent ATP-a2PPi exchange; (3) amino acid-dependent ATP-[I~C]AMP exchange; (4) thioesterbonding of the individual amino acids, (5) ATP-dependent racemization of phenylalanine. 1. Synthesis o] Gramicidin S. The incubation mixture contains in a volume of 1 ml:100 /~moles of triethanolamine.HC1 adjusted to pH 7.6 with NaOH, 4 ~moles of dithiothreitol (DTT), 2.5 ~moles of ATP, 50 2S. G. Laland and T.-L. Zimmer, Essays Biochem. 9, 31 (1973).
[43]
GRAMICIDIN S SYNTHETASE
569
t~moles of Mg acetate, 0,25 ~moles of EDTA, 0.05 ~mole of each of the constituent amino acids in the z-form, one of which is l~C-labeled (0.5 ~Ci), and enzyme. When using purified enzyme preparations of low protein concentration, 1 mg of bovine serum albumin is added to facilitate precipitation and isolation of gramicidin S. The mixture is incubated at 37 ° for 5 rain. The reaction is stopped by adding 1 ml of ice cold 10% trichloroacetic acid (w/v) containing 0.5% Na,_,WO4 (w/v) and 1 mmole of the amino acid which was used in labeled form. After standing for 15 rnin at 0 °, the mixture is centrifuged and the precipitate is washed twice with 5% trichloroacetic acid (w/v) containing 0.25% Na2WO~ and once with 2% Na2SO~(w/v). Gramicidin S is then extracted from the precipitate with 2 ml of ethanol/0.2 N HC1 (9:1, v/v) at room temperature for at least 3 hr. After centrifugation the precipitate is washed twice with 1 ml of the same reagent, and the combined extracts evaporated to dryness. The residue is dissolved in a small volume of the ethanol-HC1 mixture, plated, and counted in a windowless gas-flow counter. 2. Amino Acid-Dependent ATP-3~-PPj Exchange. The procedure used is modified after Itoh et al. 3 and Calendar and Berg. 4 The incubation mixture contains in a volume of 1 ml : 100 ~moles of triethanolamine. HC1 adjusted to pH 7.6 with NaObI, 4 ~moles of DTT, 2.5 ~moles of ATP, 50 t,moles of Mg acetate, 0,25 /~mole of EDTA, 2 t~moles of one of the substrate amino acids, 1 ~mole of ~P-labeled Na4P,_,07 (0.02-0.03 t~Ci), and enzyme. The mixture is incubated at 37 ° for 5 rain; the reaction is stopped by adding 1 ml of ice cold 10% trichloroacetic acid (w/v) containing 0.1 mmole of Na~P207 and 15 mg of activated charcoal. After standing for 15 rain at 0 °, the mixture is filtered on Millipore filters. The charcoal is washed with 5 ml of 5% trichloroacetic acid and 25 ml of water. The filter containing the charcoal is transferred to a planchette with the charcoal facing down and dried. The filter is then dissolved with acetone, the charcoal is fastened with glue, and the radioactivity is determined in a Geiger counter. 3. Amino Acid-Dependent ATP-[I~C] A M P Exchange. The procedure described is modified after Yamada and Kurahashi. ~ The incubation mixture contains in a volume of 1 ml:100 ~moles of triethanolamine.HC1 adjusted to pH 8.6 with NaOH, 2.5 ttmoles of KF, 2.5 t,moles of DTT, 2.5 t~moles of ATP, 2.5 t,moles of MgCI.,, 0.25 tmmle of EDTA, 1 t,mole of Na~P..O~, 1 ~moie of [~C]AMP (1 ttCi) and enH. Itoh, M. Yamada, S. Tomino, and K. Kurahashi, g. Biocltem. 64, 259 (1968). R. Calendar and P. Berg, Biochemistry 5~ 1681 (1966). M. Yamada and K. Kurahashi, J. Biochem. 66, 529 (1969).
570
ANTIBIOTIC BIOSYNTHESIS
[43]
zyme. The mixture is incubated for 20 min at 37 °. The reaction is stopped by placing the tube in ice water for 15 min. Aliquots of 100 ~l are applied on a polyethyleneimine-treated Whatman No. 1 paper and chromatographed ascendingly using 2% NH4HCO~ as the solvent for 2 hr. 6 The paper is dried at 50 ° and the nucleotides detected under the UV lamp. The spots are cut out and suspended in 10 ml of scintillation liquid for counting. The scintillation liquid used contains 12.5 mg of POPOP, 1 g of PPO, 27.5 ml of Methyl Cellosolve, and 225 ml of toluene. 4. Measurement o] Thioester-Bound Amino Acids. The incubation mixture is identical to that used when measuring gramicidin S synthesis except that only one amino acid is used. The reaction is stopped by adding 1 ml of 10% ice cold trichloroacetic acid (w/v) containing 0.5% Na2W04 (w/v) and 1 mmole of the amino acid, which is used in labeled form. After centrifugation, the precipitate is washed twice with 3 ml of 5% trichloroacetic acid containing 0.25% Na._,W04, once with 3 ml of 2% Na.~SO~ (w/v). The precipitate is finally washed with 3 ml of methanol and dried in vacuum for 1 hr. The protein fraction may either be dissolved in 99% HCOOH (250 ~l per milligram of protein) and counted in the scintillation counter, or the labeled thioester-bound amino acid split off by the following procedure and then counted. The protein is dissolved in formic acid as above. After 10 min at 0 °, 100 ~l of performic acid is added per milliliter of formic acid. 7 The reaction is allowed to proceed for 2.5 hr at 0 °, and the mixture is diluted 10 times with ice cold water and freeze-dried. The residue is extracted three times with 2 ml of ethanol/0.2 N HC1 (9:1, v/v). The protein residue is dried in vacuum and submitted twice more to performic acid oxidation. The combined extracts are evaporated to dryness, dissolved in a small volume of ethanol/HC1, plated, and counted. The performic acid used is prepared by mixing 1 ml of 30% H202 and 9 ml of 99% formic acid and allowing the mixture to stand in a dark closed bottle at room temperature for 2 hr before use. When incubations containing more than one amino acid are used, thioester-bound intermediate peptides may be liberated in the same way by performic acid oxidation, separated and identified by thin-layer chromatography according to FrOyshov et al. s It should be noted that the ethanol-HC1 reagent used for extracting gramicidin S, will also liberate thioester-bound dipeptide D-phenylalanylproline (as the diketopiperazine) and ornithine. 6j. M. Gilliland, R. E. Langman, and R. H. Symons, Virology 30, 716 (1966). 7C. H. W. Hits, J. Biol. Chem. 219, 611 (1956). s 0. FrCyshov, T.-L. Zimmer, and S. G. Laland, FEBS Lett. 7, 68 (1970).
[43]
GRAMICIDIN S SYNTHETASE
571
5. Racemization o] Phenylalanine. The procedure is modified after Yamada and Kurahashi2 The incubation mixture contains in a volume of 1 ml:100 ~moles of triethanolamine.HC1 adjusted to pH 8.6 with NaOH, 2.5 ~moles of ATP, 2.5 ~moles of MgC12, 1 ~mole of AMP, 1 ~mole of NapPy07, 10 ~moles of D T T , 0.1 ~mole of 14C-labeled D- or L-phenylalanine (0.5 #Ci) and enzyme. The final pH of the incubation mixture is 8.45. The mixture is incubated at 37 ° for 15 min, and the reaction is stopped by heating in a boiling water bath for 1.5 min. To 500 td of the reaction mixture is added 0.1 t~mole of phenylalanine and 25 ~g of amino acid oxidase (D-phenylalanine and D-amino acid oxidase if L-phenylalanine was present during incubation, and L-phenylalanine and L-amino acid oxidase if D-phenylalanine was present), and 25 ~g of catalase (Boehringer). The L- and D-amino acid oxidase were crystalline preparations from Boehringer and Sigma, respectively, and were found not to be contaminated with enzyme which would oxidise the other isomer. The mixture is incubated at 37 ° for 1 hr and 100 ~1 of a solution of 0.1% (w/v) dinitrophenylhydrazine in 2 N HC1 is added to the amino acid oxidase treated as well as to an aliquot which had not been treated with amino acid oxidase and both incubated at 37 ° for 5 min. One milliliter of ethyl acetate is added to the samples, which are then centrifuged after thorough mixing; 100 td of the organic phase is plated and counted in a windowless gas-flow counter. Purification of Gramicidin S Synthetase
Step I. Cultivation of the Microorganism. STOCK CULTURES. Bacillus brevis ATCC 9999 is maintained at 4 ° on agar slants containing 3 g of Bacto beef extract, 5 g of Bacto peptone, and 15 g of Bacto Agar per liter of medium. Every 6 months an inoculum from the stock culture is subcultured by growing it with aeration overnight at 37 ° in the following medium: 10 g of Bacto Tryptone, 5 g of Bacto Yeast Extract, 0.5 g of KH2PO~, 0.5 g of K2HP04, 0.2 g MgSO4.7H20, 10 mg of NaC1, l0 mg of MnSO4.4H2O, 22 mg of CaCl~.2 H20, 10 mg of FeSO~-7H~O in a total volume of 1000 ml and the pH is adjusted to 7.2. The culture is streaked out on agar plates and after incubation at 37 ° for 24 hr, single colonies of the R form are transferred to agar slants. The agar slants are incubated for 48 hr at 37 ° and stored at 4 °. GROWTH OF THE ORGANISM FOR THE PREPARATION OF THE ENZYME.
Bacillus brevis from agar slants is transferred to two 250-ml flasks each M. Yamada and K. Kurahashi, J. Biochem. 63, 59 (1968).
572
ANTIBIOTIC BIOSYNTHESIS
[43]
0.~ "4 I
o D. U
0.3 E
.c "o 0D E
o
o C
o
ol
0.2
.c
o ._>
"5 o .o
1
Z
3
~
Growth,
5
5
?
S
hours
FIG. 1. Relationship between growth curve and gramicidin S synthesizing activity of crude extracts. O O, Absorbance at 650 nm; • O, synthesis of gramicidin S. Reproduced from S. G. Laland and T.-L. Zimmer, Essays Biochem. 9, 31 (1973). containing 60 ml of the tryptone yeast medium described in the previous section. The flasks are incubated with aeration at 37 ° overnight and the cultures transferred to 10 liters of medium in the Microferm L a b o r a t o r y Fermenter (New Brunswick Scientific Co.). The medium contains per 10 liters: 136 g of KH2P04, 20 g of (NH4)~S04, 100 mg of CaC12.2H20, 2 g of MgSO~.7 H20, 5 mg of FeSO4.TH~O, 50 g of monosodium glutamate, and 1.5 g of each of the amino acids in gramicidin S. The p H is adjusted to 6.5 with K O H . The culture is aerated at a rate of 5 liters per minute at a pressure of 10 psi with stirring at 300 rpm. The culture is harvested after about 5 hr at an absorbance of 0.27 at 650 nm in a Spectronic 20 spectrophotometer after dilution of the culture 9 times with water. The culture is cooled in the fermentor to about 15 °, and the cells are then harvested by centrifugation at 0% All further procedures are carried out at 2-4 °. The cells are washed once with a solution of 5 m M MgC12. The washed cells are either processed at once or m a y be stored at - - 2 0 ° without any significant loss of enzyme activity. Figure 1 shows the relationship between growth curve and gramicidin S synthesizing activity of the crude extract (see next section).
[43]
GRAMICIDIN S SYNTHETASE
573
Step 2. Preparation o] Crude Extract. The cells are suspended in 300 ml of a solution of 5 mM MgCI~ containing per milliliter 10 t~moles of reduced glutathione and 0.25 t~mole of EDTA, with pH adjusted to 7.5 and disintegrated by ultrasonic treatment with a Branson Sonifier Cell Disruptor S 75 at 75 W and 20 KHz for 7 rain under cooling. The resulting suspension is centrifuged for 15 min at 3000 g and then 15 rain at 20,000 q. The supernatant after the last centrifugation is called the crude extract and contains approximately 2.5 g of protein. It may be stored at --20 ° without loss of activity. Step 8. Streptomycin Sul]ate Precipitation. To the crude extract is added dropwise under stirring a neutral solution of streptomycin sulfate, 50 mg/ml, to a final concentration of 5 mg/ml. The mixture is allowed to stand for 30 rain before it is centrifuged for 15 rain at 20,000 g. The precipitate is discarded. Step ~. Ammonium Sul]ate Precipitation. Solid (NH~).,SQ is added (final concentration 0.29 g/ml). After standing for 30 rain, the mixture is centrifuged for 15 rain at 20,000 g, the supernatant discarded and the precipitate dissolved in 50 ml of buffer A [50 mM KH~PO~, 0.25 mM EDTA, 1 mM DTT, 20% glycerol (v/v), pH 7.5]. The preparation contains approximately 10 mg of protein per milliliter and is stable at --20 °. Step 5. Chromatography on DEAE Sephadex A-50. The ammonium sulfate fraction from step 4 is applied on a DEAE Sephadex A-50 column (2.5 X 40 cm) previously equilibrated with buffer A containing 105, glycerol (instead of 205,) and 0.2 M KC1; 60 ml of the equilibrating buffer is then run through the column. The concentration of KC1 in the eluent is then increased linearly from 0.2 to 0.5 M. The light and the heavy enzyme are partially separated by this procedure (Fig. 2) in contrast to what was achieved with a former procedure where a linear gradient of phosphate buffer was used. ~° The fractions rich in the light and the heavy enzyme, respectively, were combined and precipitated by ad(lition of ammonium sulfate to 60% saturation. The precipitates were then dissolved in 5-10 ml of buffer A. Step 6. Chromatography on Sephadex G-200. Of the protein from the fraction rich in the heavy enzyme from step 5, 7.7 mg are apt)lied to a column of Sephadex G-200 (2.5 X 30 cm) equilibrated with buffer A. A separation of the light and heavy enzyme is achieved (Fig. 3) upon elution with buffer A. Fractions 13-18 are combined and contain essentially pure heavy enzyme. Similarly, light enzyme free of the heavy enzyme may be obtained by Sephadex G-200 chromatography of the light enzyme-rich fraction from step 5. The fractions containing the light and ~oj. E. Bredesen, T.-L. Berg, K. J. Figenschou, L. O. Fr0holm, and S. G. Laland, Ear. J. Biochem. 5, 433 (1968).
574
[43]
ANTIBIOTIC B I O S Y N T H E S I S 2.0
8
1.5
0
0.5
1.0 o
'>
< a: O.g 2
L~ 10
40
Fraction
60
80
100
120
{12
number
Fro. 2. Chromatography of ammonium sulfate fraction on DEAE-Sephadex A-50. Of the protein from step 4, 450 mg was applied to the column (2.5 X 40 cm). Fractions of 4.5 ml were collected upon elution with a linear 0.2-0.5 M KC1 gradient in buffer A containing 10% glycerol at a rate of 25 ml/hr. - - , Absorbance at 280 n m ; • • , D-phenylalanine stimulated ATP-~PP~ exchange; 0 - - 0 , L-ornithine stimulated ATP-'PP~ exchange. 1600
120o
E
e
>
?, "a o
0.3 400
~"
02
0.1 '~
lO
20 Fraction
30
1,0
number
FIG. 3. Chromatography on Sephadex G-200 of the fraction rich in heavy enzyme from step 5. Protein, 7.7 mg, was applied to the column (2.5 X30 cm) and eluted with buffer A. Fractions of 3 ml were collected at a rate of 10 ml/hr. - - , Absorbance at 280 rim; • 0 , D-phenylalanine stimulated ATP-~PPI exchange; O O, t-ornithine stimulated ATP-~PPI exchange.
[43l
GRAMICIDIN S SYNTHETASE
575
7 E
o. u
._> O o ¢~
] J,\ 0
10
~
30
Fraction number
FIG. 4. Affinity chromatography of gramicidin S synthetase. Protein, 0.5 mg in 3 ml from step 5, to which was added 7.5 #moles of ATP, was applied to a column (1 X 8 cm) of 3,'3-diaminodipropylamine-substituted Sepharose with L-proline as ligand. The column was eluted with buffer A at a rate of 7 ml/hr, and 2.5-ml fractions were collected. At the points shown by arrows 0.1 M and 0.5 M KCI, respectively, were added to the elution buffer. • O, D-phenylalanine stimulated ATP-32PPI exchange; 0 O, L-ornithine stimulated ATP22PPt exchange.
the heavy enzyme, respectively, are concentrated against buffer A using Sartorius collodion bags to concentrations of approximately 1 mg/ml. The light enzyme has been purified by Yamada and Kurahashi ~ to a nearly homogeneous state by a different purification procedure.
Step 7. Alternative Method ]or the Separation o] Light and Heavy Enzyme: Affinity Chromatography. 3,3'-Diaminodipropylamine substituted Sepharose to which one of the amino acids in gramicidin S is coupled through its carboxyl group, has been used to study the affinity between the light and the heavy enzyme of gramicidin S synthetase. 11 The column with proline (1 ffmole of proline per milliliter of Sepharose) attached provides a rapid and convenient method for the separation of the two enzymes (Fig. 4). It should be noted that when a DEAE-Sephadex preparation rich in light enzyme is used, a substantial amount of light enzyme is not absorbed and is consequently eluted before the addition of KCl. " L. Pass, T.-L. Zimmer, and S. G. Laland, Eur. J. Biochem. 40, 43 (1973).
576
ANTIBIOTIC BIOSYNTHESIS
[43]
TABLE I GRAMICIDIN S SYNTHESIZING ACTIVITY AND ATP-a2PPI EXCHANGE ACTIVITY DURING PURIFICATION OF GRAMICIDIN S SYNTHETASE
Fraction
Protein (mg)
Gramicidin S synthesizing activity (%)
Crude extract Ammonium sulfate fraction DEAE Sephadex A-50 Sephadex G 200
3000 500 25 20
100" 90 30 3
ATP-3~PPi exchange activity (%) --100 80
Under the conditions used for measuring gramicidin S synthesis, the crude extract from 10 liters of Bacillus breviswill incorporate about 10 to 15 × 106 cpln into gramicidin S.
Comments on the Purification of Gramicidin Synthetase. The biosynthesis of gramicidin S is the result of the concomitant functioning of a large number of catalytic activities. For instance, in the case of the heavy enzyme it has been estimated that about 18-20 different catalytic activities are involved. 1~ During the purification of gramicidin S synthetase which gives an almost homogeneous preparation of heavy enzyme, some 97% of its ability to synthesize the antibiotic is lost (Table I). The loss is particularly great during fractionation on the D E A E Sephadex G-50 and Sephadex G-200. Of the many catalytic activities involved in the biosynthesis, the catalytic activities responsible for amino acid activation are remarkably stable. As the crude extract and the ammonium sulfate fraction are unsuitable for determining the absolute levels of specific amino acid stimulated activation due to the high level of background ATP-32PPi exchange, the degree of purification of the synthetase cannot be determined by this method.
Properties of Gramicidin S Synthetase Purity o] the Enzymes. The heavy enzyme is found to be essentially pure as determined by polyacrylamide gel electrophoresis. The same technique reveals that the light enzyme preparation (by our procedure) is contaminated to a degree of about 10% by other proteins. 1~S. G. Laland, ~. Fr0yshov, C. Gilhuus-Moe, and T.-L. Zimmer, Nature (London) New Biol. 239, 43 (1972).
[43]
GRAMICIDIN S SYNTHETASE
577
TABLE II PHYSICAL PROPERTIES OF GRAMICIDIN S SYNTHETASI~ Properties MW s2o,w (S)
Heavy enzyme 240,000 280,000 b 11.6
Light enzyme 100,00() 6.1"
H. Yamada and K. Kurahashi, J. Biochem. 66, 529 (1969). b It. Kleinkauf, W. Gevers, and F. Lipmann, Proc. Nat. Acad. Sci. U.S. 62, 226 (1969).
Stability. The purified enzyme preparation may be stored at --20 ° for several months without loss of amino activating as well as gramicidin S synthesizing activities. Physical Properties. Our recent results have shown the molecular weight and the sedimentation Constant to be 240,000 and 11.6 S, respeelively as determined by sucrose gradient centrifugation (Table II). Polyacrylamide disc electrophoresis confirms this molecular weight. Presence o] 4'-Phosphopantetheine. Treatment of the essentially pure heavy enzyme with 0.2 M KOH at 37 ° for 1 hr 1~ liberates 1 mole of 4'-phosplIopantetheine from 1 mole of enzyme1'-' as determined by a microbiological assay using Lactobhcillus helveticus 80 (ATCC 12046)." 4'-Phosphopantetheine is believed to play an essential role in the stepwise polymerization process leading to gramieidin S. ~ Specificity. (a) AMINO ACID ACTIVATION. (i). The light enzyme. The light enzyme will activate both the L- and the D-isomers of phenylalanine, the former giving the highest rate of reaction. Phenylalanine analogs such as p-fluorophenylalanine, fl-2-thienylserine, and fl-phenylserine stimulate the ATP-:t~PPi exchange reaction, '~ whereas tyrosine and tryptophan as well as phenylpyruvic acid do not. (ii). The heavy enzyme. The following amino acids have been found in our laboratory to stimulate the ATP-:'~PP~ exchange reaction: azetidine-2-carboxylic acid, isoleucine, norvaline, norleucine 'rod D-leucine. Lysine and arginine do not stimulate the reaction. The D-isomers of proline, valine, and ornithine are not activated. '~ (t)) SYNTHESIS Of GRAMICIDINS. The following amino acids may be incorporated into gramicidin S: p-fluorophenylalanine, azetidine-2-carboxylic acid, norvaline, norleucine, and isoleucine. ~P. W. Majerus, A. W. Alberts, and P, R Vagelos, Proc. Nat. Acad. Sci. 1;.S. 53, 410 (1965). ~J. A. Craig and E. E. Snell, J. B~wteriol. 61, 283 (1951). '~ D. C. Leung and R. Baxter, Biochim. Biophgs. Acta 279, 34 (1972).
578
[43]
ANTIBIOTIC BIOSYNTHESIS TABLE I I I KINETIC CONSTANTS OF GRAMICIDIN S SYNTHETASE Amino acid
K ~ for amino acid (raM)
L-Phe D-Phe L-Pro I,-Val I,-0rn L-Leu
0.0450. 042" 0.23 0.31 0.17 0.10
K m for A T P (raM)
0.71, 0.711.40 2.33 1.72 3.10
Ki for A M P a (raM)
1.2 0.08 0.36 0.54 0.38 0.42
a T. Kristensen, C. C. Gilhuus-Moe, T.-L. Zimmer, and S. G. Laland, Eur. J. Biochem. 84, 548 (1973).
Inhibitors o] Gramicidin S Synthesis. The heavy enzyme contains several SH-groups essential for activity. Reagents known to react with sulfhydryl groups are strong inhibitors of gramicidin S synthesis. Amino acid derivatives which show affinity for the activation sites, such as the amino acid amides, are also inhibitory. The D-isomer of leucine which is activated and becomes thio ester bound to the heavy enzyme ~6 cannot be incorporated into gramicidin S and therefore inhibits total synthesis. Ammonium ions inhibit gramicidin S synthesis presumably by reacting with the activated amino acids to form the corresponding amides. 17 Kinetic Constants. The Km values for the ATP-S-~PPi exchange reaction with respect to ATP and the individual amino acids, as well as the Ki for AMP, have been determined (Table III).ls Characteristics o] the Light Enzyme. The light enzyme catalyzes an ATP-dependent racemization of phenylalanine. The enzyme does not require pyridoxal phosphate or FAD for activity and is not influenced by known inhibitors of pyridoxal-containing enzymes such as hydroxylamine, KCN, or isonicotinic hydrazide. 9 The results seem to indicate that racemization takes place after the amino acid has become thioester-bound to the enzyme 19 ATP L-Phe ~ L - P h e - A M P PPi
,~b-Phe-S
enzyme ~ D-Phe-S enzyme
AMP
~H. Saxholm, T.-L. Zimmer, and S. G. Laland, Eur. J. Biochem. 30, 138 (1972). ~7S. Tomino, M. Yamada, H. Itoh, and K. Kurahashi, Biochemistry 8, 2552 (1967). 1ST. Kristensen, C. C. Gilhuus-Moe, T.-L. Zimmer, and S. G. Laland, Eur. J. Biochem. 34, 548 (1973). 19H. Takahashi, E. Sato, and K. Kurahashi, J. Biochem. 69, 973 (1971).
[44l
POLYMYXIN SYNTHETASE
579
The biosynthesis of tyrocidine, a related cyclic decapeptide produced by a different strain of B. brevis, is also initiated by phenylalanine in a similar reaction. It has been found in this laboratory and by others 2° that the light enzyme of gramicidin S synthetase may substitute for the light enzyme of tyrocidine synthetase and vice versa in antibiotic formation. ~°K. Fujikawa, Y. Sakamoto, and K. Kurahashi, Y. Biochem. 69, 869 (1971).
[44] P o l y m y x i n S y n t h e t a s e : L - 2 , 4 - D i a m i n o b u t y r a t e Activating Enzyme By HENRY PAULUS
L-2,4-Diaminobutyrate -t- ATP ~- i-2,4-diaminobutyryl-AMP + PPI The sequence of reactions leading to the biosynthesis of polymyxin has not yet been elucidated. However, the following lines of evidence implicate an enzyme that activates L-2,4-diaminobutyrate in the biosynthetic process: (1) The level of the L-2,4-diaminobutyrate activating enzyme parallels the rate of polymyxin B production during the growth cycle of Bacillus polymyxa Pfizer 24591; (2) the L-2,4-diaminobutyrate activating enzyme is also found in B. polymyxa ATCC 10401, which produces polymyxin D, and in B. circulans ATCC 14040, which produces circulin, while it is absent from mutants of B. polymyxa Pfizer 2459 which have lost the ability to produce polymyxin B1; and (3) the enzyme is highly specific for L-2,4-diaminobutyrate which constitutes 5 or 6 of the 10 amino acid residues of polymyxin and circulin (Fig. 1) but is absent from other cellular constituents of B. polymyxa. 1,2 Assay Method 1
The assay is based on that of the aminoacyl RNA synthetases 3 and relies on the L-2,4-diaminobutyrate-dependent exchange of a2PPi with ATP, measured by the formation of charcoal-adsorbable radioactivity. In order to avoid interference by amino acid acyl RNA synthetases, the L-2,4diaminobutyrate should be free of contaminating amino acids, and crude bacterial extracts should be freshly dialyzed. 1K. Jayaraman, J. Monreal, and H. Paulus, Biochim. Biophys. Acta 185, 447 (1969). 2M. Brenner, E. Gray, and H. Paulus, Biochim. Biophys. Acta 90, 401 (1964). F. H. Bergmann, this series, Vol. 5, p. 708.
[44l
POLYMYXIN SYNTHETASE
579
The biosynthesis of tyrocidine, a related cyclic decapeptide produced by a different strain of B. brevis, is also initiated by phenylalanine in a similar reaction. It has been found in this laboratory and by others 2° that the light enzyme of gramicidin S synthetase may substitute for the light enzyme of tyrocidine synthetase and vice versa in antibiotic formation. ~°K. Fujikawa, Y. Sakamoto, and K. Kurahashi, Y. Biochem. 69, 869 (1971).
[44] P o l y m y x i n S y n t h e t a s e : L - 2 , 4 - D i a m i n o b u t y r a t e Activating Enzyme By HENRY PAULUS
L-2,4-Diaminobutyrate -t- ATP ~- i-2,4-diaminobutyryl-AMP + PPI The sequence of reactions leading to the biosynthesis of polymyxin has not yet been elucidated. However, the following lines of evidence implicate an enzyme that activates L-2,4-diaminobutyrate in the biosynthetic process: (1) The level of the L-2,4-diaminobutyrate activating enzyme parallels the rate of polymyxin B production during the growth cycle of Bacillus polymyxa Pfizer 24591; (2) the L-2,4-diaminobutyrate activating enzyme is also found in B. polymyxa ATCC 10401, which produces polymyxin D, and in B. circulans ATCC 14040, which produces circulin, while it is absent from mutants of B. polymyxa Pfizer 2459 which have lost the ability to produce polymyxin B1; and (3) the enzyme is highly specific for L-2,4-diaminobutyrate which constitutes 5 or 6 of the 10 amino acid residues of polymyxin and circulin (Fig. 1) but is absent from other cellular constituents of B. polymyxa. 1,2 Assay Method 1
The assay is based on that of the aminoacyl RNA synthetases 3 and relies on the L-2,4-diaminobutyrate-dependent exchange of a2PPi with ATP, measured by the formation of charcoal-adsorbable radioactivity. In order to avoid interference by amino acid acyl RNA synthetases, the L-2,4diaminobutyrate should be free of contaminating amino acids, and crude bacterial extracts should be freshly dialyzed. 1K. Jayaraman, J. Monreal, and H. Paulus, Biochim. Biophys. Acta 185, 447 (1969). 2M. Brenner, E. Gray, and H. Paulus, Biochim. Biophys. Acta 90, 401 (1964). F. H. Bergmann, this series, Vol. 5, p. 708.
580
H
MOA
[44]
ANTIBIOTIC BIOSYNTHESIS
NH
~ L-DBA
~ L-Thr~p
1
NH z
]----~L-DBA~L-DBA
2
3
4
NH 2
~'{ 5
.
[~[
6
NH~
]~L-DBA~-DBA~L-Thr 7
8
9
10
FI¢. 1. Structure of some polymyxins. MOA, (+)-6-methyloctanoate; DBA, 2,4-diaminobutyrate. Antibiotic Polymyxin Polymyxin Polymyxin Polymyxin Circulin A
A, B1 D1 E~
Residue 3
Residue 6
Residue 7
D-DBA L-DBA L-ser L-DBA L-DBA
D-Leu D-Phe D-Leu D-Leu D-Leu
L-Thr L-Leu L-Leu L-Leu L-Ile
Reagents Potassium phosphate, 0.1 M, pH 7.5. 32PPi, 20 raM, obtained commercially or prepared by the pyrolysis of [a2P]K~HP04 in a platinum crucible2 The presence of small amounts (5-10%) of [32p]orthophosphate will not interfere with the assay, and purification by anion exchange chromatography is therefore not necessary. ATP, 20 mM MgCI~, 50 mM L-2,4-Diaminobutyrate, 10 raM, Commercial preparations are frequently contaminated with other amino acids ~ and must therefore be further purified by chromatography on Dowex 50 or by crystallization as the dipicrate and then as the monohydrochloride2 AIternatively, L-2,4-diaminobutyrate can be conveniently prepared by the Schmidt degradation of L-glutamic acid and crystallized as above2 ,~ Enzyme. Ultrasonic or lysozyme extract as described below. HCIO~, 1 N Norit A charcoal suspension in water (10 mg/ml)
Procedure. The incubation mixtures contain 0.1 ml of phosphate buffer, 0.1 ml of 3~PPi, 0.1 ml of ATP, 0.1 ml of MgCI~, 0.1 ml of L-2,4-diaminobutyrate, enzyme and water to give a final volume of li0 ml. The reaction is carried out for 30 min at 37 ° for the assay of the 4 M. DiGirolamo, O. Cifferi, and A, B. DiGirolamo, J. Biol. Chem. 239, 502 (1964). D. W. Adamson, J. Chem. Soc. London, p. 1564 (1939). 6 H. Paulus and E. Gray, J. Biol. Chem. 239, 865 (1964).
[44l
POLYMYXIN SYNTHETASE
581
solubilized enzyme or at 25 ° with the membrane-bound enzyme and is terminated by the addition of 1 ml of cold HCI04 and 1 ml of Norit A charcoal suspension. After 2 hr at 0 ° with occasional shaking, the mixtures are filtered on Millipore filters (0.45 t~m) and the charcoal is washed with five 10-ml portions of water. The filters are transferred to glass vials and dried at 100°; their radioactivity is determined in a liquid scintillation spectrometer with 5 ml of toluene containing 0.4% Omnifluor (New England Nuclear). The results are corrected by subtracting the radioactivity observed in controls from which L-2,4-diaminobutyrate had been omitted. The assay is linear with respect to enzyme concentration and time for at least 45 min if the exchange does not exceed 0.2 umole. A unit of activity is defined as the amount of enzyme that catalyzes a 3~PP~-ATP exchange of 1 umole per minute under these conditions.
Enzyme Preparation Reagents Medium for starter cultures. Per liter: glucose, 10 g; Bacto yeast extract, 5 g; ammonium sulfate (Schwarz-Mann enzyme grade), 20 g; K,,HPQ, 2.6 g; MgSO~-7H20, 0.5 g; NaC1, 50 rag; and FeSO.7H20, 10 mg Growth medium. Per liter: ammonium sulfate (Schwarz-Mann enzyme grade), 42 g; MgSO~.7H,_,O, 0.2 g; NaC1, 0.1 g; CaCI~, 0.1 g ; FeSO4- 7H~O, 10 mg; ZnSQ 10 mg; MnSO~. H..,O, 7.5 mg; biotin, 0.5 ug; 0.5 M potassium phosphate, pH 7.5, 40 ml; and glucose (500 g/liter), 10 ml (added separately as a sterile solution to the autoclaved medium) Buffer A: 10 mM Tris.HC1, pH 7.5; 10 mM KC1; 2 mM EDTA and 2 mM 2-mercaptoethanol Buffer B: 10raM Tris.HC1, pH 7.5; 10 mM KC1; 2 mM MgCI~ and 2 mM 2-mercaptoethanol Pancreatic deoxyribonuclease I (crystalline) Egg white lysozyme (twice crystallized) Potassimn phosphate, 5 raM, pH 7.5 Growth of Bacteria. 1,6 The bacterial strain used is a well sporulating derivative of B. polymyxa Pfizer 2459, selected by passing the culture through several sporulation cycles. The strain is best maintained at --15 ° as a suspension of heat-activated spores. Starter medimn is inoculated with spores and, after overnight growth, 5 ml are transferred to 1 liter of growth medium in a 2-liter Erlenmeyer flask on a rotary shaker at
582
[441
ANTIBIOTIC BIOSYNTHESIS SUBCELLULAR D~STmBUTION OF AMINO ACID ACTIVATING ENZYMI~;S
Activation of Method of c e l l disruption Assayed at 25° Procedure A (lysozyme lysis) Assayed at 37° Procedure B (sonication)
Subcellular fraction
L-Diaminobutyrate
Soluble Particles Washed particles
1.6 1.8 1.1
Soluble Particles
8.7 1.5
L-Leucine L-Methionine
Units per g of cells 6.2 1.3 0.06 27 2.3
0.5 0.7 0.05 18 0.7
37 ° . Near the end of the exponential phase of growth, the cells are harvested by centrifugation at 4 ° and washed once with the buffer to be used for cell disruption. Preparation o] Extracts. 1 The subcellular distribution of the L-2,4diaminobutyrate activating enzyme depends on the method of cell disruption. Mild lysis of protoplasts prepared with lysozyme (Procedure A) yields primarily membrane-bound enzyme, which can be easily separated from most other amino acid activating enzymes by washing of the membrane fragments. In contrast, ultrasonic disruption (Procedure B) leads to almost complete solubilization of tile L-2,4-diaminobutyrate activating enzyme, which can then be purified further by conventional enzyme fractionation procedures. Procedure A. Freshly harvested cells of B. polymyxa (1 g) are suspended in 50 ml of buffer A and treated with lysozyme (25 mg) at 25 ° for 20 min. The suspension is cooled to 4 ° and centrifuged at this temperature for 20 min at 30,000 g to yield a soluble fraction and a particulate fraction. The latter is resuspended in 25 ml of buffer A and centrifuged again at 30,000 g for 20 min. The particles are then suspended in 25 ml of buffer B and treated with 1 mg of pancreatic deoxyribonuclease for 10 min at 25 °. The sample is again cooled to 4 ° centrifuged and washed (with buffer B) as before, and the final membrane preparation is then resuspended in a small volume of buffer B. As shown in the table, this fraction contains about one-third of the total L-2,4-diaminobutyrate activating activity and only very low levels of other amino acid activating enzymes. Procedure B. All steps are carried out at 4 °. Washed cells of B. polymyxa, either freshly harvested or after storage at --15 °, are suspended in 3 volumes of 5 m M potassium phosphate buffer, p H 7.5, and
[44l
POLYMYXIN SYNTHETASE
583
sonicated in 10-ml portions for 10 min each with an MSE model 60 ultrasonic disintegrator. After centrifugation at 30,000 g for 20 min, most of the L-2,4-diaminobutyrate activating activity, together with other amino acid-activating enzymes, is found in the supernatant fraction (see the table). A 3-fold purification can be achieved without loss of activity by centrifugation at 100,000 g for 90 rain, followed by precipitation of the enzyme by addition of solid ammonium sulfate (25 g/100 ml). When the ammonium sulfate precipitate is subjected to gel filtration on Sephadex G-200 in 5 mM potassium phosphate, pH 7.5, the a-2,4-diaminobutyrate-activating enzyme emerges near the void volume of the column while most other amino acid-activating enzymes elute somewhat later. The preparation so obtained is essentially free of the activating enzymes for basic amino acids and is therefore suitable for the assay of L-2,4-diaminobutyrate in mixtures of basic amino acids. 2
Properties Stability. 1 The particulate enzyme preparation obtained by Procedure A is quite unstable at elevated temperatures. In buffer B, its half-life is 5 min at 37 ° and several hours at 25 °. The solubilized enzyme (Procedure B) is relatively stable, even at 37% and has been stored in 40% ethylene glycol at --15 ° for a year without loss of activity. This contrasts with the extreme lability of the enzyme from other polymyxin-producing organisms (B. polymyxa ATCC 10401 and B. circulans ATCC 14040), where sonication leads to complete destruction of enzyme activity and enzyme preparations obtained by milder methods of cell disruption (alumina grinding or Hughes' press treatment) are completely inactivated upon overnight storage at 4 ° . Molecular Properties. 1 On isopycnic centrifugation in sucrose gradients, the particulate L-2,4-diaminobutyrate-activating enzyme obtained after protoplast lysis (Procedure A) bands at a density of 1.20-1.28 g/ml, indicative of an association with the cytoplasmic membrane. The soluble enzyme (Procedure B) appears polydisperse upon zone sedimentation in sucrose gradients, with an average molecular weight of about 300,000. After treatment with streptomycin, the soluble enzyme yields a sharper peak on sucrose gradients at a position corresponding to a molecular weight of about 100,000, but, on account of its instability, this form of the enzyme has not been studied further. Catalytic Properties. 1,~ The apparent K,,, for L-2,4-diaminobutyrate is 0.6 mM for both the solubilized and the membrane-bound enzyme. No
H. Paulus, unpublished experiments.
584
ANTIBIOTIC BIOSrNTHESlS
[44]
reaction is observed with related compounds such as D-2,4-diaminobutyrate, b-ornithine, and L-lysine. L-2,4-Diaminobutanol is a competitive inhibitor with a K~ of 1.5 raM. No pyrophosphate exchange is observed with GTP, UTP, or CTP in the place of ATP. The enzyme has an absolute requirement for Mg 2÷ ion, with maximum activity at 5-10 mM Mg 2+. Its pH optimum is at 7.0. Other Considerations Since other steps in the biosynthesis of polymyxin have not yet been elucidated, it is not possible to define the place of the L-2,4-diaminobutyrate activating enzyme in the biosynthetic scheme at this time. Nevertheless, it is possible to make a comparison with the biosynthesis of gramicidins and tyrocidine,9 which also involves the activation of the constituent amino acids by specific enzymes. These enzymes catalyze the formation of the appropriate aminoacyl adenylates, from which the amino acid is then transferred to a sufhydryl group on the enzyme. The activating enzymes thus have two catalytic functions, the formation of aminoacyl adenylates and of aminoacyl thiol esters, which are reflected in the amino acid-dependent exchange of ATP with PPi and of ATP with A M P . ~° While the L-2,4-diaminobutyrate activating enzyme catalyzes an ATP-PPi exchange, attempts to demonstrate a diaminobutyrate-dependent exchange of ATP with AMP have been unsuccessfulJ The failure to observe such an exchange reaction might imply that thiolester formation does not occur on the activating enzyme itself but, rather, that the enzyme-bound diaminobutyryl-AMP is the substrate for a separate diaminobutyryltransferase. In this case, the role of the L-2,4-diaminobutyrate activating enzyme would be analogous to that of the D-alanine activating enzyme in the synthesis of teichoic acid by L a c t o b a c i l l u s c a s e i Y
8This volume [43]. This volume [45]. 1oF. Lipmann, Science 173, 875 (1971). 1~R. Linzer and F. C. l'Teuhaus,J. Biol. Chem. 248, 3196 (19737.
[45[
TYROCIDINE SYNTHETASE SYSTEM
5S;~
[45] Tyrocidine Synthetase System B y Su~'G G. LEE and FRITZ LIPMANN
I. Biosynthesis of Tyrocidine During our work on the biosynthesis of gramicidin S by another Bacillus brevis (see Zimmer and Laland, this volume [43]), the synthesis of which is, in prineiple, quite analogous to that of tyrocidinc, it was first observed t h a t the activation of amino acids in this antibiotic polypeptide synthesis was a two-phase process. 1,2 Pyrophosphate exchange with A T P t h a t was dependent on amino acid activation had already been described by Kurahashi, :~ but we soon realized t h a t the exchange reaction indicating an aminoaeyl-adenylate formation, as in the activation for ribosomal synthesis, was followed by a secondary transfer. I t was then found t h a t using [ 3 H ] A M P and 14C-labeled amino acids for the amino a e i d - A T P reaction, the Sephadex G-50 filtrate contained two kinds of amino acids, only half of which was obviously bound to 13H]AMP as it was lost on triehloroaeetie acid precipitation together with [:~H]AMP. "-,4 The trichloroaeetie acid precipitate, however, contained the other half of the ['~C]amino acid, and tests for its binding to the protein showed it to be linked in thioester linkage. This observation seems to have been the first clear indication of the amino acids being linked by thioester bonds to the enzyme protein before polymerization. Polymerization apparently occurred without release of the terminal amino acid, since enzyme-bound polypeptides, isolated first by Laland and his collaborators ~ and then by Gevers et at.,'-' were shown to be linked by thioester bonds to the enzymes. The similarity between this type of activation and t h a t of acetate and of growing f a t t y acid chains in f a t t y acid synthesis to protein-bound pantetheine, 6,~ suggested ~H. Kleinkauf, W. Gevers, and F. Lipmann, Proc. Nat. Aead. Sci. U.S. 62, 226 (1968). z W. Gevers, H. Kleinkauf, and F. Lipmann, Proc. Nat. Acad. Sci. U.N. 63, 1335 (1969). H. Itoh, M. Yamada, S. Tomino, and K. Kurahashi, d. Biocl~ern. (Tokyo) 64, 259 (1968). W. Gevers, H. Kleinkauf, and F. Lipmann, Proc. Nat. Acad. Sci. U.S. 60, 269 (1968). 5T. Ljones, O. Vaage, T. L. Zimmer, L. O. FrOholm, and S. G. Laland, FEBS Lctt. 1, 339 (1968). " P. N. Majerus, A. W. Alberts, and P. R. Vagelos, Proc. Nat. Acad. Sci. U.S. 53, 410 (1965). F. Lynen, D. Oesterhelt, E. Sehweizer, and K. Willeeke, i~* "Cellular Compartmentalization and Control of Fatty Acid Metabolism" (F. C. Gran, ed.), p. 1. Universitetsforlaget, Oslo, 1968.
586
ANTIBIOTIC BIOSrNTHESIS
~ediate
@
enzyme
/ D~-Phe ,---! I
\,,o
•
[45]
q.Tjs
DLPhe\ ,, \ . . ~
./]
eovy enz~0 FIG. 1. Structure of tyrocidine. that pantetheine might possibly be involved in the polymerization reaction of amino acids in antibiotic synthesis. Using an inefficient procedure for pantetheine determination, our early attempts to find it were frustrated2 However, in the meantime, Laland and his group 9 had found pantetheine in the gramicidin S system; they made the important observation that, only in the larger enzyme, 1 mole of pantetheine was present. Most likely, then, pantetheine was involved in the polymerization reaction since this enzyme had been found to thioesterify four amino acids which only polymerized after induction by the smaller phenylalanineactivating enzyme. Such a function of pantetheine in polymerization was preliminarily indicated by experiments with gramicidin S and tyrocidine by comparing the location of amino acid marker with that of pantothenic acid in enzyme systems charged with single amino acids and polypeptides. Fragmentation of the charged enzymes with pepsin after the reaction showed coincidence of pantothenic acid in the fragments containing peptides but not in those containing single amino acids. 1° More definitive experiments on the functioning of pantetheine and the isolation of pantetheine-peptidyl-carrier proteins from the two polyenzymes involved in tyrocidine synthesis has now confirmed the peptidyl carrier function. 11 The isolation procedure of a peptidyl carrier fragment from the intermediate and heavy enzymes for tyrocidine synthesis will be described in the second part of this report; the tyrocidine-synthesizing system will be described in detail in the first part. In summary, the synthesis of the cyclic decapeptide (Fig. 1) is obtained by the assembly of three separable 8It. Kleinkauf, W. Gevers, R. Roskoski, Jr., and F. Lipmann, Biochem. Biophys. Res. Commun. 4, 1218 (1970). gS. G. Laland, O. Frfyshov, C. Gilhuus-Moe, and T. L. Zimmer, Nature (London) New Biol. 239, 43 (1972). 1oI-I. Kleinkauf, R. Roskoski, Jr., and F. Lipmann, Proc. Nat. Acad. Sci. U.S. 68, 2069 (1971). 11S. G. Lee and F. Lipmann, Proc. Nat. Acad. Sci. U~. 71, 607 (1974).
[45]
TYROCIDINE SYNTHETASE SYSTEM
587
enzymes with molecular weights of 100, 230, and 440 X 103. They activate, respectively, one, three, and six amino acids, indicated by the brackets in Fig. 1, which are added in the direction of the arrows to form a thioester-linked growing polypeptide chain beginning with D-phenylalanine. The decapeptide is released from the enzyme by ring closure between Leu ~ S- E and the H~N. Phe at the N-terminal end of the enzymebound chain.
Growing Procedure for Obtaining Enzyme-Rich B a c i l l u s b r e v i s Materials 1. Germination medium: 1% skim milk, 0.1% yeast extract, 0.05% K2HPQ, 0.05% KH~PO4, 0.05% MgSO4.7H20, 0.01% NaC1, 0.002% CaCI2"2H20, 0.001% MnSO4.4H~O, and 0.001% FeSQ. 7H20 in distilled water. 2. Growth medium: 1% Bacto-peptone (Difco), 1% beef extract (Difco), and 0.25% NaC1 in distilled water. The pH of the medium is adjusted to 7.0 with KOH. 3. [l~C]Ornithine (New England Nuclear Corp.)
General Procedures. A batch of about 2 mg of spores of B. brevis (ATCC 8185), either in lyophilized form or in colonies on an agar slant, is transferred to 200 ml of the germination medium in a 1000-ml flask and incubated for 8-10 hr at 37 ° in a New Brunswick Controlled Environment Incubator Shaker at 300 rpm to convert the spores into vegetative cells. For small-scale growth, 2 ml of the vegetative cell culture is transferred to several 2-liter flasks, each containing 500 ml of growth medium, and incubation is continued at 37 ° in the rotary shaker at 300 rpm. For larger scale growth, a New Brunswick Fermentor, model CMG-314, is used. About 50 ml of the vegetative cell culture inoculum is used per 10 liters of growth medium, and the cells are grown at 37 ° with vigorous stirring and aeration. For a good yield of active enzymes it is crucial to harvest the organism at the time of maximum enzyme production, and keen attention should be paid to the detection of this time. For this purpose, the growth of the organism is monitored by measuring the optical density (OD) of the bacterial suspension, usually diluted 10-fold with 1% NaC1, and the density is measured at 30-min intervals at 600 nm. Beginning at the time when the OD of the culture reaches 1.5, 2 ml of tile culture is taken at 30-min intervals and incubated in a 50-ml flask with 1 tLCi of [14C]ornithine for 10 rain at 37 ° in the rotary shaker at 300 rpm. A
588
ANTIBIOTIC BIOSYNTHESIS
[45]
l0 8 6 4 E c 3 O
$2 a o
1.0 - 0.8 / i 0.6
~ o.4 ~ 0.3 0.2 0.12 i
4
6
8
IO
12
Time o f f e r inoculefion, hr
FIG. 2. Cell growth (E] [~), tyroeidine synthesis (0' ornithine uptake (O---O, epm X 10-').
O, cpm X 10-s), and
0.5-1.0-ml aliquot is withdrawn and the cells are collected on a 0.80 ~m pore Millipore filter. The filter is then washed in three 2-ml portions of the growth medium and dried quickly, and the radioactivity is counted. As shown in Fig. 2, the uptake of [14C]ornithine by the cells remains near background level, more or less in parallel with the cell density, until the appearance of tyrocidine-producing enzymes. When these enzymes appear, the capacity for ornithine uptake increases abruptly. At that time, the cells are chilled rapidly with crushed ice and harvested by centrifugation. Any delay in the chilling process causes loss of active enzymes. Generally, about 30-40% of the total ornithine is absorbed in 10 min at the peak of uptake; a maximum yield of enzymes is obtained when the cells are harvested at the time when about 10-15% of the total ornithine is taken up in 10 min. At first, the ornithine-dependent PPi-ATP exchange was measured in a cell homogenate as indicator for tyrocidine synthesis, until the abrupt upshift of the ornithine uptake was found to present a faster assay. The Enzymic Synthesis of Tyrocidine
Reagents 1. Amino acid stock solution: 20 mM D-phenylalanine, L-proline, and L-ornithine to test for the 100,000, 230,000, and 440,000 molecular weight enzymes, respectively
[451
TYROCIDINE SYNTHETASE SYSTEM
589
2. 32PPi stock solution: 10 mM PP~ (pH adjusted to 7.7) containing 20 ~Ci of ~PP~ per milliliter 3. ATP-buffer stock solution: 10 mM ATP, 50 mM MgCl~, 2.5 mM EDTA, 5 mM dithiothreitol, 100 mM triethanolamine buffer (pH 7.7), and 25% sucrose 4. Bovine serum albumin solution: 0.4% bovine serum albumin in 0.01 N NaOH 5. Charcoal suspension: mix 50 ml (wet volume) of acid-washed activated charcoal with 250 ml of 14% perchloric acid containing 0.4 M PPi ; add water to make up 1 liter. 6. PP~ washing solution: 0.1 M PP~, pH adiusted to 8.0 with HCI 7. Tyrocidine-constitutivc amino acid mixture: 1 mM each of L-phenylalanine, L-asparagine, L-glutamine, L-tyrosine, L-valine, L-ornithine, L-leucine, and [~4C]proline (20 ~Ci/ml)
Assay Procedures
AYP-PP~ Exchange. The reaction medium is prepared by mixing one part each of amino acid stock solution, 32PPi stock solution, bovine serum albumin solution, and ATP-buffer stock solution. When mixing these solutions, the 32ppi solution is added immediately before use because precipitation of Mg2P207 occurs from the medium after a long period of standing. The exchange reaction is carried out for 15 min by incubating 0.025 ml of enzyme with 0.1 ml of reaction medium at 37°; the reaction is stopped by adding 0.5 ml of charcoal suspension to the reaction vessel. The charcoal is collected on a glass filter; the filter is then washed with five 3-ml portions of 0.1 M pyrophosphate washing solution, followed by two 3-ml portions of water, and is dried and the radioactivty counted. For crude preparations, dilute solutions should be used since the exchange activity tends to decline in concentrated preparations. This is probably due to ATPase because a new addition of ATP reestablishes the exchange. Tyrocidine Synthesis. After Sephadex G-200 filtration (see below), a 0.15-ml mixture of the three complementary enzymes of tyrocidine synthesis is added to a reaction vessel containing 0.05 ml of tyrocidine-constitutive amino acids, including 1 ~Ci of [14C]proline (reagent 7) and 0.05 ml of ATP-buffer stock solution (reagent 3). The mixture is incubated for 30 rain at 37°; at the end of the reaction, 5% triehloroacetic acid (TCA) is added, and the precipitate is collected on a Millipore filter (0.45 t~m pore). The filter is washed with five 2-ml portions of 5% TCA, then dried, and the radioactivity is counted. For the blank, in place of the complete reagent 7, [14C]proline only is used in the reaction. In the latter case, the radioactivity incorporated into the TCA precipitate in
590
ANTIBIOTIC BIOSYNTHESIS
[45]
the blank represents the small amount of [~4C]proline thioesterified to the intermediate enzyme. This method is good for orientation, but for a more reliable measure, tyrocidine should be separated from the reaction mixture by extracting it with an equal volume of n-butanol:chloroform (4:1, v/v) and chromatographing it on a silica thin-layer plates using ethyl acetate:pyridine:acetic acid:water (90:30:16:9, v/v). The exact proportion of the three enzymes for a maximum rate of tyrocidine synthesis has not been established. It is observed, however, that a combination of a catalytic amount of the light enzyme and an excess of intermediate over heavy enzyme gives a much higher rate of tyrocidine synthesis than an equimolar mixture of the enzymes.
Purification of Tyrocidine-Synthesizing Enzymes
Preparation o/Extracts A batch of 80 g of frozen cells is broken up and thawed by blowing air over it at room temperature. The paste is suspended in 300 ml of 20 mM triethanolamine buffer, pH 7.7, containing 0.5 mM EDTA and 1 mM dithiothreitol (buffer B), and to this is added 80 mg of lysozyme in 20 ml of buffer B. The cells are lysed by incubation of the mixture for 5-10 min. The cells harvested at the peak of tyrocidine-synthesizing activity lyse within a few minutes, but older cells require a longer time. To the viscous mixture of the cell lysate, 50 tLg of DNase and 2 ml of 1 M MgC12 are added, and it is incubated for 1-2 min at room temperature until the disappearance of viscosity. Prolonged incubation of the lysate, especially after the addition of DNase, should be avoided since this causes dissociation of polyenzymes into inactive subunits. The lysate is chilled rapidly to 4-6 ° and centrifuged for 15 rain at 20,000 g. Solid ammonium sulfate is added to the supernatant to 33% saturation, and the precipitate is removed by centrifugation at 20,000 g for 10 min. More ammonium sulfate is added to 45% saturation, and the precipitate is again collected by centrifugation at 20,000 g for 10 min. The pellets are dissolved in buffer B (about 100-150 mg of protein per milliliter of buffer), and insoluble material is removed by centrifugation. When a cell lysate prepared from older cells is subjected to centrifugation, much of the enzyme activity precipitates with cell debris. This activity can be partly recovered by repeating washing.
Sephadex G-200 Filtration The 33-45% ammonium sulfate fraction is applied to a Sephadex G-200 column equilibrated with buffer B containing 0.1 M KC1 and is
[45]
TYROCIDINE
¢~
IOO
."'.~,
d;
,~
¢~ x:J
:
;
?
:
SYNTHETASE
A
~--Protine
:"i
•..
.."
* L I
k
D
,.'"%.,
".
"'.
Ormthine
:
I
~o
ohenylo~onine ".
"', ",
t~
! ].j
591
SYSTEM
0CO
'.
°'z x.
f
°
50
IO0 Froction
IbO
200
number
Fro. 3. Separation of the three complementaryfractions, heavy (HE), intermediate (IE), and light (LE) enzymes,on Sephadex G-200 chromatography. eluted with the same solution. For 2 g of protein, a column of 60 X 7 cm gives a satisfactory resolution of enzymes and an adequate flow rate. As shown in Fig. 3, the heavy and intermediate enzymes are located in the first and second peaks by measuring L-ornithine- and L-proline-dependent ATP-PPi exchange activities; the third and fourth peaks are assayed by D-phenylalanine-dependent ATP-PPi exchange. The third peak corresponds to the light enzyme, and the fourth peak, which may be bigger than shown in the figure, contains dissociation products of the polyenzymes. To the heavy, intermediate, and light enzyme peak fractions, solid ammonimn sulfate is added to 50% saturation, and precipitates are collected by centrifugation. The pellets are dissolved in a small volume of buffer B and passed through G-50 columns equilibrated with the same buffer. Sucrose is added to the enzyme solutions eluted from the columns to 5% ; the solutions are stored in liquid nitrogen until used for further purification.
Purification o] the Light Enzyme The light enzyme peak from G-200 chromatography is applied to a 12 X 2 cm DEAE-cellulose column equilibrated with buffer B containing 5% sucrose. The column is eluted with 100 ml of buffer B containing 5% sucrose and 0.1 M KC1, then further eluted with 500 ml of a KC1 gradient (0.1-0.4 M) in buffer B containing 5% sucrose. The light enzyme, which elutes between 0.16 and 0.18 M KC1, is assayed by measuring D-phenylalanine-dependent ATP-PPi exchange activity, and the enzyme
592
ANTIBIOTIC BIOSYNTHESIS
[45]
solution is concentrated with a Diaflo ultrafiltration apparatus to a few milliliters. The enzyme, which is about 30% pure at this stage, may be purified to homogeneity by hydroxyapatite chromatography and sucrose gradient centrifugation. The enzyme elutes at 0.03-0.04 M phosphate when applied to a hydroxyapatite column (30 X 1 cm) and eluted with a phosphate gradient (0.005-0.1 M), pH 7.3, containing 5% sucrose and 1 mM dithiothreitol. The enzyme solution is concentrated to 0.5 ml by means of the Diaflo apparatus, and 0.1 ml is layered on each of three 10-30% sucrose gradients in buffer B and 0.1 M KC1, which are then centrifuged for 7 hr at 50,000 rpm in a Beckman SW 50 rotor. Under these conditions, the peak activity of this enzyme sediments a little more than one quarter from the top.
Purification of the Intermediate Enzyme The intermediate enzyme from Sephadex G-200 chromatography is applied to a hydroxyapatite column equilibrated with 10 mM phosphate buffer, pH 7.3, containing 5% sucrose and 1 mM dithiothreitol, and the column is eluted with phosphate gradient (0.01-0.15 M), pH 7.3, containing 5% sucrose and 1 mM dithiothreitol. For 200-800 mg of protein, a 50 X 2 cm-column and a l-liter gradient are used. The intermediate enzyme elutes at 0.09-0.11 M phosphate when a 50-cm column is used, but at 0.07-0.08 and 0.05-0.06 M, respectively, when 20-cm and 12 cm columns are used, indicating that the elution concentration depends on the length of the column. The intermediate enzyme eluted from the column is located by measuring L-proline-dependent ATP-PPi exchange activity and is concentrated to a few milliliters by means of the Diaflo apparatus; this is then passed through a Sephadex G-50 column equilibrated with buffer B and applied to a DEAE-cellulose column (12 X 2 cm) equilibrated with buffer B containing 5% sucrose. The column is eluted with 50 ml of buffer B containing 5% sucrose and 0.1 M KC1, and then further eluted with 500 ml of KC1 gradient (0.1-0.5 M) in buffer B and 5% sucrose. The enzyme that comes off the column between 0.23 and 0.25 M KC1 is concentrated with the Diaflo apparatus to 0.5 ml, and 0.1 ml of this is layered on each of three 10-30% sucrose gradients in buffer B and 0.1 M KC1; the gradients are centrifuged for 7 hr at 50,000 rpm using a Beckman SW 50 rotor. Under these conditions, the intermediate enzyme sediments a little less than half way from the top.
Purification o] the Heavy Enzyme The heavy enzyme from G-200 chromatography is purified by hydroxyapatite chromatography in a manner identical to that described for
[45]
TYROCIDINE SYNTHETASE SYSTEM
dE
0.01M
[1_
%
50
~-
7D
o
593
4O
0.15M •
" ~ - - O r nithine
I/
~, 50
Io E
c © CO (M
05 5
2o !
I
I
©
10,
o E
20
I00 Froclion
150 number
00
FIG. 4. Hydroxyapatite chromatography of heavy enzyme fraction from Sephadex G-200 chromatography.
the intermediate enzyme. The heavy enzyme elutes at 0.05-0.06 M phosphate (Fig. 4). The elution concentration does not seem to be dependent on the column length. The enzyme solution is concentrated to about 1 ml and subjected to sucrose gradient centrifugation as described for the intermediate enzyme. Under such conditions, the heavy enzyme sediments a little less than two-thirds from the top (Fig. 5). The heavy enzyme eluted from the G-200 column is often turbid. Since the presence of much
,%
£3[3_ c~
to
-?
c c
g
100
_o × E Q.
~~I.
50
0rnithJne
i
"o
~/~/ t. ph~nylalonine
Froction
20 number
50
FIG. 5. Sucrose gradient centrifugation of heavy enzyme fraction from hydroxyapatite chromatography.
594
ANTIBIOTIC BIOSYNTHESIS
[45I
TABLE I PURIFICATION OF THE ~NTERMEDIATE AND HEAVY ENZYMES OF TYROCIDINE SYNTHESIS
Stages
Protein (mg)
Intermediate 1. 20,000 g supernatant 2. Ammonium sulfate (33-45% saturation) 3. Sephadex G-200 4. Hydroxyapatite 5. DEAE-cellulose 6. Sucrose gradient
Specific activity (cpm/mg protein)
Yield (%)
Proline-dependent 26,000 7,400 780 72 11 3.8
Heavy 1. 20,000 g supernatant 2. Ammonium sulfate (33-45% saturation) 3. Sephadex G-200 4. Hydroxyapatite 5. Sucrose gradient
Amino aciddependent ATP-3~PPi exchange (cpm)
3.1 X l0 g 2.4 X 109
1.2 X 105 3.2 X 105
77
1.8 9.3 5.1 2.8
2.3 1.3 4.6 7.4
106 107 107 107
58 30 16 9
1.1 X 109 7.2 >< 108
4.2 X 104 9.7 X 104
65
5.9 X 108 3.1 X 108 1.5 X 108
1.3 X 106 1.2 X 107 2.6 X 107
54 28 14
X X X X
109 108 108 108
X X X X
Ornithine-dependent 26,000 7,400 460 26 "5.8
turbid material interferes with hydroxyapatite chromatography, it is precipitated with ammonium sulfate (50% saturation) and the pellets obtained by centrifugation are dissolved in about 5 volumes of buffer B; the enzyme is passed through 15% sucrose and layered on 40% sucrose by high-speed centrifugation. Usually, 5 ml of 40% sucrose in buffer B and 0.1 M KC1 is layered on the bottom of 38-ml tubes, 10 ml of 15% sucrose in the same buffer solution is layered next, and then the enzyme solution is layered on the top. The tubes are centrifuged for 6 hr at 60,000 rpm using a Beckman 60 titanium fixed-angle rotor. The enzyme sediments through the 15% sucrose and is layered on the 40% sucrose cushion. Table I shows the progress of purification. Acrylamide gel electrophoresis (Fig. 6) indicates the purity of the stage 6 intermediate and stage 5 heavy enzymes to be approximately 90%. In Table II, the amino acids which are activated by the purified enzymes indicate their specificity. With regard to aromatic amino acids, 12 the heavy enzyme shows 12R. Roskoski, Jr., H. Kleinkauf, W. Gevers, and F. Lipmann, Biochemistry 9, 4846 (1970).
[45]
TYROCIDINE SYNTHETASE SYSTEM
595
IE HE
FIG. 6. Gel electrophoresis of intermediate (IE) and heavy (HE) enzymes from sucrose gradient centrifugation. a preference for tyrosine and the intermediate for phenylalanine and tryptophan (data not included). II. Dissociation Products of the Polyenzymes of Tyrocidine Synthesis Of the three complementary enzymes of tyrocidine synthesis, the intermediate and heavy enzymes are polyenzymes, each composed of amino-acid activating subunits of 70,000 molecular weight and of a 4'phosphopantetheine-containing protein of 17,000 molecular weight which carries the growing nascent peptides. The intermediate enzyme (230,000 molecular weight), which activates the second, third, and fourth amino acids of tyrocidine, contains three amino acid activating subunits; the heavy enzyme (440,000 molecular weight), which activates the fifth to tenth amino acids of tyrocidine, contains six amino acid activatin~ subunits. The dissociation of these polyenzymes may be achieved either by
596
ANTIBIOTIC BIOSYNTHESIS
[45]
TABLE II AMINO ACID-DEPENDENT ATP-a~PPi EXCHANGE WITH THE PURIFIED ENZYMES OF TYROCIDINE SYNTHESIS a
[3*P]ATP formed (nmoles) Amino acids
Light
Intermediate
Heavy
~Phenylalanine D-Phenylalanine I~Proline L-Asparagine L-Glutamine L-Tyrosine ~Valine L-Ornithine L-Leucine
34.3 22.4 0 0 0 NM b 0 0 0
12.3 2.1 30.2 0 0 2.7 0 0 0
2.2 0 0 20.0 11.6 13.6 38.2 15.4 24.8
The last stages of purified light enzyme (1.6 ~g), intermediate enzyme (2.4 ug), and heavy enzyme (4.1 ~g) were incubated for 15 min at 37° with 4 mM of the designated amino acids and 2 mM ATP, 2 mM ~2PPi (0.32 #Ci), and 5 mM KC1 in a reaction volume of 0.1 ml. The exchanges were assayed as described in the text. The activation of L-tyrosine by the intermediate, and L-phenylalanine by the heavy enzyme, indicate the interehangeability of aromatic amino acids, although with ~ selective affinity [Reprinted with permission from R. Roskoski, Jr., H. Kleinkauf, W. Gevers and F. Lipmann, Biochemistry 9, 4846 (1970) Copyright by the American Chemical Society]. b Not measured. extended incubation of crude bacterial extracts or by incubating the purified polyenzymes with a Triton extract made from the insoluble fraction of the bacterial lysate.
Procedure/or Dissociating Polyenzymes 13 Dissociation o/Polyenzymes in Crude Extracts. A batch of 5-100 g of frozen B. brevis (ATCC 8185) cells is thawed and suspended in 4 volumes (w/v) of buffer B, and the cells are lysed by incubation at 37 ° for 5-10 min with lysozyme (1 m g / g cells). To the lysate are added D N a s e (1/~g per gram of cells) and MgCl~ (5 m M final concentration), and the mixture is incubated for 30-60 min. Autolysis of the polyenzymes is more pronounced after addition of the DNase. Figure 7 shows a sucrose gradient analysis of the progress of autolysis of the h e a v y enzyme with 1'S. G. Lee, R. Roskoski, Jr., K. Bauer, and F. Lipmann, Biochemistry 12, 398 (1973).
[45]
SYNTHETASE SYSTEM
TYROCIDINE
597
70
o x: o
D
m
60
a_: ?
13-
"n
'
50 ?%" - -
8-
×
<
E
E
~
g c
g
40
so
2o
'~ .
{J
/'"~ c
(
e
, l", ,=..:, , . , .L
i
~c
!!
;!
l
io
\
~.: .... ' /
5 rain incub
\ ",'.
,5
....
•
6
....
I0
20 Fr0cti0n
30
number
FIG. 7. Dissociation of heavy enzyme. To the lysate of B. brevis cells, DNase and MgCI~ were added and the mixture was incubated for 5, 15, and 40 additional minutes. From the incubation mixtures taken at various times, 20,000 g supernatant fractions and 50% ammonium saturation ammonium sulfate cuts were made. These were dissolved in buffer B and subjected to 10-30% sucrose gradient centrifugation at 50,000 rpm for 7 hr using the SW 50 rotor. Forty-three fractions of 0.13 ml were collected (top at left, bottom at right) and 50 #1 from each was assayed for L-ornithine-dependent ATP-PPI exchange as described. Peaks A, B, C, and D correspond to approximate molecular weights of 440,000, 230,000, 150,000, and 70,000, respectively.
time of incubation after addition of DNase. During the autolysis, the 440,000 polyenzyme, peak A, is progressively transformed to trimers, dimers, and monomers, corresponding to molecular weights of 230,000, 150,000, and 70,000 (peaks B, C, and D). Prolonged incubation eventually converts most polymers to the monomeric forms of the subunits, as shown by the increase of peak D at the expense of A, B, and C. Dissociation of Purified Polyenzymes. In contrast to crude homogenates, the incubation of purified polyenzymes at 37 ° does not elicit separation into subunits. They can be dissociated, however, by addition of a factor extracted with Triton X-100 from the 20,000 g pellet of crude extract. To obtain this factor, B. brevis cells are lysed and the crude extract is treated with DNase. The resulting homogenate is centrifuged at 20,000 g as described above. To extract the dissociating factor, the pellet is suspended in 5 ml of buffer B made 2% in Triton X-100 per gram of cells. The suspension is stirred for 30-60 min and then centrifuged for 20 min at 20,000 g. The resulting supernatant is used as a source of polyenzyme-dissociation factor. Table III shows dissociation of purified heavy enzyme at different concentrations of dissociation factor.
598
ANTIBIOTIC BIOSYNTHESIS
[451
TABLE III DISSOCIATION OF HEAVY ENZYME BY TRITON EXTRACT OF CELL DEBRIS a Ornithine-dependent ATP-~2PPI exchange (cpm)
Volume Triton extract (~I) 0 10 50
Subunit fraction (70,000 MW) 0 13,500 27,500
Heavy enzyme fraction (440,000 MW) 67,500 48,500 29,000
About 70 ~g of heavy enzyme, purified as described in Part I, was incubated at 37 ° in 0.1 ml at p H 7.4 with different amounts of Triton extract. After incubation, the mixtures were layered in 10-30% sucrose gradients and centrifuged for 7 hr at 50,000 rpm using the SW 50 rotor to separate subunits and heavy enzyme.
Purification o] Dissociation Products Purification o] Amino Acid Activating Subunits. The amino acid activating subunits obtained by either of the two methods described above may be purified by chromatography on Sephadex G-200, DEAE-cellulose, or hydroxyapatite in a manner similar to that described for purification of the polyenzymes. The subunits elute from Sephadex G-200 at about 2.5 times void volume (see Fig. 3), and from DEAE-cellulose and hydroxyapatite at 0.13 M KCI and 0.03 M phosphate, respectively. These subunits are located throughout by measuring ATP-PP~ exchange activity as described earlier. In all of the above procedures, the different subunits elute together, suggesting their similarities in physical and chemical properties, although slight differences in their molecular weights were observed on sodium dodecyl sulfate electrophoresis (Fig. 8). They activate amino acids and bind them in thioester linkage to enzymic thiol groups, but their mixture is unable to polymerize the bound amino acids. Purification o] Peptidyl Carrier Protein ]rom Crude Extracts. As this protein contains 4P-phosphopantetheine and carries the growing nascent peptides, it may be followed during purification by radioactively marking pantetheine and/or nascent peptides. Enzyme-bound pantetheine is labeled by growing the organism with pantothenic acid or fl-alanine. Good incorporation of these markers into pantetheine is achieved only when they are added to the culture at the onset of development of the enzymes of tyrocidine synthesis, which is recognized by the abrupt development in the uptake of ornithine, as shown in Fig. 2. The pantetheine-
[451
TYROCIDINE SYNTHETASE SYSTEM
rOaltonsl
599
I
Cytochromec pY#2Z~11,700
~m,~ Carrier protein
T-Globulin ,v~,~ 23,500 light chain
Ovalbumin
~ 430,00
Serurnalburnin ",,'"~ bovine
68,GO0
~Subunits i
Standards
m lJ Undissociated 250,000 440,000 polyenzyrne
Fro. 8. Sodium dodecyl sulfate gel electrophorems of purified intermediate and
heavy enzymes. The polyenzymes partly dissociate to subunits of approximately 70,000 molecular weight, corresponding to the number of amino acids activated (see Fig. 1). The intermediate enzyme yields three evenly stained bands, and the heavy enzyme five bands, one of which is more heavily stained indicating overlap of the two, or a total of six bands.
labeled cells are chilled quickly, harvested as described above, and lysed with lysozyme. To label the carrier protein with nascent peptides, the lysate is incubated for 30-60 rain at 37 ° with the tyrocidine-constitutive amino acids, including one radioactive amino acid (reagent 7) but omitting leucine to retain enzyme-bound polypeptide. DNase is added to this mixture, and it is incubated for an additional 30-60 min to dissociate the polyenzymes. The lysate is then centrifuged at 20,000 g for 20 min, and an ammonium sulfate cut between 33 and 77% saturation is made from the supernatant. The ammonium sulfate fraction is dissolved in buffer B and applied to a Sephadex G-100 column. As shown in Fig. 9, three doubly marked peaks are generally obtained. The excluded peak A presumably contains undissociated polyenzymes, and peaks B and C, with respective molecular weights of 90,000 and 35,000, contain aggregates of peptidyl carrier protein. Peak D, which contains only pantothenic acid and has a molecular weight of about 10,000, is possibly the acetyl carrier protein of Vagelos. m14 Peaks B and C can be more highly purified by DEAE14p. Goldman, A. W. Alberts, and P. R. Vagelos, Biochem. Biophys. Res. Commun.
5, 280 (1961).
600
ANTIBIOTIC
BIOSYNTHESIS
[45]
A: Mulfienzyrnes B: Peptidyl corrier protein, polymer C: Pepfidyl terrier protein, dimer D: Acyl corrier protein ? A
300
. . . . . [14C]Pontothenote •
* 3H-Iobeled pepfide
200 3
E Q.
I00
C
~d
,"tl
2'0
Id
I 40
I
I 60
o ~
I 80
groction number
Fie. 9. Sephadex G-100 chromatography of peptidyl carrier protein prepared from crude homogenates. Carrier protein was labeled with ["C]pantothenic acid during growth of the organism and then labeled with [3It]proline peptides. Polyenzymes were dissociated by autolysis, after which a 20,000 g supernatant fraction was made from the lysate and an ammonium sulfate cut between 33 and 70% saturation was made from the supernatant. The ammonium sulfate fraction was then subjected to Sephadex G-100 chromatography.
cellulose chromatography. Figure 10 shows the D E A E - c h r o m a t o g r a p h y of peak C; little protein is eluted with buffer B alone, but this peak contains over 80% of both pantothenate and nascent peptide labels. Most of the protein, but little of the markers appear on subsequent elution with KCI. The low affinity of carrier protein for DEAE-cellulose indicates it to be a basic protein, which is confirmed by the observation that it migrates toward the cathode on paper electrophoresis at neutral pH. Isolation o] Carrier Protein ]rom Purified Polyenzymes. To follow the peptidyl carrier protein during isolation and purification, the polyenzymes are labeled with radioactive nascent peptides in the following manner: (1) purified intermediate enzyme, free of heavy enzyme, is incubated for 1 hr at 37 ° with a catalytic amount of light enzyme in a reaction mixture containing buffer B and [14C]proline, 0.2 m M phenylalanine, 2 m M ATP, and 10 m M MgC12; (2) purified heavy enzyme, combined
TYROCIDINE SYNTHETASE SYSTEM
[45]
601
. . . . . OD
300
=
~ 3H-labeled peptide
.....
[14C]Pon.lothenole
2OO E o_
0.4
-,, I00
--
..
,,
,5
G
0,',
3d
~
"~',
0
,
0,,
+
',
+;
-
io
0
"
+'"
'
O,
03
c
o
C:~
+~
O.I o
i
20 F r o c t i o n number
3'0
FIG. 10. DEAE-cellulose chromatography of peptidyl carrier protein. The peak
of the C fraction of Fig. 9 was applied to a DEAE-cellulose column equilibrated with buffer B containing 5% sucrose. The elution was started with 45 ml of buffer B containing 5% sucrose and continued stepwise with 30 ml each of the same buffer containing 0.1, 0.2, 0.3, and 0.6 M KCI.
with intermediate and light enzymes, is incubated for 1 hr at 37 ° with buffer B, 2 mM ATP, 1 mM MgC12, [14C]asparagine or [14C]glutamine, and 0.2 mM each of the remaining tyrocidine-constitutive amino acids (reagent 7), with the exception of leucine. Low molecular weight substances are then removed by filtering the reaction mixtures through Sephadex G-50 columns. To dissociate them, the labeled polyenzymes eluted from the G-50 columns are incubated for 30-60 min with the Triton X-100 extract described earlier (about 1 ml of Triton extract for up to 50 mg of polyenzymes in 10 ml of reaction volume), and ammonium sulfate is added to 70% saturation. Upon centrifugation, presumably because of the presence of Triton, the labeled carrier protein floats on top of the centrifuge tubes. It is then further purified by Sephadex G-100 and DEAE-cellulose chromatography as described above. When subjected to the former, the labeled carrier protein appears mostly as 90,000 molecular weight aggregates, in contrast to a prominent amount of 35,000 molecular weight aggregates in the Sephadex G-100 chromatography of carrier protein from crude extracts. This may be due to a high concentration of carrier protein in the preparation from purified polyenzymes. Sodium Dodecyl Sul]ate Gel Electrophoresis o] Carrier Protein. Peptidyl carrier protein exhibits a great tendency for form aggregates. These are partially converted to the 17,000 molecular weight monomeric form
602
ANTIBIOTIC BIOSYNTHESIS
[45]
3000
-
2000
70
I I
\
- 50
\
E
1 I
\ \
(9
--
\
rO
\
I
\
-30
_o x
I000
20 ~
_t,
. . . . . . . . .
....
7
IO
20
3O
Slice number
Fro. 11. Sodium dodecyl sulfate gel electrophoresis of peptidyl carrier protein. Purified heavy and intermediate enzymes were labeled with [14C]glutamine- and [l~C]proline-nascent peptides, respectively, and the peptidyl carrier proteins were isolated from polyenzymes. The mixture of the labeled peptidyl carrier proteins from the two polyenzymes was incubated with 2% sodium dodecyl sulfate and 100 mM dithiothreitol in 0.05 M phosphate buffer, pH 7.0, for 1 hr at 100°. The mixture was then subjected to a standard sodium dodecyl sulfate gel electrophoresis [K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969)].
under standard conditions of sodium dodecyl sulfate electrophoresis. 15 Incubation with 8 M urea does not disaggregate high molecular weight carrier proteins. However, preincubation of the aggregates with 2% sodium dodecyl sulfate and 100 mM dithiothreitol at 100 ° for 1 hr converts them quantitatively to the 17,000 molecular weight monomeric form (Fig. 11).
~K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969).
[46]
TETRACYCLINE METHYLTRANSFERASE
603
[45] S-Adenosylmethionine: Dedimethylamino-4aminoanhydrotetracycline N-Methyltransferase By PHILIP A. MILLER and JOHN H. HASH CH3
NH 2 •
2
S-adenosytrnethionine
CONH2 OH
OH
0
0
dedirnethylamlno-4 -amino- anhydrotetmcycllne
CH3
NICH3)~ •
2
S - adenosylhornocysteine
CONH~ OH
OH
0
0
anhydrotetracycline
This is one of a group of enzymes involved in one of the terminal steps in the biosynthesis of the tetracycline group of antibiotics. It is found in various strains of Streptomyces aureo]aciens and S. rimosus, including a wide variety of mutants in which antibiotic production is blocked. Dedimethylamino-4-amino-anhydrotetracycline and a monomethylated intermediate, dedimethylamino-4-methylaminoanhydrotetracycline, can be detected in reaction mixtures, particularly when S-adenosylmethionine is made limiting.1 It is assumed that a single enzyme introduces both methyl groups, but supporting evidence is lacking. The N-methyltransferase activity has been studied only in crude cell-free extracts.
Assay Method
Principle. The presence of N-methyltransferase activity in crude cell extracts can be determined by measuring incorporation of label from [14C]S-adenosylmethionine into the product by the reaction, anhydrotetracycline. The anhydrotetracycline can be readily separated from residual [14C]S-adenosylmethionine by extracting it into o-chlorophcnol after precipitation of proteins with perchloric acid. Unless 0~ is excluded 1p. A. Miller, A. Saturnelli, J. H. Martin, L. A. Mitscher, and N. Bohonos, Biochem. Biophys. Res. Commun. 16, 285 (1964).
604
ANTIBIOTIC BIOSYNTHESIS
[46]
from the reaction mixture, other enzymes present in crude extracts will convert the [14C]anhydrotetraeycline to [~4C]tetracycline. However, the synthesis of [~4C]tetraeycline does not interfere with the determination of N-methyltransferase activity, since the o-ehlorophenol will extract any [~C] tetracycline formed.
Reagents Tris.HCl buffer, 0.2 M, pH 7.0 [14CH3]S-Adenosylmethionine 0.5 mM Dedimethylamino-4-aminoanhydrotetracycline, 0.25 mM in methanol Perchloric acid, 3.5~ Phosphate buffer, 0.1 M, containing 0.1~ of the disodium salt of ethylenediaminetetraacetic acid.
Procedure. A reaction mixture is prepared by combining 0.1 ml of the 0.25 mM solution of the substrate, dedimethylamino-4-aminoanhydrotetracycline, 0.1 ml of the [14CH3]S-adenosylmethionine, 0.3 ml of distilled water, and 0.5 ml of a 20 mg/ml solution of the lyophilized extract in 0.2 M Tris buffer. The reaction mixture is incubated at 28 ° for 2 hr. Proteins are removed by addition of 0.2 ml of 3.5% perchloric acid, and the soluble fraction is then extracted with 0.5 ml of o-chlorophenol. The o-chlorophenol is extracted two times with water and then evaporated to dryness. The dry residue is dissolved in methanol to facilitate transfer to a paper chromatogram or to a vial prepared for liquid scintillation counting. Any standard liquid scintillator is suitable. The radioactivity in the extract is related directly to the extent of transfer of methyl groups from S-adenosylmethionine to dedimethylamino-4-aminoanhydrotetracycline. The identity of the labeled product can be established by paper chromatography using the system described in the section on preparation of dedimethylamino-4-aminoanhydrotetracycline. Preparation of Dedimethylamino-4-aminoanhydrotetracycline. The substrate for the N-methyltransferase can be isolated from cultures of Streptomyces rimosus NRRL 3098. A vegetative inoculum of S. rimosus is prepared in a medium containing in grams per liter: corn steep liquor, 20.0; sucrose, 30.0; (Ni-I~)2SO~, 2.0; and CaCO~, 7.0. Fifty milliliters of this medium in a 250-ml Erlenmeyer flask are sterilized, inoculated with spores of NRRL 3098 and then placed in a reciprocating shaker operating at 110 strokes per minute at 28 ° for 32 hr. Two milliliters of this vegetarive inoculum are used to inoculate 25 ml of the following medium conrained in a 250-ml Erlenmeyer flask: (in g/liter); KC1, 1.23; H3P04 (85%), 0.24; NH4C1, 1.50; (NH4)~SO~, 8.0; CaC03, 10.0; starch, 55.0; MgC12.6H20, 2.0; FeS04.7H20, 0.06; MnS04.4H20, 0.05; COC12.6H20,
[46]
TETRACYCLINE METHYLTRANSFERASE
605
0.005; L-histidine, 0.10; and lard oil, 20.0. The inoculated flask is incubated at 180 rpm on a rotary shaker at 28 ° for 48 hr, at which time 2.0 mg of L-ethionine are added. The fermentation is continued for an additional 48 hr and then harvested. An ethyl acetate extract of the whole fermentation broth is prepared by mixing equal volumes of broth, ethyl acetate, and 1.4% perchloric acid. The organic phase is separated from the mixture and applied to Whatman No. 1 paper strips previously buffered with 0.1 M Na~HP04 containing 0.1% of the disodium salt of ethylenediaminetetraacetic acid and dried. The strips are developed with n-butanol saturated with water for 12 hr. Three orange fluorescent zones are visible under ultraviolet illumination. The dedimethylamino-4-aminoanhydrotetracycline is present at an R r of 0.12. A slower moving component, dedimethylamino-4-methylaminoanhydrotetracycline is found at Rf 0.24 and dedimethylamino-6-demethyl-4-aminoanhydrotetracycline at Rr 0.05. The desired component at Rf 0.12 is eluted from the paper strip with methanol. The yield from 25 ml of fermentation broth is about 10 rag. Methods for the preparation of gram quantities of dedimethylamino-4-aminoanhydrotetracycline have been described. 2 Preparation of the Crude Extract. Streptomyces aureo]aciens ATCC 13,192 is grown ill a corn steep liquor, sucrose, (NH,).2SO,, CaC0:~ medium under conditions identical to those described above. The mycelial growth is separated from the medium by slow speed centrifugation and washed in sufficient distilled water to permit pouring of the mycelial suspension into the cell of a Raytheon 10 ke sonic oscillator. After a 2-min sonic treatment, a soluble fraction is obtained by eentrifugation at 40,000 g for 30 min. Lyophilization of this soluble fraction provides a powder that retains its activity for months if stored at --10% The temperature in all of the above operations is not permitted to exceed 4 ° .
Properties Stability. The lyophilized cell extract is stable for months if stored at --10%
Substrate Specificity. The enzyme methylates the following dedimethyl-amino-4-aminoanhydrotetracycline derivatives: the 7-chloro-, 6demethyl-, 2-decarboxamido-2-nitrile-, and 4-methylamino-. Source of Enzyme. The enzyme can be prepared from any tetraeyeline-producing strain of Streptomyces aureus or S. rimosus as well as various mutants blocked at points prior to the methylation reaction. The latter are preferred since cell extracts from them do not contain any of 2 U.S. P a t e n t 3,265,732 (1966).
606
ANTIBIOTIC BIOSYNTHESIS
[47]
the substrate for the methylation reaction, and controls containing no substrate will therefore show little or no incorporation of label from [ 14CH~] S-adenosylmethionine.
[47] N A D P :Tetracycline 5a (I la)Dehydrogenase By PHILIP A. MILLER and JOHN H. HASH NADPH+H
oH
cl ~,
HO
O
O
N/CH.e.
O
7- Chloro-5a (1la)dehydrotetracycline
+
NADP+
OH
Cl ~.
HO
O
O
N¢C..~.
O
7- Chlorotetracycline
Enzymic reduction of the 5 a - l l a double bond has been shown to be the final reaction in the biosynthesis of the tetracycline group of antibiotics. 1,2 The reaction occurs in antibiotic-producing strains of Streptomyces aureo]aciens as well as in various blocked mutants derived therefrom. Of the various 5 a ( l l a ) - d e h y d r o t e t r a c y c l i n e s which have been prepared, only 7-chloro-5a(lla)-dehydrotetracycline is chemically stable; we have therefore chosen it as the substrate to illustrate the reaction described here. Assay
Method
Principle. The presence of 5 a ( l l a ) dehydrogenase activity can be determined by measuring an increase in antibacterial activity due to the synthesis of 7-chlorotetracycline from its inactive precursor, 7-chloro5 a ( l l a ) - d e h y d r o t e t r a c y c l i n e . Any tetracycline-sensitive bacteria can be used, and either a turbidimetric or agar-diffusion type assay is suitable2 Reagents T r i s . H C l buffer, 0.2 M, pH 7.0 N A D P H , 2 m M in water 7-Chloro-5a(lla)-dehydrotetracycline, 1 m M in methanol 1p. A. Miller, J. H. Hash, M. Lincks, and N. Bohonos, Biochem. Biophys. Res. Commun. 18, 325 (1965). : P. A. Miller, Develop. Ind. Microbiol. 8, 96 (1967). "This volume [4].
606
ANTIBIOTIC BIOSYNTHESIS
[47]
the substrate for the methylation reaction, and controls containing no substrate will therefore show little or no incorporation of label from [ 14CH~] S-adenosylmethionine.
[47] N A D P :Tetracycline 5a (I la)Dehydrogenase By PHILIP A. MILLER and JOHN H. HASH NADPH+H
oH
cl ~,
HO
O
O
N/CH.e.
O
7- Chloro-5a (1la)dehydrotetracycline
+
NADP+
OH
Cl ~.
HO
O
O
N¢C..~.
O
7- Chlorotetracycline
Enzymic reduction of the 5 a - l l a double bond has been shown to be the final reaction in the biosynthesis of the tetracycline group of antibiotics. 1,2 The reaction occurs in antibiotic-producing strains of Streptomyces aureo]aciens as well as in various blocked mutants derived therefrom. Of the various 5 a ( l l a ) - d e h y d r o t e t r a c y c l i n e s which have been prepared, only 7-chloro-5a(lla)-dehydrotetracycline is chemically stable; we have therefore chosen it as the substrate to illustrate the reaction described here. Assay
Method
Principle. The presence of 5 a ( l l a ) dehydrogenase activity can be determined by measuring an increase in antibacterial activity due to the synthesis of 7-chlorotetracycline from its inactive precursor, 7-chloro5 a ( l l a ) - d e h y d r o t e t r a c y c l i n e . Any tetracycline-sensitive bacteria can be used, and either a turbidimetric or agar-diffusion type assay is suitable2 Reagents T r i s . H C l buffer, 0.2 M, pH 7.0 N A D P H , 2 m M in water 7-Chloro-5a(lla)-dehydrotetracycline, 1 m M in methanol 1p. A. Miller, J. H. Hash, M. Lincks, and N. Bohonos, Biochem. Biophys. Res. Commun. 18, 325 (1965). : P. A. Miller, Develop. Ind. Microbiol. 8, 96 (1967). "This volume [4].
[47]
NADP :TETRACYCLINE 5a(11 a)I:)EttYDROGENASE
607
Procedure. A reaction mixture is prepared by combining 0.1 ml of the substrate, 0.1 ml of NADPH, 0.3 ml of water, and 0.5 ml of a 20 mg/ml solution of lyophilized cell-free extract in Tris buffer. The reaction mixture is incubated at 28 ° for 2 hr and assayed for antibiotic activity using a Staphylococcus aureus turbidimetric method. ~ The product, 7-chlorotetracycline, can be identified using the following paper chromatographic system: Whatman No. 1 paper buffered with 0.3 M phosphate, pH 3.0, and developed with n-butanol saturated with water. Preparation o] 7-C hloro-5a (11a )-dehydrotetracycli~w. This substrate can be prepared by fermentation using a blocked mutant of S. aureofaciens, ATCC 12748. A vegetative inoculum of this strain is prepared by inoculating spores into a medium containing, in grams per liter: sucrose, 30; ammonium sulfate, 2; calcium carbonate, 7; and corn steep liquor, 16.5 ml. After incubation on a reciprocating shaker for 24 hr at 28 °, the resulting growth is used to inoculate a medium containing, in grams per liter: ammonium sulfate, 5; calcium carbonate, 9; ammoniun chloride, 1.5; magnesium ehloride.6H,O, 2; ferrous sulfate.7H20, 0.04; magnesium sulfate. 4H.,O, 0.05 ; cobalt chloride. 6H,,O, 0.001 ; zinc sulfate. 7H,_,O, 0.1; corn steep liquor, 25; starch, 55; lard oil, 2; and riboflavin, 0.002. Fermentation is carried out for 120 hr at 25 ° on a rotary shaker operating at 100 rpm. The yields of 7-ehloro-5a(lla~-dehydrotetracycline are in the range of 7-10 g/liter. The product can be isolated by partition chromatography on a Celite eolunm. The charge is prepared by acidifying the fermentation broth to pH 1.5 with cone. HCI, filtering, extracting the filtrate with n-butanol after saturating the filtrate with NaC1, and finally concentrating the butanol extract to a small volume. The Celite column is developed with a mixture of n-butanol/chloroform (80/20) saturated with 0.01 N HCI. Details of the chromatography and crystallization of the product have been described? Preparation o] Crude Enzyme Extract ]rom S. aureo]aciens AT('C 13,192. The crude extract is prepared the same as for S-adenosylmethionine :dedimethylamino - 4 - amino-anhydrotetracycline - N - methyltransferase."
D. C. Grove and W. A. Randall, "Assay Methods of Antibiotics," 238 pp. Medical Encyclopedia, New York, 1955. 5 U.S. Patent 3,007,965 (1962).
eThis volume [46].
[48]
AMINOGI.,YCOSIDE-MODIFYING ENZYMES
611
[48] Aminoglycoside-Modifying Enzymes B y MICHAEL J. HAAS and JOHN E. DOWDING
This article describes the isolation and assay procedures currently used in the study of the nine aminoglycoside-modifying activities listed in the table. It should be emphasized that very little information is available for some of the enzymes listed in the table and for this reason independently isolated activities catalyzing the same reaction have been grouped together (e.g., the three kanamycin acetyltransferases) pending studies showing them to be substantially different enzymes. These enzymes, which modify the aminoglycoside or aminocyclitol antibiotics, have been detected in a wide variety of resistant bacteria. In many clinical isolates they are known to be plasmid-coded, and in certain strains the enzymes appear to be located near the cell surface) a fact which is used to advantage in some of the enzyme isolation techniques described below. Among Eubacteria the three known aminoglycoside-modification mechanisms are acetylation of amino groups and phosphorylation and adenylylation of hydroxyl groups; these mechanisms have recently been reviewed by Benveniste and Davies. 1 Other modified compounds have been detected among antibiotic-producing organisms,2, 3 and it is possible that in these strains these may be biosynthetic intermediates or inactivation products. The structures of the aminoglycoside antibiotics (with positions of modification) are shown in Figs. 1-7. Although an indication of the modification site may often be obtained by examination of the substrate specificity of an enzyme, it has been determined in most cases by chemical and/or spectroscopic analysis of the modified antibiotic (table). The study of these enzymes is particularly valuable for two reasons. First, determinations of the type and site of modification have allowed the design and synthesis of semisynthetic antibiotics not modified by the enzyme and therefore active against many resistant isolates. Second, the enzymes provide a very sensitive, rapid and often specific assay for the aminoglycoside antibiotics2, ~ It is important to remember that many R ]actor-containing strains
1R. Benveniste and J. Davies, Annu. Rev. Biochem. 42, 471 (1973). "~M. K. Majumdar and S. K. Majumdar, Biochem. J. 120, 271 (1970). M. Kojuma, S. Inouye, and T. Niida, J. Antibiot. 26, 246 (1973). 4j. Davies, M. Brzezinska, and R. Benveniste, Ann. N.Y. Acad. Sci. 182, 226 (1971), 5M. J. Haas and J. Davies, Antimicrob. Ag. Chemother. 4, 497 (1973).
612
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
/b HO,--x--.~C,H,~O\
o"
I- b
d
7
~
C H o ~ N H R
3
0
~o~H~ H O ~
Kanamycin A Kanamycin B Kanamycin C BB-K8 (amikacin)
Rl
R~
NH~ NH~ OH NH~
OH NH2 NH: OH
OH OH R3
H H H --CO---CH(OH)CH:--CH2NH~
Fro. 1. Structure of the kanamycins. Arrows indicate sites of O-phosphorylation (a), N-acetylation (b, c, d), and O-adenylylation (e).
are pathogens or potential pathogens. Care should be exercised when handling cultures and all used media and glassware should be sterilized be]ore disposal or cleaning.
Assay Procedures General Protocol. Enzyme activities are assayed by means of the phosphocellulose paper binding assay devised by Davies et al. 4 which measures transfer of radiolabel from a suitable cofactor to the antibiotic. The general assay procedure used is outlined below and is followed by details for each type of reaction. The reaction mixture consists of buffer, labeled cofactor, enzyme prepa s
I
0 NH,7
~
NH~
O OH
t
d
Fxo. 2. Structure of tobramycin (nebramycin factor 6). Arrows indicate sites of N-acetylation (a, b, c) and O-adenylylation (e).
[48]
AMINOGLYCOSIDE-MODI FYI NG ENZYMES
.R, -
613
/b
S(~H-R~,-'I~O \
d
o R~
/
e
Gentamicin Gentamicin Gentamicin Gentamicin Gentamicin
A B CI~ C~ C1
R1
R~
R3
R4
R5
R6
R7
H H H CH3 CHa
OH NH2 NH~ NH~. NHCHa
OH H H It H
OH OH H H H
NH2 OH NH2 NH~ NH..
H OH OH OH OH
OH CHa CHa CH3 CH3
FxG. 3. Structure of the gentamicins. Sisomicin is 4,5-dehydrogentamicin C1, (ring I). Arrows indicate sites of O-phosphorylation (a), N-acetylation (b, c, d), and O-adenylylation (e). a r a t i o n a n d a n t i b i o t i c . A c r u d e e n z y m e e x t r a c t (S100 or o s m o t i c shocka t e ) is of s u i t a b l e p u r i t y for t h e a s s a y . F o r large n u m b e r s of a s s a y s it is c o n v e n i e n t to d i s t r i b u t e p r e m i x e d buffer, c o f a c t o r , a n d e n z y m e (or buffer, cofaetor, a n d a n t i b i o t i c ) . Such m i x t u r e s s h o u l d n o t be r e - u s e d b
HO
6CHL'N/H O.
R3
Ribostamycin Butirosin A Butirosin B
OH
R,
1{2
R3
H - - C O--C H (O H) - - C H r--C H..,NH.., --CO--CH(OH)--CH2--CH~NH2
H OH H
OH H OH
FIG. 4. Structures of ribostamycin and the butirosins. Arrows indicate sites of O-phosphorylation (a) and N-acetylation (b, c, d).
614
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
/b 0 ,-.~,^
c
o"7" ~ - - - ~ --.~'. %
I).o\?-..y6.
'~ '2
R,~
Rl
R~
Neomycin B Neomycin C
NH2 NH2
OH OH
Paromomycin
OH
OH
Lividomycin A Lividomycin B
OH OH
H H
0
---~-
=
OH
R8
R4
H CH2NH2 H + {CH.NH~ H H
R~
CH~NH2 H
H H
CH~NH2}
H
CH2NH2 CH:NH2
mannose H
FIe. 5. Structures of the neomycins and lividomycins. Neamine (and paromamine) are rings I and II. Arrows indicate sites of O-phosphorylation (a) and N-acetylation (b, c, d). NH II HNCNH2
NH CNH2
0
H
R~
OH
Ii"
OH ~) R=O C~H~HO4 /OH
Streptomycin Dihydrostreptomycin Mannosidostreptomycin
RI
R2
CHO CH2OH C HO
H H mannose
Fro. 6. Structure of the streptomycins. Ring I is streptidine, ring II is streptose, and ring III is N-methyl4.-glucosamine. The arrow indicates the site of O-adenylylation or phosphorylation.
[481
AMINOGLYCOSIDE-MODIFYIN G ENZYMES
~
()l;~
CH3
0 FIG. 7. Structure of speetinomycin. The arrow indicates the site of O-adenylylation. after refrigeration or freezing. Control (blank) assays lacking either antibiotic or enzyme should be included in each set of assays. The components of the assay are mixed in chilled 75 X 10 mm tubes and the reaction is started by incubation at 30 ° or 35 °. After 15-20 rain of incubation the tubes are returned to the ice bath and a 10-20 ~1 sample of each reaction mixture is pipetted onto a numbered piece (ca. 1 cm ~) of W h a t m a n P-81 phosphocellulose paper raised above a block of styrofoam on a pin. After 30 sec at room temperature papers and pins are placed in about 500 ml of hot (70--80 °) distilled water for 3-4 min. The liquid, which contains unused labeled cofactor, is poured off and disposed of accordingly. The papers are rinsed 3 or 4 times with 500-600 ml of distilled water and dried under heat lamps (avoid charring) or using a hot-air dryer. Papers (with or without pins) are placed in glass scintillation vials containing 10 ml of toluene scintillation fluid (3 g PP(), 0.1 g P O P O P per liter of toluene) and counted. A time course of inactivation may be determined by simply scaling up the reaction mixture and removing samples for counting at various times. Descriptions of large-scale inactivations for chemical analyses m,~y be found among the references in the table. Assay o.f Kanamycin and Gentamicin Acetyltrans]erases. Acetyltransferases with acid pH optima are assayed in a buffer prepared by adding to 30 ml of deionized water: 12.75 ml of 0.6 M citric acid, 27.25 rnl of 0.6 M tripotassium citrate, 2.4 ml of 1.0 M magnesium acetate, and 1.6 ml of 0.5 M D T T . The pH is adjusted to the desired value at 30 ° with potassium hydroxide or glacial acetic acid and the volume is adjusted to 80 ml with deionized water. Acetyltransferases with neutral or slightly basic pH optima are assayed in a buffer prepared by adding to 40 ml of deionized water: 25 ml of 1 M Tris base, 5 ml of 1 M MgCl~, 1 ml of 0.5 M D T T and 10 ml of 1 M NH4C1. The pH is adjusted to the desired value at 30 ° with HC1 or glacial acetic acid and the volume is adjusted to 100 ml with deionized water.
616
ANTIBIOTIC INACTIVATION AND MODIFICATION A M I N O G L Y C O S I D E - M ODIFYING
Enzyme-
[48]
ENZYMES
Synonyms
Substrates b
Kanamycin acetyltransferase (KAT)
--
Gentamicin acetyltransferase I (GATI)
--
Neomycins, kanamycins A and B, gentamicin Cla, tobramycin, butirosins, ribostamycin, sisomicin, BB-K8 (gentamicin C2) Gentamicins, sisomicin (kanamycin B, tobramycin)
Gentamicin acetyltransferase II (GATH)
Gentamiein acetyltransferase I I I (GATm) Gentamicin adenylyltransferase (GAdT) Streptomycin-spectinomycin adenylyltransferase (SAdT) Neomycin phosphotransferase I (NPTI) Neomycin phosphotransferase II (NPT~) Streptomycin phosphotransferase (SPT)
Gentamicin adenylate synthetase (GAS), kanamyein nucleotidyltransferase (KNT) k Streptomycin adenylate synthetase (SAS) Kanamyain phosphotransferase I (KPTI) n, possibly also lividomycin phosphotransferase (LPT) Kanamycin phosphotransferase II (KPTII) n
Kanamycin C, gentamicins, sisomicin, tobramycin, butirosins (neomycins, ribostamycin kanamycin B) Kanamycins, gentamicins, sisomicin, ribostamycin, tobramycin, lividomycins Kanamycins, gentamicins, tobramycin Streptomycin, spectinomycin, dihydrostreptomycin (mannosidostreptomycin) Neomycins, kanamycins, lividomycins, ribostamycin, gentamicins A and B Neomycins, kanamycins, butirosins, ribostamycin, gentamicins A and 'B Streptomycin, dihydrostreptomycin
"Enzyme names and abbreviations are currently under review. b Poor substrates are shown in parentheses. c Aminoglycoside structures are shown in Figs. 1-7. d H. Umezawa, M. Okanishi, R. Utahara, K. Maeda, and S. Kondo, J. Antibiot. 20, 136 (1967). M. Yagisawa, H. Naganawa, S. Kondo, T. Takeuchi, and H. Umezawa, J. Antibiot. 25, 495 (1972). I R. Benveniste and J. Davies, Proc. Nat. Acad. Sci. U.S. 70, 2276 (1973). g M. Brzezinska, R. Benveniste, J. Davies, P. J. L. I)aniels, and J. Weinstein, Biochemistry 11, 761 (1972). h M. Chevereau, P. J. L. Daniels, J. Davies, and F. Le Goffic, Biochemistry 13, 598 (1974). i S. Biddlecome, P. Daniels, J. Davies, M. Haas, and D. Rane, in preparation. i H. Naganawa, M. Yagisawa, S. Kondo, T. Takeuchi, and H. Umezawa, J. Antibiot. 24, 913 (1971).
[48l
AMINOGLYCOSIDE-MODIFYING ENZYMES
Cofactors
Modificationc
617
Representative strains ~
Acetyl coenzyme A
6-Amino group of aminohexose I is aeetylated a,e,/
Acetyt coenzyme A
3-Amino group of deoxystreptamine (II) is acetylatedg
Acetyl coenzyme A
2-Amino group of aminohexose I is acetylated/,h
Acetyl coenzyme A
3-Amino group of deoxystreptamine (II) is acetylated ~
P. aeruginosa PST 1*
Ribo- or deoxyribonucleoside triphosphates (ATP preferred)
2-Hydroxyl group of aminohexose I I I is adenylylatedi
E. coli JR66/W677
ATP or dATP m
3-Hydroxyl group of Nmethyl-I~glucosamine is adenylylated z 3-Hydroxyl group of aminohexose I ° or 5-hydroxyl group of pentose I I I ~ is phosphorylated 3-Hydroxyl group of aminohexose I is phosphorylated
E. coli RlOO/W4354," NR73/W677
ATP
ATP ATP
3-Hydroxyl group of Nmethyl-L-glucosamine is phosphorylated~
E. coli R5/W677~; NR79/ W677, P. aeruginosa GN315/3796, Streptomyces kanamyceticus ATCC 12853/ P. aeruginosa 1 3 0 / P . aeruginosa 2 0 9 / E . coli R135/C600 Providencia 164,h Streptomyces spectabilis UC 2472/
E. coli JR35/W677p
E. coli JR66/W677,q P. aeruginosa Ps49q E. coli J R 3 5 / W 6 7 7 / P. aeruginosa H-9, r Staphylococcus aureus B294," Streptomyces griseus ATCC 10971t
k S. Kondo, K. Iinuma, M. Hamada, K. Maeda, and H. Umezawa, J. Antibiot. 27, 90 (1974). z T. Yamada, D. Tipper, and J. Davies, Nature (London) 219, 288 (1968). " R. Benveniste, T. Yamada, and J. Davies, Infection Immunity 1, 109 (1970). " H. Umezawa, H. Yamamoto, M. Yagisawa, S. Kondo, T. Takeuchi, and Y. A. Chabbert, J. Antibiot. 26, 407 (1973). ° S. Kondo, M. Okanishi, R. Utahara, K. Maeda, and H. Umezawa, J. Antibiot. 21, 22 (1968). p B. Ozanne, R. Benveniste, D. Tipper, and J. Davies, J. Bacteriol. 100, 1144 (1969). q M. Brzezinska and J. Davies, Antimicrob. Ag. Chemother. 3, 266 (1973). r O. Doi, M. Ogura, N. Tanaka, and H. Umezawa, Appl. Microbiol. 16, 1276 (1968). O. Doi, M. Miyamoto, N. Tanaka, and H. Umezawa, Appl. Microbiol. 16, 1282 (1968). t j. Walker and M. Skorvaga, J. Biol. Chem. 248, 2435 (1973). " Cultures not available from culture collections may usually be obtained from authors of relevant publications.
618
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
Lyophilized [1-14C]acetyl coenzyme A (50-60 Ci/mole) is resuspended in deionized water to an activity of 25 ~Ci/ml and diluted with 2 parts of 0.88 mg/ml trilithium acetyl coenzyme A before use. Each assay contains: 10 t~l of buffer, 10 t~l of [14C]acetyl-CoA solution, 5 ~l of enzyme preparation, 5 ~l of antibiotic (1 mg/ml). The mixture is incubated at 30 ° for 15 min and 20-~1 samples are counted. If diluted or less active enzyme preparations are being assayed, up to 20 t~l may be used in each assay. Assay o] Neomycin and Streptomycin Phosphotrans]erases. To prepare 30 ml of buffer (sufficient for 3000 assays), mix 15 ml of distilled water with 2 ml of 1 M Tris, 1.25 ml of 1 M MgCl~, 6 ml of 2 M NH4CI, and 0.1 ml of 0.5 M DTT. Adjust the pH to 7.1 at 5 ° with 1 M maleic acid and make up to 30 ml with deionized water. [~2P]ATP solution consists of 15 ~l of 50 mM sodium ATP (adjusted to pH 7.2 with NaOH) and 10-100 ~1 of [~/-~P]ATP depending on specific activity (initially-~ 20 Ci/mole, 0.75 mCi/ml) made up to 1 ml with deionized water. Each assay contains: 10 ~l of buffer, 10 ~l of [3-°P]ATP solution, 10 t~l of enzyme preparation, 2 ~l of antibiotic (1 mg/ml). The mixture is incubated at 35 ° for 20 min, and 20-~1 samples are counted. COMMENTS. Owing to the short half-life of 32p, ATP solutions should be used within 2-3 weeks of preparation. Assays o/ Gentamicin and Streptomycin Adenylyltrans]erases. The assay buffer is the same as for phosphotransferase assays except that it is brought to pH 7.8 at 5 °. Uniformly labeled [~C]ATP (400-500 Ci/mole) is purchased in 50% ethanol, lyophilized to dryness and resuspended in deionized water to an activity of 50 ~Ci/ml. To 1 ml of this are added 50 ~l 0.1 M ATP and 3.95 ml of deionized water, yielding a final cofactor preparation of approximately 10 t~Ci/tLmole/ml. Each assay contains: 10 ~l of buffer, 10 ~l of [~4C]ATP solution, 10 ~l of enzyme preparation, 2 ~l of antibiotic (1 mg/ml). The mixture is incubated at 30 ° for 30 min and 20- or 25-~1 samples are counted. COMMENTS. [14C]ATP may be purchased in 2% ethanol, when it is used directly after dilution. [a-32P]ATP may also be used as an adenyl donor but the short half-life of 32p is usually a disadvantage. A number of other methods have been used to assay inactivation by aminoglycoside-modifying enzymes, the most common being a microbiological assay which measures residual antibiotic activity by disc assay against a sensitive test organism2 Although this assay is convenient and T. Gavan, E. Cheatle, H. McFadden, Jr., Eds., "Antimicrobial Susceptibility Testing." American Society of Clinical Pathologists, Chicago, Illinois (1971).
[481
A M I N O G L Y C O S I D EODIFYI -M NG ENZYMES
619
simple, it may not be sufficiently quantitative for some experiments (e.g., time courses of inactivation). In addition, it cannot measure modification of a compound that is not an antibiotic. Furthermore, modification may not be detected if it does not result in complete inactivation (e.g., 6'-Nacetylneomycin B retains considerable antimicrobial activity). N-Acetylation may be monitored by means of a colorimetric assay using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). 7 Free eoenzyme A formed during acetylation reacts with DTNB to form a mixed disulfide; this is detected by an increase in A41_~. The assay is useful for certain antibiotics which do not bind quantitatively to phosphocellulose paper or if radiolabeled acetyl CoA is unavailable, but it requires the use of at least partially purified enzyme preparations. Sulfhydryl reagents interfere with the DTNB assay. The products of modification reactions may also be identified by cellulose-acetate strip electrophoresis 7 and thin-layer chromatography ;~ residual antibiotic can be determined by high-pressure liquid chromatography, s Growth of Cells
Drug-resistant bacteria are grown at 37 ° in medium containing, per liter: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride. Ten grams of glucose are added separately, as a sterile 50% solution, after sterilization of the medium. One of the antibiotics to which the R factor confers resistance is routinely added to the medium after sterilization (to a final concentration of 20 ~g/ml) to prevent possible segregation of the R factor. Streptomycetes are grown for 18-36 hr at 30 ° in Difco Nutrient Broth supplemented after sterilization with glucose (final concentration 1%).
Buf]ers Buffer I: 10 mM Tris-chloride, 10 mM magnesium chloride, 25 mM ammonium chloride, 0.6 mM fl-mercaptoethanol, pH 7.8 at 4 ° Buffer II: 20 mM Tris-chloride, 10 mM magnesium acetate, 25 mM ammonium chloride, 10 mM potassium chloride, 2 mM DTT, pH 7.5 at 4 ° Buffer III: 10 mM Tris-acetate, 10 mM magnesium acetate, 1 mM fl-mercaptoethanol, pH 7.4, at 4 °. pH is adjusted with acetic acid. Buffer IV: 20 mM Tris-chloride, 10 mM magnesium chloride, 10 mM potassium chloride, 30% glycerol, pH 7.8 R. Benveniste and J. Davies, Biochemistry 10, 1787 (1971). s H. Umezawa, H. Yamamoto, M. Yagisawa, S. Kondo, T. Takeuchi, and Y. Chabbert, J. Antibiot. 26, 407 (1973).
620
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
Buffer V: 10 mM Tris-chloride, l0 mM magnesium chloride, 3 mM, fl-mercaptoethanol, pH 7.8
Isolation of Crude Enzyme Preparations For those species (Escherichia, Shigella, Enterobacter, Serratia, Salmonella, and some strains of Pseudomonas) which release their drugmodifying activity when subjected to an osmotic shock, the procedure of Neu and Heppel 9 is used to obtain crude enzyme preparations. This technique solubilizes essentially all the aminoglycoside activity of an R ÷ E. coli while releasing only 4-6% of the total cell protein. One to two percent of the cells are lysed duriag such treatment. Where purification of an enzyme is desired, it is obviously an advantage if the R factor can be transferred to a strain susceptible to osmotic shock. 1° Aminoglycoside-modifying enzymes may be isolated from strains not susceptible to osmotic shock (notably Proteus, Providencia, Staphylococcus, Streptomyces, and some strains of Pseudomonas) either by sonication, French pressing, or enzymic lysis of cells. Osmotic Shock. Late log phase cells are harvested by centrifugation and resuspended in 100 volumes of 10 mM Tris-chloride, 30 mM sodium chloride, pH 7.8 at room temperature. The cells are again harvested by centrifugation, resuspended in 35 volumes of 3 mM EDTA, 33 mM Trischloride, pH 7.8, in 20% sucrose and stirred for 15 min at room temperature (a magnetic stirring bar is convenient). The cells are centrifuged at 16,000 g for 15 min in a refrigerated centrifuge, and the supernatant is removed. The centrifuge tube is inverted, and the pellet is allowed to drain in the cold for 10 min. It is important to remove all traces of sucrose or the cells will not shock properly. Therefore all excess fluid is removed from the cell pellet with cotton or other absorbent material. The ceils are resuspended in 40 volumes of ice-cold 0.5 mM m~gnesium chloride, stirred at 4 ° for 10-20 min and then centrifuged at 26,000 g for 30 min at 4 °. The pellet is discarded; the supernatant ("osmotic shockate") contains the aminoglycoside-modifying enzymes. Sonication. Washed log phase cells are resuspended in an appropriate buffer (0.6 g wet cells to 8 ml of buffer). The suspension, cooled in an ice bath, is subjected to 30-see bursts from a sonicator at 100 W output. Bursts are alternated with 2-min cooling periods, and 3-5 bursts are usually adequate (cell breakage may be monitored by phase-contrast microscopy). Some staphylococci cannot be broken by sonication; others may reH. C. Neu and L. A. Heppel, J. Biol. Chem. 240, 3685 (1965). 1, j. Davies, this volume [3].
[4sl
AMINOGLYCOSIDE-MODIFYING ENZYMES
621
quire the use of glass powder and longer sonication times for efficient breakage. Streptomycetes are resuspended in a minimum volume of Buffer I and sonicated as above. Sonicated cell suspensions are centrifuged at 30,000 g for 20-30 min, and the supernatant is dialyzed. The extract may be further clarified by preparing a 100,000 g supernatant (S100); deoxyribonuclease I is added to the 30,000 g supernatant to a final concentration of 4 ~g/ml, and the mixture is centrifuged at 100,000 g for 2 hr at 4 °. The supernatant is dialyzed against an appropriate buffer (usually buffer I or buffer II--see above). French Pressure Cell. The French pressure cell is the most convenient method of breaking large quantities of cells. The cells from 6 liters of a log phase culture are harvested by centrifugation, resuspended in 1 liter of 10 mM phosphate buffer, pH 7, and pelleted by centrifugation. The cells are resuspended in 30-40 ml of buffer II and passed twice through a French pressure cell at a pressure in excess of 12,000 psi into a cold receptacle. The resulting suspension is centrifuged and dialyzed as described for sonication. Streptomycetes are resuspended in a minimum quantity of buffer ][, sonicated for 15 sec to disperse mycelial clumps and treated as described above. Enzymic Lysis by Lysozyme. Escherichia coli can be lysed by resuspending a washed cell pellet in 3 ml (per gram) of 0.1 M K~PQ (pH 7.5 at room temperature) containing 0.5 mg/ml EDTA and 1 mg/ml egg white lysozyme and incubating for 1 hr at room temperature. The resulting suspension is then centrifuged and dialyzed as described above. Lysozyme may also be used to lyse streptomycetes as described by Walker and Walker. 11 Frozen 2.5-day mycelial pads are dispersed and digested for 1 hr at room temperature in 3 volumes of 0.1 M potassium phosphate buffer (pH 7.4) containing 5 mg/ml EDTA and 1 mg/ml lysozyme. The supernatant is centrifuged at 30,000 g for 30 min at 4 °, dialyzed against 1 mM phosphate buffer-EDTA containing 0.1 ml fl-mercaptoethanol per 4 liters and finally dialyzed against 25 mM Tris (pH 7.4) containing fl-mercaptoethanol. A similar procedure has been described by Hey and Elbein. 12 Enzymic Lysis by Lysostaphin. Staphylococci are refractory to the action of lysozyme but can be lysed with lysostaphin. The washed cell pellet from a 100-ml early stationary phase culture is resuspended in 4 ml of buffer I, lysostaphin is added to a final concentration of 50 ~g/ml 1, j. B. Walker and M. S. Walker, Biochemi, try 6, 3821 (1967). ~ A. Hey and A. D. Elbein, J. Bacteriol. 96, 105 (1968).
622
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
and the mixture is incubated for 30 min at 37 °. The suspension is centrifuged and dialyzed as described for sonication. P o l y m y x i n B. Polymyxin treatment may be used to release aminoglycoside-modifying enzymes from E. coli '~ and some strains of Pseudomonas. A culture growing exponentially in glycerol-salts medium (per liter: 2.5 g glycerol, 7 g K2HPO4-3H20, 3 g KH~P04, 1 g (NH4)2SO~, 0.1 g MgSO4.7H~O, 0.5 g sodium citrate.5.5H.~O, 0.1 ml of a 0.335% solution of ferric citrate, and 0.1 ml of a 0.163% solution of MnS04, pH 7.0-7.2) is harvested by centrifugation at an A~oo of 1.0. The cells are washed once in one-third culture volume of 0.14 M NaC1, pH 7.3, centriftLged, resuspended in one-tenth of the original culture volume of 0.14 M NaC1 (pH 7.3) and treated for 1 min at 37 ° with 200 ~g/ml polymyxin B sulfate (Sigma). The residue is pelleted by centrifugation at 10,000 g or by filtration through cellulose acetate filters (pore size 0.45 ~m). The filtrate or supernatant contains the aminoglycoside-modifying enzymes. This technique has been reported to release periplasmic enzymes with a 50-100% yield while releasing only about 5% of the total cellular proteinJ 3 Purification
Despite several efforts, using many different techniques, 1~-1~ there is no satisfactorily documented account of the successful purification of any of the enzymes to homogeneity. In a number of cases, however, partial purification has been achieved, 17,18 usually by a combination of streptomycin sulfate and ammonium sulfate precipitations followed by DEAE-cellulose chromatography. In general, all operations are carried out at 4 ° . The normal starting point for partial purification is an osmotic shockate or a whole-cell lysate S100. Streptomycin sulfate is added to the cold enzyme solution to a final concentration of 1.5%. After the streptomycin has dissolved, the mixture is mixed gently for 15 rain and then centrifuged at 15,000 g for 15 rain. The enzyme is precipitated from the supernatant by a suitable ammonium sulfate cut (this usually lies between 30 and 60% saturation with ammonium sulfate); the precipitate from the final cut is resuspended in and dialyzed against a suitable buffer and absorbed 13G. Cerny and M. Teuber, Arch. Mikrobiol. 78, 166 (1971). 1'O. Doi, S. Kondo, N. Tanaka, and H. Umezawa, J. Antibiot. 22, 273 (1969). ~Y. Sakagami, N. Takaishi, and A. Hachimori, J. Antibiot. 27, 248 (1974). ~"M. J. Haas, unpublished data. 17A. L. Smith and D. H. Smith, g. Inject. Dis. 129, 391 (1974). ~8M. Brzezinska and J. Davies, Antimicrob. Ag. Chemother. 3, 266 (1973).
[48]
AMINOGLYCOSIDE-M ODIFYING ENZYMES
603
to a DEAE-eellulose column. The column is washed with a small volume of buffer, and protein is eluted with buffer containing a linear sodium chloride, ammonium chloride, or potassium acetate concentration gradient from 0 to 0.5 M. Such a procedure usually results in a 25-70% increase in specific activity with a recovery of 3o-7o% of the initial activity.
Properties General Properties. The aminoglycoside-modifying enzymes are poorly characterized from a physical and chemical point of view. Most of the enzymes are inactivated by repeated freezing and thawing and are stable at --70 °. Substrate inhibition is commonly observed. The molecular weights of several of the enzymes have been estimated at 20,000-30,000. TM In general, a suitable buffer for dialysis, storage and assay contains 10 mM Tris-ehloride or acetate, 10 mM magnesium chloride or acetate, 25 mM ammonium chloride, and a sulfhydryl reagent (DTT, 0.5 raM-1 mM or fl-mercaptoenthanol, 1 mM-3 mM), pH 7.0-8.0. Kanamgcin AcetyltransJeq'ase (E. coli R5/~:6771';; NR79/W677 ~) Stability and Activity. In unbuffered osmotic shockates the enzyme loses 8% of its activity during 4.5 hr of incubation at 30 °. The presence of 5-25% glycerol increases stability but decreases activity by as much as 30%. The enzyme is stable at --70 ° and is stable to lyophilization and acetone precipitation. The pH optimmn for the acetylation of the kanamycins, neomycins, and tobramycin is approximately 5.8; the optimum for gentamicins C1 and C1,~is near 7.6. Enzyme stability and activity are reduced by concentrations of DTT above 5 mM. EC]ect o] BuJ~ers and Ions. The standard buffcr is buffer III. Tricine, MOPS, and cacodylate buffers at 10 mM are satisfactory. In phosphate, HEPES, Kolthoff-borate-phosphate, and Tris-maleate buffers the enzyme is less stable. The enzyme requires 10 mM magnesimn for activity but is inhibited by concentrations above 20 raM. The enzyme is unstable in sodium chloride but is stable in potassium acetate. Kinetic Data. The Kn, for the aeetylation of kanamyein A is 1 ~M at pH 7.0. The V..... is 1 ~mole/min using the assay conditions described. The acetylation of kanamyein is inhibited by paromomyein, paromamine, and gentamiein A. Substrate inhibition occurs at drug concentrations above 0.1 raM. ' ' J . Davfi~s and R. Benvcniste, Ann. N . Y . Acad. Sci. 235, 130 (1974).
624
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
Kanamycin Acetyltrans]erase (P. aeruginosa 37962°) Stability. KAT-3796 is stabilized by glycerol and ammonium chloride. The standard working buffer is buffer IV. Activity. The pH optimum for the acetylation of kanamycin A is between 5.6 and 6.5, and no pH optimum is exhibited between 5 and 9 for the acetylation of sisomicin and gentamicin C1~. The acetylation of kanamycin A, neomycin B and gentamicin Cla is inhibited by kanamycin C, gentamicin A, and, to a lesser extent, lividomycin.
Gentamicin Acetyltrans]erase I (E. coli R135/C6002°) Stability. The enzyme is stable at --20 ° and rapidly loses activity above 35 °. It is generally less stable than a similar enzyme isolated from P. aeruginosa 130. The standard buffer is buffer V. Kinetic Data. The Km for the acetylation of sisomicin is approximately 10 pjl//, and the V~ax is 0.9 nmole/min. The enzyme is inhibited by concentrations of sisomicin (but not of gentamicin C1) above 0.1 raM. Gentamicin Acetyltrans]erase I (P. aeruginosa 1302°) Stability. The enzyme is stable at --20 ° and is rapidly inactivated above 35 °. Buffer V is the standard buffer. pH Optimum. The pH optimum for the acetylation of sisomicin and kanamycin B is about 7.4. Gentamicin C1 acetylation exhibits a broad pH optimum (pH 5.4-8.0). Kinetic Data. The K,, for sisomicin acetylation is approximately 0.1 mM and the V~ax is about 60 nmole/min. The enzyme is inhibited by sisomicin, but not gentamicin C~, at concentrations above 0.1 mM. Gentamicin Acetyltrans]erase II (Providencia 16421) Stability. The enzyme is stable at --20 ° . Reactions are routinely supplemented with magnesium acetate and DTT although an absolute requirement for these has not been demonstrated. Activity. The pH optimum for acetylation of kanamycin C is 6.0; for gentamicin C1 it is 6.6. The enzyme is inhibited by poor substrates and nonsubstrates. 20S. Biddlecome, M. S. thesis, University of Wisconsin,Madison, 1973. 51M. Chevereau, P. J. L. Daniels, J. Davies, and F. LeGoffic, Biochemistry 13, 598 (1974).
[48]
AMI NOGLYCOSIDE-MODIFYING ENZYMES
625
Gentamicin Acetyltrans]erase III (P. aeruginosa PST-1 ~°) Stability. The enzyme is stable at --20 ° . It is not inactivated by incubation at 30 ° for 20 min. Activity. The pH optimum for the acetylation of the kanamycins, gentamicins, and neomycins is between pH 5.5 and 6.0. Magnesium (10 mM optimum) and a sulfhydryl reagent (DTT, 3 mM optimum) are required for activity but not for stability. Substrate inhibition occurs at varying concentrations with different substrates. Gentamicin A denylytrans]erase (E. coli JR66/W67722,-~:~) Requirements Jor Activity. DTT (1-10 mM) and magnesium are necessary for the maintenance of activity. Removal of magnesium results in irreversible inactivation. The enzyme is inhibited by high salt concentrations. pH Optimum. The pH optimum for the adenylylation of the gentamicins and kanamycins is 8.0-8.2. Streptomycin-Spectinomycin AdenylyltransJerase (E. coli NR73/W67724) Stability and Activation. The enzyme is stable at --10 ° and is activated by concentrations of ammonium chloride above 1 M. Ion and Co]actor Requirements. For the maintenance of activity and stability, 10 mM magnesium is required and may be replaced by manganese (0.1 mM), zinc, or cadmium. The enzyme will use ATP or dATP as cofactors. Kinetic Data. The Km's for the adenylylation of streptomycin and spectinomycin are 20 t~M. Activity is 50% reduced by 30 t~M pyrophosphate, but not by azide, iodide, cyanide, or pCMB. The enzyme is inhibited by streptomycin or spectinomycin at 0.2 mM. Streptomycin adenylylation is inhibited by tetracycline. Streptomycin-Spectinomycin AdenylyltransJe,'ase (E. coli B/RE130 '-'~) Activity Requirements. The enzyme requires magnesium (8-10 mM) and a reducing agent for maximal activity. Magnesium cannot be re52R. Benveniste, Ph.D. thesis, University of Wisconsin, Madison, 1972. 53M. Yagisawa, H. Naganawa, S. Kondo, M. Hamada, T. Takeuchi, H. Umezawa, J. Antibiot. 24, 911 (1971). 2~R. Benveniste, M. S. thesis, University of Wisconsin, Madison, 1970. 25j. H. Harwood and D. H. Smith, J. Bacteriol. 97, 1262 (1969).
626
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
placed by nickel, cobalt, zinc, calcium, or manganese. The pH optimum for the adenylylation of streptomycin is near 8.3. The enzyme will use ATP and dATP as cofactors.
Neomycin Phosphotrans]erase I (E. coli JR35/W677:6,27; JR39/W6772s; ML162929)
Activity and Stability. The enzyme from JR39 has an absolute requirement for magnesium and is stabilized by 0.1 M ammonium chloride or 10 ~g/ml neomycin B. The enzyme from ML1629 is inactivated by 5 min of incubation at 45 °. It also requires magnesium. DTT, 10 mM, prevents denaturation and restores the activity of denatured enzyme. Inhibition. Phosphorylation of neomycin B is inhibited by gentamicin C1~ and by tobramycin. Neomycin Phosphotrans]erase H (E. coli JR66/W677~s,29; P. aeruginosa Ps49 ~8) Stability. The enzyme is stabilized by 10 ~g/ml neomycin B or 0.1 M ammonium chloride. Activity. There is no marked pH optimum between pH 5.5 and 8.0 for the phosphorylation of neomycin B. Neomycin B phosphorylation is not inhibited by tobramycin or by gentamicin C1~ but phosphorylation of kanamycin C is inhibited by tobramycin. Neomycin PhosphotransJerase (P. aeruginosa H-93°) Stability. The phosphorylation of kanamycin proceeds for 20 rain at 55 °, for 5 min at 65 ° and not at all at 75% pH and heat stability are not markedly affected by 10 mM EDTA, 10 mM magnesium acetate, or 0.4 mM kanamycin. The enzyme is stable between pH 7.5 and 9. pH and Kinetic Data. The pH optimum for the phosphorylation of kanamycin is 7.5. At this pH, the Km for the phosphorylation of kanamycin is 0.3 raM, and the Vm~xis 42 ~moles/hr/OD unit. Ion and Cofactor Requirements. The enzyme requires magnesium, manganese, zinc, or cobalt (all at 1 raM) for activity. ATP is the only ~ M. S. Okanishi, S. Kondo, R. Utahara, and H. Umezawa, J. Antibiot. 21, 13 (1967). 21B. Ozanne, R. Benveniste, D. Tipper, and J. Davies, J. Bacteriol. 100, 1144 (1969). M. Brzezinska and J. Davies, Antimicrob. Ag. Chemother. 3, 266 (1973). 20M. Yagisawa, H. Yamamoto, I-I. Naganawa, S. Kondo, T. Takeuchi, and H. Umezawa, J. Antibiot. 25, 748 (1972). 3, O. Doi, S. Kondo, N. Tanaka, and H. Umezawa, J. Antibiot. 22, 273 (1969).
[48]
AMINOGLYCOSIDE-MODIFYING ENZYMES
627
accepted phosphoryl donor when partially purified enzyme preparations are used; crude preparations will use other nucleoside triphosphates. One hundred percent of the available kanamycin is modified in the presence of equimolar ATP.
Neomycin Phosphotrans/erase (S. aureus B294 :~) pH Optimum. The pH optimum for the inactivation of kanamycin is between pH 7.0 and 7.5. Inhibition. Substrate inhibition occurs at a kanamycin concentration of 2 m M but not at 0.2 mM. Lividomycin Phosphotrans/erase (E. coli R,,~+/ML14103'-'; P. cleruginosa Ti-1333; S. aureus KW-234)
Activity Requirements. The three enzymes require A T P and magnesium for activity.
pH Optimum. The pH optimum for the phosphorylation of lividomycin is between 6.5 and 7.0 for the three enzymes. Streptomycin Phosphotrans/erase (E. coli JR35/W677 '-,T) Ion and Co/actor Requirements. Magnesium is required for maximal activity, but not for stability, and can be partially replaced by zinc or manganese but not by copper (all at 5 raM). The enzyme uses A T P or G T P (but not C T P or U T P ) . pH Optimum. The pH optimum for the phosphorylation of strei)tomycin is 8.0. Streptomycin Phosphotransferase (P. aeruginosa H-93~) pH Optimum. The pH optimum for the phosphorylation of streptomycin is 8.5.
Co/actor Requirements. The enzyme will use ATP, CTP, GTP, or U T P ~' O. Doi, M. Miyamoto, N. Tanaka, and H. Vmezawa, Appl. Microbiol. 16, 1282 (1968). 3;M. Yamaguchi, T. Koshi, F. Kobayashi, and S. Mitsuhashi, Antin~icrob. Ag. Chemother. 2, 145 (1972). ~F. Kobayashi, M. Yarnaguchi, and S. Mitsuhashi, Antimic~'ob. Ag. Chemother. 1, 17 (1972). ~ F. Kobayashi, T. Koshi, J. Eda, Y. Yoshimura, and S. Mitsuhashi, Antimicrob. Ag. Chemother. 4, 1 (1973). '~ O. Doi, M. Ogura, N. Tanaka, and H. Umezawa, Appl. Microbiol. 16, 1276 (1968).
628
[49]
ANTIBIOTIC INACTIVATION AND MODIFICATION
as cofactors; maximal activity is observed at an ATP concentration of 2O mM. Acknowledgment We thank Dr. Julian Davies for many useful contributions to this work.
[49] ATP:Streptomycin 6-Phosphotransferase: By JAMES B. WALKER Dihydrostreptomycin +
MgATP
'l
NH,
MgADP
L~---o ,\
.,c~/, OH
C:NH + I NH
+
OH
I
J - - - - t ~,=H~
, "Kc.,o. I
NH z
/,'-O.~oH
,
(1)
)~
oPO;-
I
Dihydrostreptomycin-6-P
H~N--C--NH
Streptidine
+
MgATP
,
NH2 I + C=NH,
~ MgADP
(2) tt
oPofSt reptidine - 6 - P
Streptomycin 6-kinase is present in high concentration in extracts of streptomycin-producing strains of Streptomyces. 2-~ This enzyme can also 1EC 2.7.1.72 (mistakenly called 5-kinase). "J. B. Walker and M. S. Walker, Biochim. Biophys. Acta 148, 335 (1967). 3 A. L. Miller and J. B. Walker, J. BacteTiol. 99, 401 (1969). , M. S. Walker and J. B. Walker, J. Biol. Chem. 245, 6683 (1970). O. Nimi, G. Ito, Y. Ohata, S. Funayama, and R. Nomi, Agr. Biol. Chem. 3~, 850 (1971).
628
[49]
ANTIBIOTIC INACTIVATION AND MODIFICATION
as cofactors; maximal activity is observed at an ATP concentration of 2O mM. Acknowledgment We thank Dr. Julian Davies for many useful contributions to this work.
[49] ATP:Streptomycin 6-Phosphotransferase: By JAMES B. WALKER Dihydrostreptomycin +
MgATP
'l
NH,
MgADP
L~---o ,\
.,c~/, OH
C:NH + I NH
+
OH
I
J - - - - t ~,=H~
, "Kc.,o. I
NH z
/,'-O.~oH
,
(1)
)~
oPO;-
I
Dihydrostreptomycin-6-P
H~N--C--NH
Streptidine
+
MgATP
,
NH2 I + C=NH,
~ MgADP
(2) tt
oPofSt reptidine - 6 - P
Streptomycin 6-kinase is present in high concentration in extracts of streptomycin-producing strains of Streptomyces. 2-~ This enzyme can also 1EC 2.7.1.72 (mistakenly called 5-kinase). "J. B. Walker and M. S. Walker, Biochim. Biophys. Acta 148, 335 (1967). 3 A. L. Miller and J. B. Walker, J. BacteTiol. 99, 401 (1969). , M. S. Walker and J. B. Walker, J. Biol. Chem. 245, 6683 (1970). O. Nimi, G. Ito, Y. Ohata, S. Funayama, and R. Nomi, Agr. Biol. Chem. 3~, 850 (1971).
[40]
ATP :STREPTOMYCIN 6-PHOSPHOTRANSFERASE
629
phosphorylate the corresponding position of dihydrostreptomycin [Eq. (1) ] free streptidine [Eq. (2)], 2-deoxystreptidine, and presumably the dephosphorylated derivatives of various intermediates in the biosynthetic conversion of streptidine-6-P to streptomycin (Fig. 2 of a previous article2 Mature mycelia of all strains of Streptomyces tested thus far, including many strains not known to synthesize streptomycin, accumulate streptidine-6-P during several hours following the addition of streptidine to the culture medium. 7 It therefore appears that the gene coding for streptomycin 6-kinase activity has a wide distribution among Streptomyces, but high activity can normally be detected in vitro only in extracts of streptomycin-producing strains. The function of this enzyme other than in detoxification of a potent inhibitor of protein biosynthesis is not known, but it might participate in regulation of protein biosynthesis during the growth cycle or perform a function in membrane synthesis or transport, s Assay Methods Method I Principle. [3'a-'~H]Dihydrostreptomycin is incubated with MgATP and a dialyzed extract of a strain of streptomycin-producing Streptomyces, and the labeled product [Eq. (1)] is separated by paper chromatography and counted." This assay can be used with crude dialyzed extracts and purified enzyme preparations. Nondialyzed extracts should not be used, since they also contain a labile dihydrostreptomycin 3'a-kinase. TM Reagents
[3'a-~H]Dihydrostreptomycin (700-3000 Ci/mole) from AmershamSearle Dialyzed extract of mature mycelia of Streptomyces bikiniensis ATCC 11062 or other streptomycin-producing strain 11 Tris C1, 0.5 M, pH 9.0, containing 40 mM MgC1._. ATP, 36 mM, pH 6.8-7.0 Procedure. The complete incubation mixture contains: [3H]dihydrostreptomycin, 5gl (e.g., 60,000 cpm); Tris Mg, 5 t~l; ATP, 5 ~l; and dialyzed extract, 10 ~1. After incubation in a stoppered 13 X 100 mm This volume [21]. 7M. S. Walker and J. B. Walker, J. Biol. Chem. 241, 1262 (1966). 8j. B. Walker and M. Skorvaga, J. Biol. Chem. 248, 2435 (1973). This volume [24]. ,OThis volume [51]. 11This volume [25].
630
[49]
ANTIBIOTIC INACTIVATION AND MODIFICATION
test tube at 35 °, a 10-~1 aliquot is spotted, and the labeled components are separated by paper chromatography with ammoniacal phenol and counted2 For mobilities, see the table of a previous article2 More reliable kinetic data can be obtained by adding nonlabeled dihydrostreptomycin before incubation.
Method II Streptidine
mature myeelia 3-I0 h r i n v i v o
streptidine-6-P (intracellular)
~
L- (guanidino- 14C) Arginine +
NH2 I
NH~ NH
C, NH2+ NH2 NH K
NH L-ornithine
H
(3)
(4)
+ H
oPo~-
opo~-
This method is used only to detect the presence of streptomycin 6-kinase activity in vivo in strains of Streptomyces not known to synthesize streptomycin. Principle. The intraeellular streptidine-6-P which accumulates in mature mycelia following streptidine feeding [Eq. (3)] is detected by incorporation of label from L-[guanidino-~C]arginine by an exchange transamidination 3,m2 in the presence of inosamine-P amidinotransferase [Eq. (4) ].11
Reagents Mature (3- to 4-day) culture of Streptomyces growing in complex medium on a rotary shaker at 24-28 ° Dialyzed extract of Streptomyces bihiniensis ATCC 11062 as source of amidinotransferase2 S. glebosus ATCC 14607 can also be used. Streptidine, sulfate salt 9 L- [Guanidino-'4C] arginine (12-30 Ci/mole) Tris C1, 0.5 M, containing 13 mM EDTA, pH 7.4 2-Mercaptoethanol, 0.3 M
Procedure. A culture of the Streptomyces strain to be assayed is grown on a rotary shaker as described. ~3 After 3-4 days of growth from a slant 12A. L. Miller a n d J. B. Walker, J. B a c t e r i o l . 104, 8 (1970). ,3 This volume [22].
[49]
ATP :STREPTOMYCIN 6-PHOSPHOTRANSFERASE
631
inoculum, or at least 1 day after approximately 50% of the maximal mycelial yield has been attained, streptidine is added to the culture to a final concentration of 1 mg/ml as the sulfate salt. After 3-10 hr of further shaking, the mycelia are harvested by filtration, and a hot water extract is made as described ~ for natural amidine acceptors. This extract is then assayed enzymically for streptidine-6-P. The complete incubation mixture contains: dialyzed S. bikiniensis extract, 10 td; tHC]arginine (33 uCi/ml), 5 ~1; Tris-EDTA, 5 td; mercaptoethanol, 1 ~l; and hot-water extract of streptidine-fed mycelia, 10 t~l. After incubation in a stoppered 13 X 100 mm test tube at 35 ° for 120 rain, a 10-ul aliquot is spotted, and the components are separated by paper chromatography with ammoniacal phenol and counted." A broad single or double peak of radioactivity, centering around RI 0.40, indicates incorporation of label into streptidine-6-P by exchange transamidination [Eq. (4)]. This exchange is facilitated by trace amounts of ornithine present in the extract.
Properties The 6-kinase from S. bikiniensis is relatively stable, particularly in the presence of streptomycin, and can be easily purified on a DEAE-celhflose column as described. 11 The 6-kinase from S. griseus HUT 6037 has been purified 200-fold by DEAE-Sephadex A-25 and Sephadex G-100 column chromatography. 5 Biological Distribution. Streptomycin 6-kinase activity is highest in mature mycelia of strains of Streptomyces which are known to synthesize streptomycin. 11 Enzymic activity is usually low during the early phase of rapid mycelial growth. Evidence provided by the assay of Method II suggests that 6-kinase activity occurs in a very large number of Streptomyces strains, but this observation should be confirmed by purification and characterization of the activities observed in vivo. Specificity and Alternate Assays. The ATP requirement can he met by dATP but not by other nucleosidetriphospbates; Mn '-,~ can replace Mg 2÷. This enzyme appears to require the presence of guanidino groups at positions 1 and 3 and will tolerate a number of substitutions at position 4. The most active phosphate acceptors include streptomycin, dihydrostreptomycin, and 3'-deoxydihydrostreptomycin. Dihydrostreptomycin3'a-P and dihydrostreptomycin-3"-P can tie phosphorylated by this enzyme, but less readily than dihydrostreptomycin. At higher concentrations streptidine and 2-deoxystreptidine can serve as phosphate acceptors. ~ An acid-labile derivative of streptidine-6-P which accumulates in the culture medium only during streptomycin secretion in production media ~t can be enzymically dephosphorylated with alkaline pbosphatase ~4A. L. Miller and J. B. Walker unpublished results.
632
ANTIBIOTIC INACTIVATION AND MODIFICATION
[50]
or streptomycin-6-P phosphatase and then rephosphorylated with ATP and the 6-kinase. It would therefore appear that any of a number of intermediates in the biosynthetic pathway between streptidine-6-P and streptomycin-6-P which might become dephosphorylated by streptomycin-6-P phosphatase 1~ can be rephosphorylated in a salvage process by the 6-kinase. Identification of a product of 6-kinase activity as a derivative of streptidine-P can be established by mobilities (the table of a previous article 6 before and after treatment with (a) streptomycin-6-P phosphatase 1~ and (b) 2 N HC1 at 50 ° for 3 hr to hydrolyze the glycosidic linkage at position 4. 4 Related compounds which are not good phosphate acceptors include guanidinodeoxy-scyllo-inositol, 1D-l-guanidino-3amino-l,3-dideoxy-scyUo-inositol, 1D-l-amino-3-guanidino-l,3-dideoxyscyllo-inositol, streptamine, and 2-deoxystreptamine. Alternate assays for 6-kinase activity include a bioassay of crude incubation mixtures with a streptomycin-sensitive bacterium 5 or an assay employing [y-3~P]ATP as the labeled substrate, followed by passage of the incubation mixture through a small Dowex 1 (C1-) column, or adsorption of labeled [6-32P]streptomycin on a circle of cation-exchange paper. 16 These latter assays cannot distinguish among the various dihydrostreptomycin kinases.lO.17 ~ This volume [28]. ~, R. B e n v e n i s t e and J. Davies, Proc. Nat. Acad. Sci. U.S. 70, 2276 (1973). ~7This v o l u m e [50].
[50]ATP:Strcptomycin 3"-Phosphotransfcrase By J A M E S B. W A L K E R Streptomycin
and M A R G A R E T +
S. W A L K E R
MgATP
Nil 2
I
MgADP
C--_NH~+ t NH
I-------A
I\ .
HsCN/1
oPo~-
-
I
[
HO
]
Streptomycin-3 " - P
OH
NH2 1
C=NH:
(1)
632
ANTIBIOTIC INACTIVATION AND MODIFICATION
[50]
or streptomycin-6-P phosphatase and then rephosphorylated with ATP and the 6-kinase. It would therefore appear that any of a number of intermediates in the biosynthetic pathway between streptidine-6-P and streptomycin-6-P which might become dephosphorylated by streptomycin-6-P phosphatase 1~ can be rephosphorylated in a salvage process by the 6-kinase. Identification of a product of 6-kinase activity as a derivative of streptidine-P can be established by mobilities (the table of a previous article 6 before and after treatment with (a) streptomycin-6-P phosphatase 1~ and (b) 2 N HC1 at 50 ° for 3 hr to hydrolyze the glycosidic linkage at position 4. 4 Related compounds which are not good phosphate acceptors include guanidinodeoxy-scyllo-inositol, 1D-l-guanidino-3amino-l,3-dideoxy-scyUo-inositol, 1D-l-amino-3-guanidino-l,3-dideoxyscyllo-inositol, streptamine, and 2-deoxystreptamine. Alternate assays for 6-kinase activity include a bioassay of crude incubation mixtures with a streptomycin-sensitive bacterium 5 or an assay employing [y-3~P]ATP as the labeled substrate, followed by passage of the incubation mixture through a small Dowex 1 (C1-) column, or adsorption of labeled [6-32P]streptomycin on a circle of cation-exchange paper. 16 These latter assays cannot distinguish among the various dihydrostreptomycin kinases.lO.17 ~ This volume [28]. ~, R. B e n v e n i s t e and J. Davies, Proc. Nat. Acad. Sci. U.S. 70, 2276 (1973). ~7This v o l u m e [50].
[50]ATP:Strcptomycin 3"-Phosphotransfcrase By J A M E S B. W A L K E R Streptomycin
and M A R G A R E T +
S. W A L K E R
MgATP
Nil 2
I
MgADP
C--_NH~+ t NH
I-------A
I\ .
HsCN/1
oPo~-
-
I
[
HO
]
Streptomycin-3 " - P
OH
NH2 1
C=NH:
(1)
IS01
A T P :STREPTOMYCIN 3H-PHOSPHOTRANSFERASE
633
Streptomycin 3"-kinase activity occurs in a nonstreptomycin-producing strain, Streptomyces griseus ATCC 10971, in a labile formF ,-° The physiological function of 3"-kinase activity in Streptomyces is not known, although a role in detoxification, transport, or biosynthesis has been suggested. ~ I t is anticipated that 3"-kinase from Streptomyces will be studied in detail in the future to ascertain whether there is an evolutionary relationship between this enzyme and the 3"-kinases of streptomycin-resistant pseudomonads and enteric bacteria carrying certain R -determinants. 1-3
Assay Method Principle. [3'a-3H]Dihydrostreptomycin is converted to [3'a-3H]dihydrostreptomycin-3"-P, and the components are separated by paper chromatography and counted. Reagents [3'a-3H]Dihydrostreptomycin (700-3000 Ci/mole) from AmershamSearle Glycylglycine, 0.5 M, containing 40 m M MgCl.,, pH 8.0 ATP, 36 mM, pH 6.8-7.0 Fresh, or freshly thawed, supernatant solution from a 50% (w/v) sonicate of mature mycelia of Streptomyces griseus ATCC 10971; a 5-fold diluted extract is often employed ~
Proced~re. The complete incubation mixture contains: [3H]dihydrostreptomycin, 5 ~l (e.g., 60,000 cpm); glycylglycine-Mg, 5 ~1; ATP, 5 ~l; and fresh sonicate supernatant of S. griseus ATCC 10971, 10 F1. After incubation in a stoppered 13 X 100 mm test tube at 35 °, a 10-~1 aliquot is spotted, and the components are separated on an ammoniacal paper chromatogram and counted. '~ For mobilities, see table of following article. ';
Properties Streptomycin 3"-kinase from S. griseus ATCC 10971 is stable in frozen mycelial pads or ill frozen extracts, but most activity is lost in thawed extracts within 8 hr at room temperature or at 2 °. Biological Distribution. Incomplete evidence in our laboratory suggests that a more stable 3"-kinase might occur ill certain other strains J. B. Walker and M. Skorvaga, J. Biol. Chem. 248, 2435 (1973). 2M. S. Walker and J. B. Walker, J. Biol. Chem. 245, 6683 (1970). 3R. Benveniste and J. Davies, Proc. Nat. Acad. Sci. U.S. 70, 2276 (1973). 4This volume [22]. 5This volume [24]. " This volume [51].
634
[51]
ANTIBIOTIC INACTIVATION AND MODIFICATION
of Streptomyces, but the precise location of the phosphate group has not yet been established. Other streptomycin 3"-kinases occur in Pseudomonas aeruginosa 7 and enteric bacteria carrying a certain R determinant2 Specificity and Alternate Assay. Streptomycin-6-P, dihydrostreptomycin-6-P, and 3'-deoxydihydrostreptomycin-6-P also can serve as phosphate acceptors. Neither dihydrostreptomycin-3'a-6-diP nor dihydrostreptomyein-3'a-P can serve as an acceptor. Presumably activity could be detected by incubating streptomycin with [),-32P]ATP and fresh extract and adsorbing the labeled product on a cation-exchange paper disk2 Location of the phosphate group at position 3" can be deduced from NMR spectra 9 or from lack of susceptibility of 3'-deoxydihydrostreptomycin-3",6-diP to periodate degradation? 7I-I. Kawabe, F. Kobayashi, M. Yamaguchi, R. Utahara, and S. Mitsuhashi, J. Antibiot. 9.4, 651 (1971). "B. Ozanne, R. Benveniste, D. Tipper, and J. Davies, J. Bacteriol. 100, 1144 (1969). 9 H. Naganawa, S. Kondo, K. Maeda, and H. Umezawa, J. Antibiot. 24, 823 (1971).
[51] A T P : D i h y d r o s t r e p t o m y c i n - 6 - P 3'a-Phosphotransferase
By JAMES B.
WALKER and MARGARETS. WALKER
D i h y d r o s t r e p t o m y e i n - 6- P
+
MgATP
I•H2 C___--NH+
MgADP
L____,,
HO/[V'
]
+
"k
NH
O
HO
NH 2
I C=NH+
1~
J
(1)
OPO~-
Dihydro streptomycin- 3'c~, 6- dip
High levels of dihydrostreptomycin-6-P 3'a-kinase activity are present in fresh nondialyzed sonicates of mature mycelia of strains of Streptomyces which produce streptomycin. 1,-~ This kinase has not so far been observed in nonstreptomycin-produeing strains. Streptomycin-6-P cannot serve as substrate, since streptomycin has an aldehyde group rather than 1M. S. Walker and J. B. Walker, Y. Biol. Chem. 245, 6683 (1970). 2j. B. Walker and M. Skorvaga, J. Biol. Chem. 248, 2435 (1973).
634
[51]
ANTIBIOTIC INACTIVATION AND MODIFICATION
of Streptomyces, but the precise location of the phosphate group has not yet been established. Other streptomycin 3"-kinases occur in Pseudomonas aeruginosa 7 and enteric bacteria carrying a certain R determinant2 Specificity and Alternate Assay. Streptomycin-6-P, dihydrostreptomycin-6-P, and 3'-deoxydihydrostreptomycin-6-P also can serve as phosphate acceptors. Neither dihydrostreptomycin-3'a-6-diP nor dihydrostreptomyein-3'a-P can serve as an acceptor. Presumably activity could be detected by incubating streptomycin with [),-32P]ATP and fresh extract and adsorbing the labeled product on a cation-exchange paper disk2 Location of the phosphate group at position 3" can be deduced from NMR spectra 9 or from lack of susceptibility of 3'-deoxydihydrostreptomycin-3",6-diP to periodate degradation? 7I-I. Kawabe, F. Kobayashi, M. Yamaguchi, R. Utahara, and S. Mitsuhashi, J. Antibiot. 9.4, 651 (1971). "B. Ozanne, R. Benveniste, D. Tipper, and J. Davies, J. Bacteriol. 100, 1144 (1969). 9 H. Naganawa, S. Kondo, K. Maeda, and H. Umezawa, J. Antibiot. 24, 823 (1971).
[51] A T P : D i h y d r o s t r e p t o m y c i n - 6 - P 3'a-Phosphotransferase
By JAMES B.
WALKER and MARGARETS. WALKER
D i h y d r o s t r e p t o m y e i n - 6- P
+
MgATP
I•H2 C___--NH+
MgADP
L____,,
HO/[V'
]
+
"k
NH
O
HO
NH 2
I C=NH+
1~
J
(1)
OPO~-
Dihydro streptomycin- 3'c~, 6- dip
High levels of dihydrostreptomycin-6-P 3'a-kinase activity are present in fresh nondialyzed sonicates of mature mycelia of strains of Streptomyces which produce streptomycin. 1,-~ This kinase has not so far been observed in nonstreptomycin-produeing strains. Streptomycin-6-P cannot serve as substrate, since streptomycin has an aldehyde group rather than 1M. S. Walker and J. B. Walker, Y. Biol. Chem. 245, 6683 (1970). 2j. B. Walker and M. Skorvaga, J. Biol. Chem. 248, 2435 (1973).
[51]
DIHYDROSTREPTOMYCIN-6-P-3' a-PHOSPHOTRANSFERASE
635
CHROMATOGRAPHIC BEHAVIOR OF MONO- AND ])IPHOSPHORYLATED DERIVATIVES OF STREPTOMYCIN
Compound Dihydrostreptomycin Streptomycin-6-P Dihydrostreptomycin-6-P 3'-Deoxydihydrostreptomycin-6-P Streptomyein-3"-P Dihydrostreptomycin-3"-P Dihydrostreptomycin-3'a-P l)ihydrostreptomycin-3'a,6-diP 1)ihydrostreptomycin-3'~,6-diP 3~-Deoxydihydrostreptomycin-3'a,6-diP 3'-Deoxydihydrostreptomycin-3",6-diP Streptomycin-3H,6-diP St reptidine-6-P
Rj ~ 0.97 0.63 0.79 0.79 -0.79 0.71 0.21 0.29 0.29 0.40 0.12 0.40
Bio- Relative HVE mobilityc Rex-70 b (M) pH 3.6 pH 10.4 2.0 0.8 0. ~ 0.8 0.8 0.3 0.8 0.1 0.1 0. I 0.1 0.1 0.3
- 1.0 -0.7 --0.7
- 0.7 -0. I - 0.3
--- 0.7
- 0.1 -- 0.2 +0.2 -
-........
--
Ascending paper chromatography, 80% phenol-20% H:O, NH4OH atmosphere (this volume [24]). b Minimum concentration of ammonium formate required to elute compound adsorbed on a Bio-Rex-70 (NH4 +) column in the stepwise sequence: 0.l M, 0.3 M, 0.8 M, and 2.0 M ammonium formate (this volume [28]). c Mobilities relative to distance traveled by a picric acid marker during highvoltage paper electrophoresis (HVE) at the indicated pH (this volume 122]). A minus sign indicates migration toward the negative electrode, and a positive sign indicates migration toward the positive electrode. Mobilities vary somewhat with point of application and temperature. a h y d r o x y m e t h y l group at position 3'a. Because of its l a b i l i t y , 3 ' a - k i n a s e a c t i v i t y has n o t y e t been s e p a r a t e d from 6 - k i n a s e a c t i v i t y ; c o n s e q u e n t l y it is n o t k n o w n w h e t h e r the acceptor m u s t be p h o s p h o r y l a t e d at position 6. T r e a t m e n t of d i h y d r o s t r e p t o i n y c i n - 3 ' a , 6 - d i P with s t r e p t o m y c i n - 6 - P p h o s p h a t a s e ~ gives d i h y d r o s t r e p t o m y c i n - 3 ' a - P . T h e physiological f u n c tion of d i h y d r o s t r e p t o m y c i n - 6 - P 3 ' a - k i n a s e is n o t y e t k n o w n . I t could be i n v o l v e d in b i o s y n t h e s i s , detoxification, t r a n s p o r t , or i n c o r p o r a t i o n of d i h y d r o s t r e p t o m y c i n into a larger structure. M o b i l i t i e s a n d c h r o m a t o graphic b e h a v i o r of v a r i o u s m o n o - a n d d i p h o s p h o r y l a t e d s t r e p t o m y c i n d e r i v a t i v e s are given in the table.
Assay Method
Principle. [ 3 ' a - . ~ H ] D i h y d r o s t r e p t o m y c i n - 6 - P or [ 3 ' a - ~ H ] d i h y d r o s t r e p t o m y c i n is i n c u b a t e d with M g A T P a n d a fresh, or freshly t h a w e d , This volume [28].
636
ANTIBIOTIC INACTIVATION AND MODIFICATION
[51]
sonicate supernatant solution prepared from mature mycelia of a streptomycin-producing strain of Streptomyces [Eq. (1)]. The labeled components are separated by paper chromatography and counted. With the 6-phosphate derivative as phosphate acceptor, only 3'a-kinase activity is measured. With [~H]dihydrostreptomycin as phosphate acceptor, both 6-kinase and 3Pa-kinase activities are utilized; monophosphorylated derivatives do not usually accumulate when fresh sonicates are employed.
Reagents [3~a-3H]Dihydrostreptomycin (700-3000 Ci/mole) from AmershamSearle, or [3%-3I-I]dihydrostreptomycin-6-P, prepared as described ~ Glycylglycine, 0.5 M, containing 40 mM MgCl~, pH 8 ATP, 36 mM, pH 6.8-7.0 Supernatant solution from fresh, or freshly thawed frozen, sonicate 4 (50% w/v) of Streptomyces bikiniensis ATCC 1062, diluted 20fold with water just before use
Procedure. The complete incubation mixture contains: [3'a-~H]dihy drostreptomycin, 5 ~l (e.g., 60,000 cpm); glyclyglycine-Mg, 5 t~l; ATP, 5 ~l; and diluted fresh, or freshly thawed, extract from a streptomycin producer, 10 ~l. After incubation in a stoppered 13 X 100 mm test tube at 35 °, a 10-~1 aliquot is spotted, and the components are separated on an ammoniacal phenol paper chromatogram and counted2 For mobilities, see the table. A similar procedure is followed when the acceptor employed is [3'a-3H ] dihydrostreptomycin-6-P. 2
Properties The 3%-kinase might be a membrane-bound enzyme, but this has not been established. This kinase is stable for months while frozen as an extract or in intact mycelial pads, but activity is rapidly lost by thawed preparations at room temperature or at 2 °, as is the case with streptomycin 3"-kinase from S. griseus ATCC 10971. 6 Little activity remains in an extract after 6 hr at 2 °. However, significant activity is retained in a 20-fold diluted extract stored overnight at 4 ° in 30% glycerol. Biological Distribution. High levels of activity can be detected in sonicates of mature mycelia of a number of streptomycin producing strains of Streptomyces. 7 As noted earlier, steptomycin-6-P is not a substrate, so the function of this enzyme remains obscure in strains that produce or encounter streptomycin rather than dihydrostreptomycin. ~ 4T h i s This eThis 7T h i s
volume volume volume volume
[22]. [24]. [50]. [25].
[$2]
MANNOSIDOSTREPTOMYCIN HYDROLASE
637
Activity has not been detected in a number of strains which do not secrete streptomycin. Specificity. ATP cannot be replaced by UTP, GTP, or CTP. [6-32p]3'-Deoxydihydrostreptomycin-6-P is also a good phosphate accepfor. 2 As noted earlier, it is not known whether acceptors must be phosphorylated at position 6. The following compounds cannot serve as phosphate acceptors: streptomycin, streptomycin-6-P, dihydrostreptomycin-3"-P, and dihydrostreptobiosamine. Phosphate is presumed to be esterified at position 3'a since 3'-deoxydihydrostreptomycin-6-P, but not streptomycin-6-P, can serve as phosphate acceptor. 2
[52]
Mannosidostreptomycin Hydrolase
By
EDWARDINAMINEand ARNOLDL. DEMAIN NH II HN-- C --NH 2
\ ! O
L/OH
Streptidine 1
l Streptose OH
Streptomycin Mannosido-
l
streptomycin
O
l
N-Methyl-Lglucosamine
/ ~H
CII2OH
l D-Mannose Mannosidostreptomycin+ H20
* streptomycin+ mannose
[$2]
MANNOSIDOSTREPTOMYCIN HYDROLASE
637
Activity has not been detected in a number of strains which do not secrete streptomycin. Specificity. ATP cannot be replaced by UTP, GTP, or CTP. [6-32p]3'-Deoxydihydrostreptomycin-6-P is also a good phosphate accepfor. 2 As noted earlier, it is not known whether acceptors must be phosphorylated at position 6. The following compounds cannot serve as phosphate acceptors: streptomycin, streptomycin-6-P, dihydrostreptomycin-3"-P, and dihydrostreptobiosamine. Phosphate is presumed to be esterified at position 3'a since 3'-deoxydihydrostreptomycin-6-P, but not streptomycin-6-P, can serve as phosphate acceptor. 2
[52]
Mannosidostreptomycin Hydrolase
By
EDWARDINAMINEand ARNOLDL. DEMAIN NH II HN-- C --NH 2
\ ! O
L/OH
Streptidine 1
l Streptose OH
Streptomycin Mannosido-
l
streptomycin
O
l
N-Methyl-Lglucosamine
/ ~H
CII2OH
l D-Mannose Mannosidostreptomycin+ H20
* streptomycin+ mannose
638
ANTIBIOTIC INACTIVATION AND MODIFICATION
[52]
Mannosidostreptomycin hydrolase is an enzyme that transforms mannosidostreptomycin to streptomycin by the hydrolytic removal of the mannose moiety. 1 The two antibiotics are made concurrently during the course of a fermentation by strains of S t r e p t o m y c e s griseus. ~ The hydrolase is an inducible enzyme whose formation is subject to catabolite repression so t h a t it is usually synthesized late in a fermentation when the repressible carbon source is near depletion. 3-~
Assay Method Principle. The a-D-mannosidase enzyme is most conveniently assayed by use of the chromogenic substrate p-nitrophenyl-a-D-mannopyranoside. The p-nitrophenol liberated is determined spectrophotometrically at 400 nm2, 4 Reagents
Potassium phosphate buffer, 0.1 M, pH 7.2 Manganese acetate, 80 mM, prepared in 50 m M potassium phosphate buffer and the p H is adjusted to 7.2 with 20% K O H p-Nitrophenyl-a-D-mannopyranoside, 16.6 mM, in water Potassium carbonate, 0.2 M and 1.0 M Procedure. A reaction mixture containing 1.0 ml enzyme source, 4.0 ml buffer, 1.0 ml of manganese acetate, and 1.0 ml of water (which can be replaced by other additives) is equilibrated at 28 ° for 5 min in a r o t a r y water bath shaker at 275 rpm. The reaction is started by the addition of 1.0 ml of the substrate. Two controls, one without enzyme and the other without substrate, are run simultaneously. After 30 min, the incubation is terminated and color is developed by the addition of 2.0 ml of 1.0 M potassium carbonate. The mixture is centrifuged at 10,000 rpm for 3-5 rain. One milliliter of the clear supernatant fluid is diluted with 9.0 ml of 0.2 M potassium carbonate and the p-nitrophenol concentration determined on a Spectronic 20 colorimeter at 400 nm against the enzyme blank. Since the substrate itself undergoes a very slow hydrolysis during the incubation period, a substrate blank correction is applied. The standard curve is constructed with reagent grade p-nitrophenol. The com-
D. Perlman and A. F. Langlykke, J. Amer. Chem. Soc. 70, 3968 (1948). ~J. Fried and E. Titus, J. Biol. Chem. 168, 391 (1947). 8D. J. D. Hockenhull, G. C. Ashton, K. H. Fantes, and B. Y. Whitehead, Biochem. J. 57, 93 (1954). E. Inamine, B. Lago, and A. L. Demain, in "Fermentation Advances" (D. Perlman, ed.), p. 199. Academic Press, New York, 1969. 5G. Kollar, Acta Microbiol. Acad. Sci. Hung. 5, 19 (1958).
[52]
MANNOSIDOSTREPTOMYCI N HYDROLASE
639
pound is dissolved in 0.2 M potassium carbonate and its optical density at 400 nm is determined over a 0-20 n m o l e / m l range. Definition o] Unit Activity. One unit of activity is defined as t h a t amount of enzyme which catalyzes the release of 1 nmole of p-nitrophenol in 30 min. Substrate Source. p-Nitrophenyl-a-D-mannopyranoside can either t)e prepared by the method of Conehie and L e v v y ~ or purchased commercially from any number of sources. The natural substrate, mannosidostreptomycin, is not commercially available.
Properties Specificity. Streptomyces mannosidase hydrolyzes phenyl-~-D-mannopyranoside, mannosidostreptomycin, mannosidodihydrostreptomyein in addition to p-nitrophenyl-a-D-mannopyranoside. ',~,4 All the available d a t a on the properties of the enzyme are derived from studies done with crude preparations. Location. The Streptomyces enzyme is bound to the cell until lysis occurs. The cell-bound enzyme can be extracted into water, but its release is inhibited by sodium chloride, phosphate, or Tris. The enzyme t h a t is released by water is still attached to particulate matter which remains suspended at 10,000 r p m but is sedimented when centrifuged at 50,000 rpm. The activity of the cell-bound enzyme is not increased by lysozyme lysis, toluene treatment, or grinding with sand, so t h a t the enzyme appears to be bound at or near the cell surface and is readily accessible to the substrate. 7 Temperature and pH Optima. Optimmn temperature for the Streptomyces enzyme is 40 ° and it is rapidly inactivated at 50 ° while the p H for o p t i m u m activity is in the range of 7.0-8.0 depending on the strain of organism used. 1,:~,s Metal Requirement. The a-D-mannosidase of Streptomyces requires a divalent cation for activity. 7 The activity t h a t is lost by E D T A treatment is completely restored by Mn '-'+while Zn '-'+ and Ca ~+ are not as effective. Metals such as Cu -°+, H g 2+, and Fe ~+ inhibit the enzyme while K +, Mg ~+, and Ba '-'+ are without effect. ~,:~ Stimulators and Inhibitors. Arsenate is reported to stimulate the hydrolysis of phenyl-a-D-mannopyranoside while phosphate is without effect. :~,s Cyanide, fluoride, azide, bisulfite, and eysteine are reported to inhibit the enzyme to various degrees. 3,s Several sugars inhibit the activ"J. Conehie and G. A. Levvy, Methods Carbohyd. Chem. 2, 345 (1963). ' E. Inamine and A. L. Demain, Biotechnol. Bioeng. 12, 159 (1970). 8 G. Kollar, Acta Microbiol. Acad. Sci. Hung. 5, 11 (1958).
640
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53a]
ity of the enzyme; the inhibition elicited by the sugars varies depending on whether the synthetic or natural substrate is used. 3,7,9 Adequate aeration is required for maximum enzyme activity2 ,s Pretreatment of enzyme-containing cells with N-ethylmaleimide is reported to eliminate the aeration requirement. 7 , D. J. D. Hockenhull, Progr. Ind. Microbiol. 2, 133 (1960).
[53a] f l - L a c t a m a s e ( B a c i l l u s c e r e u s ) By DAvm R. THATCHER
The reactions catalyzed by the fl-lactamases and the methods for assaying these enzymes are described elsewhere in this volume. 1 Bacillus cereus produces two distinct fl-lactamases in high yield. These two enzymes have been called fl-lactamase I (EC 3.5.2.6) and fl-lactamase II (EC 3.5.2.8) 2'3 and are coded by two separate genes. 4 No immunological affinity, as tested by antibody absorption or by the effect of antisera on activity, has been demonstrated between them. 5 Both enzymes are found as cellular and extracellular types and are coordinately induced in the presence of low levels of penicillin. 6 Cell bound fl-lactamase is also found in an immunologically distinguishable, iodine sensitive state (y-penicillinase)7,s This distinct fl-lactamase species must not be confused with a number of "y-state" derivatives of extracellular fl-lactamase I which have been reported in the literature. 9,1o Although displaying many of the properties of y-penicillinase, these induced conformational states of fl-lactamase I are not y-penicillinase sensu stricto. The presence of a separate cephalosporinase activity in culture supernatants and cells of B. cereus has been knbwn since 19626 and the enzyme responsible for this activity, f~-lactamase II, was isolated from fl-lac1 This volume [5]. S. Kuwabara and E. P. Abraham, Biochem. J. 103, 27 (1967). s M. R. Pollock, Ann. N.Y. Acad. Sci. 151, 502 (1968). ' M. R. Pollock and J. Fleming, J. Gen. Microbiol. 59, 303 (1969). M. R. Pollock and J. Fleming, unpublished results, 1968. 6 B. Crompton, M. Jago, K. Crawford, G. G. F. Newton, and E. P. Abraham, Biochem. J. 83, 52 (1962). M. R. Pollock, J. Gen. Microbiol. 15, 154 (1956). " N. Citri and A. Kalkstein, Arch. Biochem. Biophys. 121, 720 (1967). "M. B. Rudzik, and J. Imsande, J. Biol. Chem. 245, 3556 (1970). 10N. Citri, Biochim. Biophys. Acla 27, 277 (1958).
640
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53a]
ity of the enzyme; the inhibition elicited by the sugars varies depending on whether the synthetic or natural substrate is used. 3,7,9 Adequate aeration is required for maximum enzyme activity2 ,s Pretreatment of enzyme-containing cells with N-ethylmaleimide is reported to eliminate the aeration requirement. 7 , D. J. D. Hockenhull, Progr. Ind. Microbiol. 2, 133 (1960).
[53a] f l - L a c t a m a s e ( B a c i l l u s c e r e u s ) By DAvm R. THATCHER
The reactions catalyzed by the fl-lactamases and the methods for assaying these enzymes are described elsewhere in this volume. 1 Bacillus cereus produces two distinct fl-lactamases in high yield. These two enzymes have been called fl-lactamase I (EC 3.5.2.6) and fl-lactamase II (EC 3.5.2.8) 2'3 and are coded by two separate genes. 4 No immunological affinity, as tested by antibody absorption or by the effect of antisera on activity, has been demonstrated between them. 5 Both enzymes are found as cellular and extracellular types and are coordinately induced in the presence of low levels of penicillin. 6 Cell bound fl-lactamase is also found in an immunologically distinguishable, iodine sensitive state (y-penicillinase)7,s This distinct fl-lactamase species must not be confused with a number of "y-state" derivatives of extracellular fl-lactamase I which have been reported in the literature. 9,1o Although displaying many of the properties of y-penicillinase, these induced conformational states of fl-lactamase I are not y-penicillinase sensu stricto. The presence of a separate cephalosporinase activity in culture supernatants and cells of B. cereus has been knbwn since 19626 and the enzyme responsible for this activity, f~-lactamase II, was isolated from fl-lac1 This volume [5]. S. Kuwabara and E. P. Abraham, Biochem. J. 103, 27 (1967). s M. R. Pollock, Ann. N.Y. Acad. Sci. 151, 502 (1968). ' M. R. Pollock and J. Fleming, J. Gen. Microbiol. 59, 303 (1969). M. R. Pollock and J. Fleming, unpublished results, 1968. 6 B. Crompton, M. Jago, K. Crawford, G. G. F. Newton, and E. P. Abraham, Biochem. J. 83, 52 (1962). M. R. Pollock, J. Gen. Microbiol. 15, 154 (1956). " N. Citri and A. Kalkstein, Arch. Biochem. Biophys. 121, 720 (1967). "M. B. Rudzik, and J. Imsande, J. Biol. Chem. 245, 3556 (1970). 10N. Citri, Biochim. Biophys. Acla 27, 277 (1958).
[53a]
~-LACTAMASE (Bacillus cereus)
641
tamase I in 1967. 2 Despite this evidence for the production of more t h a n one fl-lactamase in this organism, several reports have been published since which describe the preparation of fl-lactamase I and yet do not indicate the presence or absence of fl-lactamase II. ~ As fl-lactamase I I shares a number of properties with fl-lactamase I during their purification, the homogeneity of these preparations is certainly questionable.
Purification Discussion. The two strains of B. cereus which have been used almost exclusively in the preparation of fl-lactamases are the magnoconstiturive ': m u t a n t s B. cereus 5 6 9 / H ( N C T C ) and B. cereus 5/B ( N C T C ) . These organisms are conveniently stored as spore suspensions and can readily be checked for the constitutive production of fl-lactamase by plating on Andrade indicator agar. ~3 Cell-bound fl-lactamase m a y be released by autolysis, 7 t r e a t m e n t with trypsin, TM sonication, 8 and by the formation of an acetone powder. ~'~ Approximately 90% of the fl-lactamase produced by B. cereus is extracellular and consequently most of the methods available for enzyme purification involve the manipulation of large volumes of culture supernatant. The resulting problem of enzyme concentration has been overcome by a number of methods including evaporation of the culture supernatant under reduced pressure TM followed by acetone precipitation, ~7 adsorption on glass ''~ or Celite, TM :° and a m m o n i u m sulfate fractionation. 2~ Only ammonium sulfate fractionation and adsorption on Celite ~° have been used successfully to separate fl-lactamase I and II. As fl-lactamase I I was not looked for in the other preparations, its absence either as an active enzyme or as an inactive contaminating protein cannot be ascertained. Further purification of the fl-lactamase I of strain 5 6 9 / H has been
11See references cited in footnotes 14, 16, 24, and 34. "~J. F. Collins, J. Mandelstam, M. R. Pollock, M. H. Richmond, and P. It. Sneath, Nc~ture (London) 208, 841 (1965). 13M. Kogut, M. R. Pollock, and E. J. Tridgell, Biochem. J. 62, 391 (1956). ~V. CsAnyi, I. Mile, I. Ferencz, and E. Szabo, Acta Microbiol. Acad. Sci. Hung. 18, 7 (1971). S. Kuwabara and E. P. Abraham, Biochem. J. IlS, 859 (1969). ,s G. V. Patil and R. A. Day, Biochim. Biophys. Act¢z 293, 490 (1973). 1~M. R. Pollock, A. M. Torriani, and E. J. Tridgell Biochem. J. 62, 387 (1956). ~ N. Citri, N. Garber, and M. Sela, J. Biol. Chem. 235, 3434 (1960). ~9V. CsSnyi, I. Mile, I. Ferencz, and E. Szabo, Biochim. Biophys. Acta 198, 332 (1970). :0 R. B. Davies, E. P. Abraham, and J. Melling, Biochem. J. 143, 115 (1974). :~S. Kuwabara, Biochem. J. 118, 457 (1970).
642
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53a]
achieved by the application of cellulose phosphate chromatography,9,22 diethylaminoethyl cellulose chromatography,~1 carboxymethyl cellulose chromatography,s carboxymethyl Sephadex chromatography,2° ethanol precipitation, :1 and recrystallization.13,21 A commercial preparation of 569/H fl-lactamase I, Neutrapen, purified by chromatography on Amberlite IRC-50 ion exchange resin (U.S. patent 2,982,696) is available from Riker Laboratories, Northridge, California. This preparation is of low specific activity ~ and contains at least one other enzymically inactive component. ~ The specific activity of a fl-lactamase preparation has been defined as the number of micromoles of benzylpenicillin hydrolyzed per hour per milligram of protein nitrogen at 30 ° and pH 7.0. Kogut et al. ~ obtained a value of 2.02 X 106 U/rag N for a crystalline preparation of extracellular fl-lactamase I (569/H), which was homogeneous by ultracentrifugal and electrophoretic analysis. A similar value was also published in the same year for a crystalline preparation of the same enzyme from strain 5/B27 These values have become a yardstick for several other groups, and in a number of cases the specific activity of a preparation has been the only criterion of homogeneity presented. Kuwabara 21 obtained specific activities of 5.42 X 106 U/rag N for 569/H fl-lactamase I and 2.59 X 10G U/rag N for fl-lactamase II. Davies et al. 2° using a different method of purification obtained values of 3.3 X 106 U/mg N and 9.2 X 105 U/rag N for the extracellular fl-lactamase I and fl-lactamase II of the same strain as Kuwabara. The procedure outlined below has been used successfully to purify large quantities of extracellular fl-lactamase I and fl-lactamase II from both strains 569/H and 5/B. This method overcomes in a convenient manner the problem of concentrating the enzyme and finally produces homogeneous samples of fl-lactamase I and fl-lactamase II in reasonable yield. Stage I. Production. The bacteria are first checked for the constitutive production of fl-lactamase by plating on Andrade agar and incubating overnight at 37 °. Suitable penicillinase producing colonies are then used as inocula for a number of 5-ml shake cultures (S broth).25 After a few hours, each culture is checked for the production of fl-lactamase and examined microscopically for the sole presence of gram-positive bacilli. Portions (0.1 ml) of a viable culture are then used to inoculate 5 EhrlenL. Sabath and M. Finland, J. Bacteriol. 1511 (1968). M. R. Pollock, personal communication. 24j. Imsande, F. D. Gillin, R. J. Tanis, and E. G. Atherly, J. Biol. Chem. 245, 2205 (1970). 35M. R. Pollock and C. ft. Perret, Brit. J. Exp. Pathol. 32, 387 (1951).
[53a]
~-LACTAMASE (Bacillus cereus)
643
meyer flasks each containing 1 liter of sterile casamino acid-citrate medium ( C H / C ) . 0-6 These 5 flasks are then incubated overnight in shake culture at 37 ° and will produce an actively growing culture suitable for inoculating a bulk culture. Two hundred liters of C H / C medium are sterilized in a 250-liter fermenter (the citrate in this case is sterilized separately and added later). The medium is equilibrated to a temperature of 37 °, and 5 liters of inoculum are added. Aeration is switched on, and the culture temperature is held constant. After 3 hr, sampling of the culture is initiated. Turbidity and fl-lactamase levels are determined, and the rate of growth of the culture is followed closely. Stage II. fl-Lactamase ConcentrationY ~ When an opacity equivalent to 1.5-1.8 mg/ml (dry weight) has been reached, the pH of the culture is adjusted to 7.0 with sterile 2 M hydrochloric acid. Approximately 3 kg of commercial Celite ~s (prewashed first in tap water and then distilled water) are added to the growing culture. Growth is continued for 45 rain to 1 hr before two final samples are taken. The Celite in one of the samples is removed by centrifugation, and the B-lactamase activity of the supernatant is determined. The fl-lactamase levels are determined in the other samples in the presence of Celite. As the enzymes are still active while adsorbed to the Celite, an estimation of the proportion of the enzyme left in the liquid phase may be calculated. Most of the extracellular B-lactamase produced should be adsorbed to the Celite (__90%). The fermenter is now switched off, and the Celite is allowed to settle out over a period of 15 min. The culture supernatant containing most of the cellular material may be drained from the bottom of the fermenter without disturbing the Celite layer. The Celite can then be rinsed with 50 liters of distilled water in the fermenter using a similar method. Finally the Celite is suspended in a minimum of tap water and allowed to settle in a 200 cm X l0 cm glass column. After 12 hr the supernatant is removed from the Celite column and elution initiated with a Tris-citrate buffer (0.1 M Tris-citrate, pH 7.0; 1.5 M with respect to sodium chloride and 1 m M with respect to zinc acetate). Fractions of 250 ml are collected and each is assayed for fl-lactamase activity. The fractions containing the enzymes are dialyzed exten~Oxoid casamino acids 10 g/liter, potassium dihydrogen phosphate 2.7 g/liter, 1 M trisodium citrate 50 ml/liter and trace elements. 27The basis of stage II of this procedure was developed by R. Davies and E. P. Abraham of the Department of Pathology, University of Oxford, U.K., in conjunction with Dr. J. Melling of the Microbiological Research Establishment, Porton Down, Wilts. U.K. 2sCelite 545 is obtainable from Johns-Manville, New Jersey.
644
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53a]
sively against 5 mM N-ethylmorpholine acetate buffer, pH 7.0 (1 mM with respect to zinc acetate) and lyophilized. Stage III. Separation o] fl-Lactamase I and fl-Lactamase II. The freeze dried protein from the previous step is carefully dissolved in a minimum of 10 mM N-ethylmorpholine acetate buffer, pH 7.0 (1 mM zinc acetate). After adjusting the conductivity of the protein solution to that of the original buffer by dilution with distilled water, the pH is checked. A column of Whatman CM-52 carboxymethyl cellulose (H. Reeve, Angel & Co. Ltd., London EC4 6AY) (10 cm X 2.5 cm) is equilibrated with the same buffer and the protein solution applied at a flow rate of 40 ml/hr. The column eluate is checked for the absence of fl-lactamase activity before eluting with a 600-ml buffer gradient, increasing linearly in sodium chloride concentration to a molarity of 0.4 M; buffer and zinc concentrations remaining constant. fl-Lactamase II of B. cereus 5/B elutes first, well separated from a peak of fl-lactamase I. Traces of fl-lactamase I activity may be removed from the fl-lactamase II fractions by heat treatment (60 ° for 10 min). B. cereus 569/H gives a more complicated separation pattern. This strain produces a heterogeneous form of extracellular fl-lactamase I so that fl-lactamase II cannot be completely resolved in one step, as is the case with 5/B. Figure 1 describes a typical elution profile for the extracellular fl-lactamase of 569/H. fl-Lactamase II may be separated from the acidic species of fl-lactamase I by rechromatography on a column of CM-52 carboxymethyl cellulose using a less steep elution gradient. The relevant fractions are pooled and dialyzed against the N-ethylmorpholine buffer. The protein solution is then applied to a column of equilibrated CM cellulose (2.5 X 10 cm) and eluted with a gradient increasing in sodium chloride concentration to 0.2 M (Fig. 2). The method described above produces preparations of fl-lactamase I and fl-lactamase II which are homogeneous on polyacrylamide disc gel electrophoresis at pH 4.0 and at pH 7.0 in the presence of SDS. Table I summarizes the details of a 569/H purification carried out in this laboratory.
Properties of/~-Lactamase I Stability. Although stable for several hours at room temperature in dilute solution, handling of concentrated solutions requires care. At 37 ° a 10 mg/ml enzyme solution will be visibly denatured within 15 rain and completely denatured by treatment at 50 ° for 10 rain. The enzyme is also susceptible to surface denaturation and must never be agitated violently. Special care must obviously be taken when dissolving freeze-dried
[53a]
~-LACTAMASE
(Bacillus cereus)
645
3.0
\
! z.0
~
I
/ /~\
/
\ i.o
/
/
\ x
o.o
2
I
10
I
I
I
30
i',,
50
70
Fraction Number
FIG. 1 The elution profile obtained during the first carboxymethyl cellulose chromatography step, A column (10 X 2.5 cm) of Whatman CM 52 cellulose was eluted with a linear gradient (0 to 0.4 M) of increasing sodium chloride concentration, Conductivity measurements and total fl-lactamase activity ( 0 - - 0 ) were recorded for every third fraction. The solid line is proportional to the absorbance of the effluent at 254 nm and was monitored using a contiauous recording absorptiometer. The first peak eluted contained all the t~-lactamase II activity and some t~-lactamase I activity.
material. The fl-lactamase I of B . c e r e u s 5/B should not be exposed to solutions of high ionic strength a s this treatment appears to affect its subsequent solubility in dilute solution. The enzyme can tolerate pH 8.0
~_
~-- 6.O
E
"•2.C
/
~,
~ i
~,
~', ......... .
,,'"
0
1.0 w,-
~•
; ....~....;
2.o
,,
o z
0.~
0.0
I
20
L 4o
Fraction
60
8o
Number
FIG. 2. The elution profile obtained during rechromatography of the fractions from Fig. I containing both 18-1actamase I and fl-lactamase II. Conditions are as in Fig. 1 except that the enzymes were eluted with a linear gradient of 0 to 0.25 M sodium chloride (total volume 600 ml).
646
ANTIBIOTIC
INACTIVATION
AND
MODIFICATION
¢D
8
~z
o
~T ~x
e~ ©
I~ ~ 0
~z
~× O
•~ ? r~
8
~x
O
0 Z © [...
.~ o~
"r~
[53a]
[53a1
~-LACTAMASE (Bacillus cereus)
647
TABLE II SUBSTRATI~; PROFILE OF THE EXTRACELLULAR ~-LA('TAMASE
I OF
Bacillus cereus 569/H ~ Subst,rate
Relative rate of hydrolysis (benzylpenieillin taken as 100%)
Benzylpenieillin Methicillin Ampieillin 6-Aminopenicillanic acid Cephalosporin C Cephaloridine Benzylcephalosporin C
100.0 4,0 194.0 12.0 0.1 1.0 0.1
J. Fleming, unpublished results. ranges between 5.0 and 9.0 with no loss in enzymic activity and m a y be renatured after t r e a t m e n t with urea or guanidine hydrochloride. ~'~' Kinetic Properties (569/H). The enzyme has a broad p H optimum between 6.0 and 7.0 and displays a characteristic substrate profile (Table I I ) similar to t h a t of m a n y other gram positive fl-lactamases 3° Phenoxymethylpenicillin and ampicillin are hydrolyzed at rates similar to t h a t of benzylpenicillin, whereas the enzyme degrades methicillin, oxacillin, cloxaeillin, and cephalosporins at low rates. Cell bound ,/-penicillinase displays a different substrate profile, notably in an increased ability to hydrolyze methicillin, a' M a n y penicillins which are poor substrates for fl-lactamase I also act as competitive inhibitors of activity with benzylpenicillin or in some cases irreversible inhibitors. Michaelis constants have been determined for a number of substrates, and a value of 60 uM has been recorded for benzylpenicillin. 3 Chemical Properties (569/H). The amino acid composition of fl-lactamase [ is shown in Table I I I . The enzyme consists of a single polypeptide chain of approximately 270 residues and the partial sequence data available show t h a t the molecule is chemically homologous to the other gram-positive fl-lactamases which have been sequenced2 -~ Earlier hypotheses based on immunological and enzymological criteria have therefore been confirmed. 3a ~"R. Davies, E. P. Abraham, and D. G. Dalgleish, Biochem. J. 143, 137 (1974). ~0N. Citri, in "The Enzymes" (P. Boyer, ed.), 3rd ed., Vol. 4, pp. 23-46. Academic Press, New York, 1971. '~'E. Ron-Zenziper and N. Citri, Nature (London) 198, 887 (1963). "~D. R. Thatcher, unpublished results. ~ M. R. Pollock, Proc. Roy. Soc. London B 179, 385 (1971).
648
[53a]
ANTIBIOTIC INACTIVATION AND MODIFICATION
TABLE I I I AMINO ACID COMPOSITION OF THE EXTRACELLULAR~-LACTAMASE I OF Bacillus cereus 569/H a Asp Thr Ser Glu Pro Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg Trp
31 b 13 5 22 10 19 33 17 1 22 18 4 6 5 21 10 --
34 c 24 13 24 9 20 33 18 4 17 20 11 8 4 21 11 5
31 a 21 15 23 13 20 28 13 4 16 18 8 8 3 21 11 3
28¢ 17 9 22 6 17 27 15 3 18 16 7 6 3 19 12 2
3V 17 14 25 9 19 27 17 3 19 18 8 7 3 22 12 4
33g 22 13 25 10 21 30 15 4 22 19 9 7 4 23 13 3
a The values are residues per mole. b N. C~tri and M. R. Pollock, Advan. Enzymol. 28, 237 (1967). c j. Imsande, F. D. Gillin, R. J. Tanis, and A. G. Atherly, J. Biol. Chem. 245, 2205 (1970). d S. Kuwabara, E. P. Adams, and E. P. Abraham, Biochem. J. 118, 475 (1970). e I. Madaiah and R. A. Day, Biochim. Biophys. Acta 286, 191 (1971). ] R. Davies and E. P. Abraham, in preparation. g D. R. Thatcher, unpublished results. P e p t i d e m a p s of a n u m b e r of p r e p a r a t i o n s are n o w r e c o r d e d in t h e l i t e r a t u r e 16,24,3~ a n d t h e N - t e r m i n u s h a s been r e p o r t e d as a s p a r t i c acid a5 or lysine. 24 B o t h of th e s e results are in f a c t c o m p a t i b l e w i t h t h e sequence d a t a a v a i l a b l e . L i k e t h e e x t r a c e l l u l a r f l - l a c t a m a s e p r o d u c e d b y Bacillus licheni]ormis 7 4 9 / C , e x t r a c e l l u l a r f l - l a c t a m a s e I has a r a g g e d a m i n o - t e r m i n a l region. N H2-Lys-His-Lys-Asx-Glx-Ala-Thr-His-Lys- Glu-Phe-Glx-Ser N H r His-Lys-Asx-Glx-A1a-Thr- His-Lys- Glu-Phe- Glx-Ser N H~-Lys-Asx-Glx-Ala-Thr-His-Lys-Glu-Phe-Glx-Ser N H ~-Asx-Glx-Ala-Thr-His-Lys- Glu-Phe- Glx-Ser
.... .... .... ....
T h i s r a g g e d a m i n o - t e r m i n u s is sufficient to e x p l a i n t h e h e t e r o g e n e i t y of f l - l a c t a m a s e p r e p a r a t i o n s in p o l y a c r y l a m i d e gel electrophoresis, 24 isoelectric focusing, 2° a n d d u r i n g c a r b o x y m e t h y l cellulose c h r o m a t o g r a p h y . "~I. Madhaiah and R. A. Day, Biochim. Biophys. Acta 236, 191 (1971). Published by Pollock and Citri ~ as a personal communication to the authors from M. H. Richmond.
[53a]
~-LACTAMASE (Bacillus cereus)
649
P h y s i c a l Properties. Analytical ultracentrifugation d a t a indicate t h a t fl-lactamase I has an apparent weight average molecular weight of 28,000, an s..,o,w of ca. 2.75, and a diffusion coefficient of 8.28 X 10 -~ cm2sec-~? 6 A frictional ratio of 1.22 has also been calculated, 37 and optical r o t a r y dispersion (ORD) and circular dichroism (CD) studies show the presence of some secondary structure. ~8,24,38 Isoelectric focusing on a highly purified preparation resolved the enzyme into three components of p I 9.25, 9.5, and 9.73 o M o d i f i c a t i o n R e a c t i o n s and C o n ] o r m a t i o n a l Changes. Specific chemical modification of fl-lactamase I has been claimed by the use of 6-aminopenicillanic acid in the presence of nitrite, iodine, and tetranitromethane. As yet, there is no direct evidence to suggest t h a t the residues modified lie in the active site although the diazotized 6-aminopenicillanic acid t r e a t m e n t ~ and iodination bring about a reduction in catalytic activity. The mechanism of the inactivation of fl-lactamase I by iodine has been extensively studied by Csanyi and his group. ~4,~',3~'-~ Iodine sensitivity appears to be dependent on the conformation of the enzyme but can be controlled in two steps by regulating both the temperature of the reaction and the pH. One step, bringing about a 35-30% reduction in activity, occurs at 30 ° at p H 6.0 or at 0 ° at p H 9.0. The second step, leading to complete loss in enzyme activity, ~akes place under more rigorous conditions and reflects the nonspecific iodination of the protein in a denatured state. In the first reaction iodine reacts specifically with a single tyrosine residue. ~,:~ This is the same residue which is also sensitive to nitration by tetranitromethaneY-' The amino acid sequence around this reactive residue has been determined as: • . . Thr-Lys-Glu-Asp-Leu-Val-Asp-TYR-Ser-Pro-Val-Thr-Ghl
Lys-His-Val-Asp-Thr-Gly-Met-Lys . . . The residue which reacts specifically with tetranitromethane in B. liche~i]ormis and S t a p h y l o c o c c u s aureus, T Y R 77, is chemically homologous with the tyrosine of fl-lactamase I. ~'-' The ability of fl-lactamase I to exist in both an iodine-sensitive and an iodine-resistant state has been exploited as a probe for the induction ~ P. H. Lloyd and A. R. Peacocke, Biochem. J. 118, 467 (1970). ~ J. 1~. Hall and A. G. Ogston, Biochem. J. 62, 401 (1956). :~sD. G. Dalgleish and A. R. Peacocke, Biochem. J. 125, 155 (1971). 33I. Mile, V. Cshnyi, and I. Ferencz, Acta Biochem. Biop]~y.q. Acad. Sci. H~o~g. 5, 33 (1970). 4oV. Csanyi, I. Ferencz, and I. Mile, Biochim. Biophys. Acta 236, 619 (1971). 41V. Csanyi, I. Ferencz, and I. Mile, Biochim. Biophys. Acta 243, 484 (1971). 4 : R. P. Ambler and R. J. Meadway, Nature (London) 222, 24 (1969).
650
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53a]
of conformational changes. The conditions under which the iodine sensitive residue(s) are exposed are treatment with solutions of urea, guanidine hydrochloride, or ammonium sulfate adsorption on charged surfaces and exposure to a number of penicillin derivatives. 43 As well as changing the sensitivity of the enzyme to iodine, conformational changes induced in fl-lactamase I change immunological and optical properties as well as the protein's resistance to proteolytic attack. It is apparent that fl-lactamase I is able to exist in a number of distinct, stable conformations, the physiological significance of which is still obscure.
Properties of B-Lactamase II (569/H)
Stability. fl-Lactamase II is more thermostable than fl-lactamase I, a property which was partially responsible for its original discovery. Traces of fl-lactamase I activity, which has a much higher specific activity than fl-lactamase II may be removed by heat treatment as described earlier. No problems have been encountered during the manipulation of concentrated enzyme solutions and the enzyme may be stored either as a frozen solution or as a freeze dried powder with no loss in activity. The enzyme has a zinc cofactor and therefore must not be exposed to chelating buffers such as phosphate or ethylenediamine. Dialysis is best performed against buffers millimolar with respect to zinc acetate. Prolonged dialysis in the absence of zinc will bring about gradual inactivation. Kinetic Properties. fl-Lactamase II is a broad-spectrum fl-lactamase. In marked contrast to fl-lactamase I, the enzyme can hydrolyze cephalosporin C, cephaloridine, cephalothin, and cephaloglycin at rates approaching that of the hydrolysis of benzylpenicillin. The substrate profile for extracellular fl-lactamase II is shown in Table IV. Michaelis constants for a number of substrates are available and all are much higher than the corresponding constants of fl-lactamase I. a A constant 3.3 mM has been recorded for benzylpenicillin and one of 1.1 mM for cephalosporin C. Chemical Properties. fl-Lactamase II contains at least two zinc binding sites, the presence of zinc at the site of highest affinity being essential for enzymic activity. 44 The amino acid composition of fl-lactamase II is shown in Table V. A number of obvious differences in composition between fl-lactamase II 4~ N. Citri presents a detailed account of fl-lactamase I conformational changes in a general review of the conformational response in proteins: Advan. Enzymol. 37, 397 (1973). R. Davies and E. P. Abraham, Biochem. J. 143, 129 (1974).
f~-LACTAMASE ( B a c i l l u s cereus)
[53al
651
TABLE IV SUBSTRATE PROFILE OF THE EXTRACELLULAR ~-LACTAMASE I I OF
Bacilluscereus 5 6 9 / H •
Substrate
Relative rate of hydrolysis (benzylpenicillin taken as 100%)
Benzylpenicillin Methicillin Ampicillin 6-Aminopenicillanic acid Cephalosporin C Cephaloridine Benzylcephalosporin C
100 120 47 10 14 18 45
J. Fleming, unpublished results.
TABLE V AMINO ACID ANALYSIS OF THE EXTRACELLULAR ~-LACTAMASE I I OF
Bacillus cereus 569/H~ Asp Thr Ser Glu Pro Gly Ala Val Cys Met Ile Leu Tyr Phe His Lys Arg Trp
25 b 12 10 18 7 18 l0 22 1 2 11 23 4 3 5 19 4 5
.23 ~ 14 11 18 8 18 15 15 1 2 17 22 5 4 4 16 4 2
32 ~ 18 14 22 8 19 25 14 1 3 16 20 8 7 4 27 11 2
24 e 15 10 18 5 19 11 20 1 2 11 23 5 4 6 19 5 2
The values are residues per mole. b R. Davies and E. P. A b r a h a m , in preparation. c S. K u w a b a r a and P. H. Lloyd, Biochem. J. 124, 215 (1971). C a r b o h y d r a t e free preparation. d S. Kuwabara, E. P. Adams, and E. P. A b r a h a m , Biochem. J. 118, 475 (1970). Complexed with carbohydrate. D. R. Thatcher, unpublished results.
652
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53a]
and fl-lactamase I are evident, notably the presence of a single cysteine residue. Although the presence of cysteine has been shown in a number of gram-negative fl-lactamases this amino acid has not been shown to occur in other gram-positive fl-lactamases. 4~ fl-Lactamase I I prepared by the method of K u w a b a r a contains approximately 20% carbohydrate. 4~ This c a r b o h y d r a t e moiety dissociated on aging 47 and has not been found in homogeneous fl-lactamase I I preparations isolated by a method involving adsorption on Celite. 2° Physical Properties. The enzyme has a molecular weight of 22,50047 as determined by an analytical ultracentrifuge method. This value increases considerably when complexed with carbohydrate. Isoelectric focusing revealed a p I of 8.7 for most of the fl-lactamase II, with approximately 10% having a value of 10.0. C D spectra of fl-lactamase I I have been t a k e n in the presence and in the absence of carbohydrate28 Inhibition and Modification. Chelating agents such as E D T A and 1,10-phenanthroline inhibit the activity, but the enzyme m a y be subsequently reactivated by the addition of zinc ions. 48 This activation has been inhibited by the exposure of the zinc free enzyme to N - e t h y l m a l i mide, p-chloromercuribenzoate or Ellman's reagent. 44 The effect of p-chloromercuribenzoate and Ellman's reagent can be reversed by reduction with dithiothreitol. 2-~,44 fl-Lactamase I I therefore contains a single cysteine residue, the side chain of which is exposed only on removal of zinc from the active site. Although this essential cysteine residue need not necessarily be present at the active center of the enzyme, spectra of fl-lactamase I I whose zinc has been substituted by cobalt or cadmium indicate t h a t the thiol group could well be involved in metal binding at the site of highest affinity. 44
Acknowledgments
The author is grateful to Mrs. K. MacNaughton for technical assistance and to D. Waerclerle for typing the script. I am also indebted to Miss J. Fleming and Professor E. P. Abraham for making many unpublished results available. I also wish to express gratitude to Dr. R. P. Ambler and Professor M. R. Pollock for their criticism and encouragement during the preparation of this manuscript.
45 M.
R. Pollock and N. Cirri, Advan. Enzymol. 28, 237 (1966). 4BS. Kuwabara, E. P. Adams, and E. P. Abraham, Biochem. J. 118, 475 (1970). l~ S. Kuwabara and P. H. Lloyd, Biochem. J. 124, 215 (1971). ¢"L. Sabath and E. P. Abraham, Antimicrob. Ag. Chemother. 392-397 (1965).
~-LACTAMASE (Bacillus licheniformis)
[53b1
653
[53b] ¢~-Lactamase (Bacillus l i c h e n i f o r m i s ) By DAVID R. THATCHER Bacillus licheni]ormis is one of the most powerful producers of fl-lactamase known. W o r k on the structure and properties of this enzyme has largely been confined to two strains, B. licheniformis 749 ' and B. liche~riformis 6346. ='These strains on original isolation were classified as Bacill'us subtilis but were later reclassified as B. licheniformis, largely on their ability to produce gas on the anaerobic reduction of nitrate. Indeed, further investigation has shown t h a t in a wide range of B. subtilis isolates, if synthesis of fl-lactamase is at all detectable (it is of relatively rare occurrence) only low levels are produced. Bacillus licheniformis isolates on the other hand are magnoinducibleY The fl-lactamases synthesized by the two original strains 749 and 6346 show distinct physiological, enzymological and structural differences2 Examination of the fl-lactamases of B. licheniformis strains isolated on a global scale, has shown t h a t these two strains in fact t y p i f y two m a j o r classes of penicillinase in this organism2 Bacillus licheniformis produces both cell bound and extracellular fllaetamases, approximately 50% of the total enzyme synthesized being secreted. The enzyme is however coded by a single chromosomal gene, which has been mapped by transformation analysisY At least two other genes arc involved in the regulation of fi-lactamase synthesis.
Purification Discussion. Pollock isolated magnoconstitutive ,3 mutants of 749 and 6346, called B. licheniformis 749/C and B. licheniformis 6346/C respectively, r Cell-bound and extracellular enzyme from both 749/C and 6346/C were later purified to electrophoretic and analytical ultracentrifugal homogeneity2 The extracellular enzyme was concentrated from the supernatant by adsorption on cellulose phosphate and was further puri~Submitted to NCIB or obtainable from Miss J. Fleming, University of Edinburgh, U.K. " NCTC 6346 (ATCC 9800 and NCIB 6346). 3j. F, Collins, J. Mandelstam, M. R. Pollock, M. H. ttichmond, and P. A. Sneath, Nature (London) 208, 841 (1965). 4 M. R. Pollock, Biochem. Y. 94, 666 (1965). 5 M. t3. Pollock, unpublished observations. D. Sherratt and J. Collins, J. Gen. Microbiol. 217, 217 (1973). M. R. Pollock, Biochim. Biophys. Acta 76, 80 (1963).
654
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53b]
fled by ammonium sulfate fractionation. The most convenient and effective method of releasing cell bound fl-lactamase appears to be the treatment of lysed cells with trypsin, s However, exposure of B. licheniformis cells to the action of chymotrypsin, pronase, or an aqueous solution of butan-l-ol (4% v / v ) also releases the greater part of the enzyme attached to the cells2 ,1° Lampen found that membrane-bound fl-lactamase may also be released by the action of sodium deoxycholate2 Trypsin-released fl-lactamase was purified by Pollock by a combination of ammonium sulfate fractionation and DEAE-cellulose chromatography. 4 This method was later adapted by 1V[eadway who isolated enough material to determine the complete amino acid sequence of the 749/C enzyme, z° The use of affinity chromatography has been introduced for the small scale purification of B. licheni]ormis fl-lactamase2 ~ The affinity column was prepared by the treatment of CNBr activated Sepharose 4B with cephalosporin C. A crude preparation of fl-lactamase was applied to this column at pH 4.0 and, on elution at pH 6.0, fl-lactamase of near electrophoretic homogeneity was released. The column was found to be reusable with no apparent loss in resolution. Affinity chromatography using a column of ampicillin linked to glutaraldehyde-treated indubiose has been successfully employed in the purification of fl-lactamases from other sources and could well be applied to the B. licheni]ormis system, lz Production. Organisms are preserved as aqueous spore suspensions and are checked for the constitutive synthesis of fl-lactamase before use. ~3 Suitable penicillinase-producing colonies are then used as inocula for each of 10 Erlenmeyer flasks containing 100 ml of glutamate medium24 These flasks are then incubated at 37 ° overnight on a high speed shaker. The liter of exponentially growing bacteria produced is used as an inoculum for a 9-liter batch of sterile glutamate medium in a small commercial chemostat. The pH and nutrient levels in the culture are maintained and the degrees of aeration and foaming are controlled automatically. The bacterial growth rate and level of fl-lactamase production are carefully monitored so that the rate of removal from the fermenter is neither too slow nor too rapid. In the case of 6346/C the culture must be cooled 8D. J. Kushner and M. R. Pollock, J. Gen. Microbiol. 26, 255 (1961). 9j. O. Lampen, J. Gen. Microbiol. 48, 249-259 (1967). 1oR. J. Meadway, Ph.D. Thesis, University of Edinburgh, 1969. ~ L. J. Crane, G. E. Bettinger, and J. O. Lampen, Biochem. Biophys. Res. Commun. 50, 220 (1973). 12F. Le Goffic, R. Labia, and J. Andrillon, Biochim. Biophys. Acta 315, 439 (1973). ~sAs described in the production of Bacillus cereus fl-lactamases, this volume [53a]. ~'Monosodium glutamate 40 g/liter; ammonium sulfate 2 g/liter; potassium dihydrogen phosphate 5.5 g/liter; dipotassium hydrogen phosphate 14.6 g/liter; magnesium sulfate 0~22g/liter, and trace elements.
[53b]
~-LACTAMASE (Bacillus licheniformis)
655
immediately to 4 °, as this strain has a tendency to lyse in stationary culture. Cells are separated from the culture by the use of a continuous centrifuge. The supernatant enzyme should be processed immediately, but cells of both strains may be stored at --15 ° for some years with no detectable loss in fl-lactamase activity. Due to a large difference in specific activity between the two strains the number of units of penicillinase activity in a culture of 749/C may be much higher than 6346/C. Strain 749/C produces a total of approximately 4000 U/mg dry weight (1 unit is the number of micromoles of benzylpenicillin hydrolyzed per hour), and B. licheniJormis 6346/C about 600 U/mg dry weight. Purification o] Extracellular fl-Lactamase. The cell-free culture supernatant is dialyzed overnight against running tap water in 1.5-inch diameter Visking tubing. The glutamate culture medium is of relatively high ionic strength, and an adequate volume in the dialysis tubing must be allowed for osmotic expansion. A large column (30 X 8 cm) of carboxymethyl cellulose (Whatman CM-11 carboxymethyl cellulose, M. Reeve Angel & Co. Ltd., London EC 4, U.K.) is equilibrated with 10 mM ammonium acetate buffer, pH 4.8. The dialyzed culture supernatant is adjusted to the same pH value by the judicious addition of 2 M acetic acid and is then passed through the CM-cellulose column. The activity of the eluate is determined and should not exceed 1% of the fl-lactamase activity of the original enzyme solution. The enzyme is eluted from the column with 0.1 M ammonium acetate buffer, pH 8.5. Fractions of about 250 ml may be collected manually. The fractions containing the bulk of the enzymic activity are dialyzed extensively against l0 mM ammonium acetate buffer, pH 8.5, and lyophilized. The freeze dried enzyme is dissolved in a minimum of 10 mM ammonium acetate buffer, pH 8.5, and the concentrated protein solution is applied to a column of Sephadex G-100 (100 cm X 5 cm) equilibrated with 50 mM ammonium acetate, pH 8.5. Figure 1 describes an elution profile obtained during the purification of the extracellular fl-lactamase of 6346/C. Fractions which make up the fl-lactamase peak are pooled, dialyzed against 10 liters of distilled water and lyophilized. Purification of the Cell-Bound Enzyme. Frozen cells are thawed overnight at 4 °. They are then placed in a blender with 10 mM sodium phosphate buffer, pH 7.0 (1 liter/kg cells). Blending is conducted for l0 min before the addition of lysozyme (200 mg/kg cells), ribonuclease (50 mg/kg cells), and deoxyribonuclease (50 mg/kg cells). After 1 hr the viscosity of the solution will have noticeably decreased and enzyme release can be initiated by the addition of 50 mg of trypsin per kilogram of cells. The mixture is then stirred overnight at 37 ° in a stoppered flask.
656
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53b]
C~ 160 N
~
120
A
.£ 8o •2
40
g
I
I 40
t
1,,~
Fraction
80
120
NO.
Fla. 1. The elution profile obtained during final Sephadex G-100 chromatography of the extracellular fl-lactamase of Bacillus licheni]ormis 6346/C. The solid line represents a proportion of the absorbance at 254 nm measured with a Uvicord II, ultraviolet absorptiometer (detector unit type 8308A, LKB-Producter, Stockholm, Bromma 1, Sweden), and the dashed line ( O - - - O ) is proportional to the activity of the enzyme against benzylpenicillin. Fractions of 10 ml were collected, at a flow rate of 150 ml/hr.
The cell debris is removed by centrifugation and the supernatant cooled to 0 °. Solid ammonium sulfate is added to the protein solution to a saturation of 75% (20 °) and the mixture allowed to equilibrate to room ternperature. The precipitate which forms is removed by centrifugation at 20,000 g for 1 hr. After raising the ammonium sulfate concentration to saturation the solution is left overnight without strirring to allow flocculation of the fl-lactamase containing precipitate. The enzyme is then collected by centrifugation at 20,000 g for 5 hr, and the supernatant is discarded. The precipitate is dissolved in a minimum of 10 mM ammonium acetate, pH 8.5, and dialyzed extensively against the same. A column of diethylaminoethyl cellulose (Whatman DE-11 DEAE cellulose, H. Reeve Angel & Co Ltd., London EC4 U.K.) 20 X 8 cm is equilibrated with 10 mM ammonium acetate buffer, pH 8.5, and the dialyzed fl-lactamase solution applied. The eluate is checked for the absence of fl-lactamase activity before eluting with 0.2 M ammonium acetate, pH 8.5. The penicillinase cochromatographs with a eytochrome c component so that fractions may be collected manually. In the case of the 6346/C enzyme, elution of the DE-cellulose column with the equilibration buffer will eventually remove the enzyme. The cytochrome c comportent runs just ahead of the fl-lactamase and the two protein fractions may be separated at this stage. A final gel filtration step using a 5 X 100 cm column of Sephadex G-100, as described in the preparation of the exoenzyme, produces a homogeneous sample of the trypsin released en-
~-LA_CTAMASE (Bacillus licheniformis)
[53b1
E
cO
657
1.6
5.0
4.0
x
1.2
3.0
2~ e,-
O.8
c~ c~
2.0 0.4
1.0 ' -
0.0
60
5O
7O
8O
90
Fraction No.
FIG. 2. The elution profile obtained during Sephadex G-100 chromatography of the trypsin-released ¢Mactamase of Bacillus lichen]ormis 749/C. The absorbance of each fraction was determined at 280 n m ( 0 O ) amt 425 nm ( A A ) . The dashed line ( Q - - - G ) represents the activity of the enzyme against benzylpenicillin.
zyme. The cytochrome c component contaminating the batch eluted 749/C fl-lactamase fractions from the DE-cellulose colmnn, are separated at this stage, and Fig. 2 describes the elution profile obtained. The details of a preparation of the fl-lactamase of B. licheniformis 749/C are summarized in Table I. The fl-lactamase of both 6346/C and 749/C, isolated by the method described above, are homogeneous and show single bands on polyacrylamide disc gel electrophoresis in the presence of 8 M urea, pH 4.0. Multiple bands are observed, however, on starch gel electrophoresis at pH 8.45. Strain 749/C fl-lactamase gives three bands whereas 6346/C gives only two bands of enzymic activity which are of slightly higher mobility. The pattern of separation obtained by starch gel electroTABLE I PURIFICATION OF THE TRYPSIN-P~I,;LI,'ASED ~-LA(vPAMASE OF
Bacillus licheniformis 749/C
Fraction Crude lysate Trypsin released A m m o n i u m sulfate supernatant After D E A E cellulose After G-100 Sephadex
VolTotal ume activity (ml) (units X 10-*)
Protein concentration (mg/ml)
Specific activity (units/ttg)
Yield (%)
Purification factor
1800 1600 800
400 356 274
73.3 32.0 12.2
3.0 7.0 28.0
100 89 69
1.0 2.5 7.3
1100
196
1.3
150.0
54
50.0
200
162
2.4
337.0
40
ll3.0
658
ANTIBIOTIC INACTIVATION AND MODIFICATION
.=_
[53b]
6_
7_
O Q
o
O
A
B
C
O
E
:FIG. 3. Starch gel electrophoretic patterns of 749/C and 6346/C fl-lactamase preparations. Enzymically equivalent amounts were applied to the starch gel, and electrophoresis was performed at 48 mA for 4.5 hr in 0.33 M sodium borate buffer pH 8.45. Spots correspond to the varying intensities of dye which were liberated on adding a concentrated solution of benzylpenicillin to the gel after soaking in a saturated aqueous solution of N-phenyl-l-naphthylamine-azo-o-carboxybenzene. (A) Crude 749/C supernatant enzyme; (B) purified 749/C supernatant enzyme; (C) purified 749/C enzyme released from cells by trypsin; (D) crude 6346/C supernatant enzyme; (E) purified 6346/C from both extracellular and cellular locations. Taken from M. R. Pollock, Biochem. J. 94, 670 (1965). phoresis can be observed both by the usual protein staining techniques and by staining for penicillinase activity. The enzyme m a y be located after spraying the gel with a solution of 0.15 M benzylpenicillin, in 16 m M iodine, 60 m M with respect to potassium iodide. The stain is only transient and the pattern obtained must be photographed immediately if direct comparison with the bands obtained on protein staining is required. Differences are observed between exo- and trypsin-released enzyme, usually involving the intensification of a particular band (Fig. 3).
Properties Stability. Bacillus licheni]ormis fl-lactamases are quite thermostable and m a y be kept at room temperature for long periods without denaturation, and preparations m a y be stored frozen at - - 1 5 ° for m a n y years without loss in activity. I f the enzyme is stored at room temperature for longer than 24 hr, solutions should be made millimolar with respect to sodium azide to prevent bacterial growth. No denaturation due to surface interactions has been encountered, and the enzyme m a y be safely concentrated by r o t a r y evaporation. Kinetic Properties. With benzylpenicillin as substrate, both fl-lacta° mases have a broad p H profile. Slight differences in p H profile, however, are observed when methicillin is hydrolyzed. ~ In contrast, the substrate profiles of the two enzymes are significantly different. The data for the
[53bl
~ - L A C T A M A S(Bacillus E licheniformis)
659
TABLE II THE SPECIFIC ACTIVITIESAND RELATIVE RATES OF HYDROLYSIS OF 6346/C AND 749/C EXOPENICILLINASESa
B. licheniformis 6346/C
B. licheniformis 749/C
Specific Relative rate c activityb of hydrolysis
Specific Relative rate c activityb of hydrolysis
Substrate BenzylpeniciUin Methicillin 6-Aminopenicillanicacid Ampicillin Cephalosporin C Benzylcephalosporin C Cephaloridine
325.0 1.5 16.2 -3.5 7.9 --
100.00 0.45 5.00 68.00 1.1 3.0 36.0
54.00 0.54 7.00 -8.5 22.7 --
100.00 1.00 13.00 12.00 1.00 32.00 165.00
a Taken from M. R. Pollock, Biochem. J. 94~, 666 (1965) and J. Fleming, personal communication. b Number of micromotes of substrate hydrolyzed per microgram of enzyme per hour under conditions of enzyme saturation at 30°. c Relative rate of hydrolysis against benzylpenicillin (100). h y d r o l y s i s of a n u m b e r of p e n i c i l l i n a n d c e p h a l o s p o r i n d e r i v a t i v e s are given in T a b l e I I . Of p a r t i c u l a r i n t e r e s t is the a b i l i t y of s t r a i n 6 3 4 6 / C to h y d r o l y z e some c e p h a l o s p o r i n d e r i v a t i v e s at m u c h higher rates t h a n 749/C, a n d this provides a n i m p o r t a n t diagnostic property. As can be seen from T a b l e I I the specific activities of 6 3 4 6 / C are g e n e r a l l y m u c h lower t h a n the c o r r e s p o n d i n g v a l u e s of 749/C. I n the case of b e n z y l p e n i cillin the e n z y m e s differ in specific a c t i v i t y b y a factor of six. T h e app a r e n t M i c h a e l i s c o n s t a n t s show a n inverse r e l a t i o n s h i p ( T a b l e I I I ) , TABLE III THE APPARENT MICHAELIS CONSTANTS OF THE 6346/C AND 749/C O-LACTAMASES USING A NUMBER OF DIFFERENT SUBSTRATES a Km
(uM) Substrate
749/C
6346/C
Benzylpenicillin Methicillin 6-Aminopenicillanicacid Cephalosporin C Benzylcephalosporin C
49.00 0.93 16.60 50.00 50.00
9.50 0.23 16.70 50.00 50.00
Taken in part from M. R. Pollock, Biochem. J. 9~, 666 (1965).
660
ANTIBIOTIC
INACTIVATION
AND
[53b]
MODIFICATION
Ly s - Thr - Glu - Met - Ly s - Asp - Asp - Ph e - Ala - Ly s - Leu - Glu - Glu -Gln Phe-Asp-Ala-
Lys-Leu-Gly
- Ile -Phe-Ala-
Leu-Asp- Thr-Gly -Thr-
A s n - A r g - Th r - V al - A l a - Ty r - A r g - P r o - A s p - G l u - A r g - P h e - A l a - P h e Ala- Ser-Thr-
lle -Lys-Ala-
Leu-Thr-Val-Gly-Val-
Lea-Leu-Gln-
Gin - Lys- Set - lle -Glu - Asp- Leu-Asn- Gln- Arg - Ile - Thr - Tyr- ThrArg-Asp-Asp-Leu-Val-Asn-Tyr-Asn-Pro-
lle -Thr-Glu-
Lys-His-
V a l - A s p - T h r - G l y - M e t - T h r - L e u - L y s - G l u - L e u - A l a - A s p - A l a - Se r Leu-Arg-Tyr-Ser-
Asp-Asn-Ala-Ala-Gin-Asn-Leu-
lle-Leu-Lys-
Gin - ne - Gly - Gly - Pro - Glu - Ser - Leu- Ly s - Ly s- Glu - Leu- Arg- Ly s Ile - Gly-Asp-Glu-Val Leu-Asn-Glu Ala- Arg-Ala-
-Thr-Asn-Pro-Glu-Arg-Phe-Glu-
-Val -Asn-Pro-GIy Leu-Val-Thr-Ser-
Asp- Lys- Leu-Pro-Ser
-Glu -Thr-Gln Leu-Arg-Ala-
Pro-Glu-
-Asp- Thr-Ser Phe-Ala-
-Thr-
Leu-Glu-
-Glu - Lys-Arg- Glu - Leu-Leu - Ile -Asp- Trp-
Met-Lys-Arg-Asn-Thr-Thr-Gly-Asp-Ala-Leu-
Ile-Arg-Ala-Gly-
Val - Pro- Asp- Gly - Trp- Glu - Val - Ala - Asp- Ly s- Thr - Gly - Ala - Ala Ser-Tyr-Gly
-Thr-Arg-Asn-Asp-
Ile -Ala- ne - lle - Trp-Pro-
Pro-
L y s - G l y - A s p - P r o - V a l - V a l - L e u - A l a - V a l - L e u - Se r - Se r - A r g - A s p Lys-Lys-Asp-Ala-
Lys-Tyr-Asp-Asp-Lys-Leu-
Ile- Ala- Glu-Ala-
Thr- Ly s- Val -Val - Met- Ly s- Ala - Leu- Asn- Met- Asn-Gly - Lys- COOH FIG. 4. C o m p l e t e
licheni]ormis 7 4 9 / C . burgh,
amino
acid sequence
Taken
from
of the
R. J. Meadway,
fl-lactamase Ph.D.
produced,
by
Thesis, University
Bacillus of Edin-
1969.
6346/C having a Kin,,,. approximately six times lower than the same value for 749/C (benzylpenicillin). The ratio of V. . . . /Km for 6346/C and 749/C gives the same value with benzylpenicillin, so that the apparent first-order rate constant for the reaction of the enzymes at low substrate concentrations (i.e., Vmax/Km) are very similar. As Pollock 4 has explained, these low substrate concentrations are those likely to be encountered in nature, and the ratio V.... ~Kin or physiological efficiency may be a useful parameter of the ecological potential of a penicillinase. The different charge variants of 749/C exoenzyme observed on starch gel electrophoresis have been separated by DEAE-cellulose chromatography. TM These isozymes were found to have identical substrate profiles. Chemical Properties. The complete amino acid sequence of B. licheniformis 749/C fl-lactamase is shown in Fig. 4 and the amino acid composition in Table IV. Approximately 40% of the residues in the sequence are identical to amino acids in corresponding positions in the sequence of Staphylococcus aureus fl-lactamase. 1~ Bacillus licheni]ormis 6346/C is R .
P.
Ambler
and
R. J.
Meadway,
Nature (London) ~22, 2 4
(1969).
[53b]
fl-LACTAMASE (Bacillus licheniformis)
T A B L E IV AMINO ACID COMPOSITION OF THE fl-LACTAMASE OF B,
661
licheniformis
749/C ~
Residues/mole
Amino acid Aspartic acid b Threonine Serine Glutamic acid b Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Lysine Histidine Arginine Amides
Exoenzyme
Trypsin released
38.8 20.8 11.4 30.6 11.5 15.7 26.7 16.3 4.9 14.7 27.4 6.2 6.9
37.7 21.1 11.9 29.3 11.4 15.4 26.0 15.9 4.7 14.4 26.7 6.2 7.0
Nearest integer Co mean
Sequence
38 21 12 30 1] 16 26 16 5 15 27 6 7 3 24 1 15 1~
37 21 11 27 I1 15 26 15 5 14 27 6 7 3 24 l 15 20
2.8 24.2 1.0 15~ 0
24.1 1.0 14.5 17.5
Taken from R. J. Meadway, Ph.l). Thesis, Universit,y of Edinburgh, 1969. b Amides included.
differs from the 749/C sequence at 4 sites at least; arginine at position 163 of the 749/C sequence is substituted by glutamine, valine substitutes methionine at residue 259, glutamic acid substitutes asparagine at residue 265, and glycine at position 266 of 749/C is substituted by serine in the 6346/C sequence. At least one other amino acid substitution, near {,he amino-terminus, must be considered in order to explain a number of genetic experiments, although this difference has not yet been defined chemically. 16 The heterogeneity with respect to charge which these fl-lactamase show on electrophoresis is due to a ragged amino-terminal region. Analysis by the FDNB method gave a mixture of threonine and lysine, in the case of the exoenzyme, and a mixture of threonine and glutamic acid for the trypsin-released fl-lactamase. TM The charge variants of the exoenzyme were separated by DEAE-cellulose chromatography using a gradient elution system at pH 8.5. Analysis of these proteins, of homogeneous 1, D. Sherratt, Ph.D. Thesis, University of Edinburgh, 1969.
662
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53b]
electrophoretic mobility, by the dansyl method confirmed the hypothesis that differences in the amino acid sequence at the amino-terminus were responsible for the observed charge heterogeneity. Physical Properties. The fl-lactamase of B. licheni]ormis 749/C has a covalent molecular weight of 28,500 as calculated from the amino acid composition. On ultracentrifugal analysis, both 6346/C and 749/C enzymes have molecular weights of about 28,0001~ and gel filtration experiments indicate a molecule weight of between 27,000 and 33,000.1°,~8 An S~o,~ of 2.7 has been determined, ~ and the isoelectric point of the major component of 749/C exoenzyme is 5.0. TM Immunology. Extensive and sensitive stimulation, inhibition, absorption, and precipitation tests between both types of enzyme and their antisera in both homologous and heterologous reactions, indicate that on strictly immunological criteria they are identical29 Modification. Tetranitromethane specifically nitrates tyrosine 77 of the 749/C fl-lactamase, bringing about a 60% diminution in activity against penicillin. 1° This specific reagent reacts in an analogous manner with the fl-lactamase I of Bacillus cereus 2° and the fl-lactamase of S. aureus21 There is no evidence that this reactive tyrosine residue is part of the active site but the fact that this discrete region of the molecule has been conserved to such a high degree during the evolution of these enzymes, would indicate that it plays an essential role in the structure and function of the enzyme. The fl-lactamases of B. licheniformis differ markedly in their susceptibility to inactivation by elemental iodine. 6346/C has been shown to be considerably more resistant to denaturation by 3 mM iodine at pH 7.0 than 749/C, which is inactivated 50% under these conditions. 4 A number of B. licheni]ormis fl-lactamase enzymes which have been modified genetically by point mutation have been studied enzymologically? ~ These mutations bring about changes in thermostability and substrate profile. In one case, single amino acid substitution of 749/C fl-lactamase changed the substrate profile of the enzyme from that of a typical gram-positive fl-lactamase to that of a cephalosporinase. Although the marked change in substrate profile of this mutant indicates that the differences observed between 6346/C and 749/C could well be caused by ~TN. Citri, "The Enzymes" (D. Boyer, ed.), 3rd Ed., Vol. 4, Chapter 2. Academic Press, New York, 1971. ~T. Sawai, L. Crane, and J. 0. Lampen, Biochem. Biophys. Res. Commun. 53, 523 (1973). ~ M. R. Pollock, Immunology 7, 707 (1964). D. R. Thatcher, this volume [53a]. 21R. P. Ambler, personal communication. ~M. R. Pollock, Ann. N.Y. Acad. Sci. 151,502 (1968).
[53b]
~-LACTAMASE (Bacillus licheniformis)
663
a single amino acid change, it is still not known whether in fact more are responsible even though hybrid molecules have been constructed genetically.:s Secretion. Cell bound fl-lactamase cannot be released as exoenzyme simply by destroying the integrity of the cells. 8 The fact that the enzyme (a) while still bound to cells is partially inhibited by antibody prepared against the exopenicillinase and (b)can be released from its cellular location by treatment with trypsin, indicates that cell bound fl-lactamase is normally attached to the cell envelope in some way. Electron microscopic evidence would suggest that cell bound penicillinase consists of a plasma membrane bound fl-lactamase and a vesicle fraction which is released on protoplasting. :4,25 fl-Lactamases from both these locations may be solubilized at pH 9.0 in the presence of sodium deoxycholate or sodium taurocholate and a chelating agent. 2s Gel filtration studies in the presence and absence of bile salts have revealed the presence of a hydrophobic species of fl-lactamase which is capable of binding these salts and thereby lowering the exclusion volume of the enzyme considerably. Ordinary supernatant exoenzyme chromatographs with a molecular weight equivalent to 28,000 in the presence or in the absence of deoxycholate. Both plasma membrane and vesicle fraction fl-lactamases have an apparent molecular weight of 45,000 in the presence of bile salts. In their absence plasma membrane penicillinase aggregates to apparent molecular weights of 102,000 and 170,000, whereas the vesicle fraction enzyme has similar elution parameters to the exoenzyme,is Vesicle fraction fl-lactamase is found to revert spontaneously to a form indistinguishable from the exo-enzyme while the plasma membrane enzyme requires treatment with 25% phosphate. 2~ The cell bound B-lactamase of B. lichen& ]orrnis therefore exists in a detergent-binding hydrophobic conformation which, in the case of the enzyme bound in the vesicle fraction, can undergo a transition into the hydrophilic exoenzyme conformation with relative ease. Purification of the plasma membrane bound fl-lactamase by gel filtration and polyacrylamide gel electrophoresis enabled analytical studies to be performed on this fraction. TM Although the enzyme had a similar amino acid composition to the extracellular enzyme strong evidence was presented for the existence of a covalently bound phospholipid component. Treatment with trypsin released the phospholipid component and 23D. A. Dubnau and M. R. Pollock, J. Gen. Microbiol. 41, 7 (1965). B, :K. Ghosh, M. G. Sargeant, and J, O. Larapen, J. Bacteriol. 96, 1314 (1968). M. G, Sargeant, B. K. Ghosh, and J, O. Larapen, J. Bacteriol. 96, 1329 (1968). M. G. Sargeant and J. 0. Lampen, Arch, Biochem. Biophys. 136, 167 (1970). ~M. G. Sargeant and J. 0. Lampen, Proc. Nat. Ac(id. Sci. U.S. 65, 962 (1970).
664
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53e]
produced an enzyme identical to the exoenzyme. Trypsin release of cell bound fl-lactamase would therefore appear to involve the cleavage of a phospholipid containing peptide from the carboxyl-terminus of the molecule. I t is of interest here t h a t a frame shift mutation through the carboxyl-terminus of 749/C fl-lactamase indicates t h a t a possible termination codon in the fl-lactamase messenger could not occur until at least 5 residues past the known carboxyl-terminus. 2s The fl-lactamase of B. licheni]ormis is then p r o b a b l y somewhat larger t h a n 270 residues, as it is synthesized on the ribosome. A p p a r e n t l y both covalent and conformational modification of the enzyme occur during its secretion.
Acknowledgments The author is grateful to Dr. R. P. Ambler and Miss J. Fleming for reading
the manuscriptand MM Denise Waeckerle for typing it. I also wish to express gratitude to Professor Martin Pollock for his criticism and encouragement in the preparation of this article, during my stay in his department at the University of Edinburgh. 2sL. Kelly and W. J. Brammar, J. Mol. Biol. 80, 135-147 (1973).
[53c] ~ - L a c t a m a s e
(Staphylococcus aureus)
B y M. H. RICHMOND
The fl-lactamases of Staphylococcus aureus are similar to the majority of these enzymes found in gram-positive species of bacteria: they are inducible and predominantly extracellular in exponentially growing cultures. 1 When fully induced, staphylococcal fl-lactamase m a y constitute up to 0.5% of the dry weight of the bacterial culture, and normally more than 80% of the enzyme is to be found in the growth medium. I t follows therefore that most of the problems t h a t surround the isolation and purification of this enzyme concern methods of achieving maximal gene expression and the way in which the enzyme m a y successfully be collected from the s u p e r n a t a n t fraction of a bacterial culture. Before embarking on a detailed account of the purification of this enzyme it is important to stress that, to date, four minor variants of the enzyme have been detected among various species of S. aureus, although no strain has yet been shown to m a k e more than one type. ~,'~ 1N. Citri and M. R. Pollock, Adva~t. Enzymol. 28, 237 (1966). 2 M. H. Richmond, Biochem. J. 94, 584 (1965). 3 V. T. Rosdahl, J. Gen. Microbiol. 77, 229 (1973).
664
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53e]
produced an enzyme identical to the exoenzyme. Trypsin release of cell bound fl-lactamase would therefore appear to involve the cleavage of a phospholipid containing peptide from the carboxyl-terminus of the molecule. I t is of interest here t h a t a frame shift mutation through the carboxyl-terminus of 749/C fl-lactamase indicates t h a t a possible termination codon in the fl-lactamase messenger could not occur until at least 5 residues past the known carboxyl-terminus. 2s The fl-lactamase of B. licheni]ormis is then p r o b a b l y somewhat larger t h a n 270 residues, as it is synthesized on the ribosome. A p p a r e n t l y both covalent and conformational modification of the enzyme occur during its secretion.
Acknowledgments The author is grateful to Dr. R. P. Ambler and Miss J. Fleming for reading
the manuscriptand MM Denise Waeckerle for typing it. I also wish to express gratitude to Professor Martin Pollock for his criticism and encouragement in the preparation of this article, during my stay in his department at the University of Edinburgh. 2sL. Kelly and W. J. Brammar, J. Mol. Biol. 80, 135-147 (1973).
[53c] ~ - L a c t a m a s e
(Staphylococcus aureus)
B y M. H. RICHMOND
The fl-lactamases of Staphylococcus aureus are similar to the majority of these enzymes found in gram-positive species of bacteria: they are inducible and predominantly extracellular in exponentially growing cultures. 1 When fully induced, staphylococcal fl-lactamase m a y constitute up to 0.5% of the dry weight of the bacterial culture, and normally more than 80% of the enzyme is to be found in the growth medium. I t follows therefore that most of the problems t h a t surround the isolation and purification of this enzyme concern methods of achieving maximal gene expression and the way in which the enzyme m a y successfully be collected from the s u p e r n a t a n t fraction of a bacterial culture. Before embarking on a detailed account of the purification of this enzyme it is important to stress that, to date, four minor variants of the enzyme have been detected among various species of S. aureus, although no strain has yet been shown to m a k e more than one type. ~,'~ 1N. Citri and M. R. Pollock, Adva~t. Enzymol. 28, 237 (1966). 2 M. H. Richmond, Biochem. J. 94, 584 (1965). 3 V. T. Rosdahl, J. Gen. Microbiol. 77, 229 (1973).
[53C]
~-LACTAMASE (Staphylococcus aureus)
665
TABLE I COMPOSITION OF 1 % C Y MEDIUM ~
Medium
Sodium ¢~-glycerophosphate MgS04-7H20 Yeast extract Acid-hydrolyzed casein Glucose Trace metal solution
0.12 M 1.0 mM 1% (w/v) 1% (w/v) 0.8% (w/v) 0.02 ml/l
Trace metal solution
CuSO4- 5H.~O ZnSQ. 7H~O FeSO4-7H20 MnCl~. 4H.~O Cone. tiC1
0.5 % (w/v) 0.5% (w/v) 0.5 % (w/v) 0.2 % (w/v) 10% (w/v)
o The glucose and t3-glycerophosphate are omitted until after autoclaving. All the variants are, however, so similar that the same purification procedure serves for all.
Bacterial Culture and Induction Growth Media. In general the yield of staphylococcal fl-lactamase increases both on a cell dry weight and on a yield per unit culture volume basis with increasing richness of the growth medium. However, since the enzyme has to be purified from this medium after the bacteria have been removed, considerable advantage lies in having it rich but without an abundance of contaminating proteins. In practice the medium that gives the highest enzyme yields per unit volume of culture but which does not at the same time contaminate the fl-lactamase with large amounts of unwanted protein is one based on casein hydrolysate. ~ The most commonly used is "1% CY medium," whose composition is shown in Table I. In general enzyme yields in synthetic media containing amino acids, vitamin B~, nicotinic acid, mineral salts, and glucose are very poor. Growth Conditions. The optimal growth conditions depend on whether it is necessary to induce the culture (see below). Maximum yields are obtained either by growth of 1.25 liter batches in a 5-liter conical flask or by growing cultures in 10-liter bench fermentor. If the latter approach is used, the pH value should be maintained at about 7.0 by continuous addition of 5 N N a O H and the culture should be aerated as much as is compatible with controlling foam production. Antifoam agents may
4 tl. P. Novick, J. Gen. Microbiol. 33, 121 (1963).
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ANTIBIOTIC INACTIVATION AND MODIFICATION
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be added without adverse effect provided they are nonionic. Molecules capable of ionization may interfere with the subsequent absorption of the enzyme onto substituted cellulose. Enzyme production by growth in a fermentor has been scaled up to the 200-liter level (Dr. J. Melling, personal communication). Optimum enzyme yields are obtained at between 35 and 37 ° regardless of the size of the growth vessel. Induction. A number of inducers and induction regimes have been used, and it is impossible to be dogmatic about which is the best. The simplest is to use a single addition of methicillin to give a final concentration of 0.5 ~g/ml. The induction kinetics of this enzyme show a considerable lag before synthesis becomes rapid, and therefore it is always necessary to collect the enzyme some time after induction. One regime that has been used successfully is to induce at a culture density of about 2X108 bacteria/ml and then crop the enzyme between 4 and 4.5 hr later. 5 At this time high-yielding strains will show a culture density of about 4 X l09 bacteria/ml and an enzyme titer in growth medium of about 1000 enzyme units/ml. Alternative induction procedures are to make a succession of methicillin additions at intervals with an increasing concentration of inducer commencing at 0.5 ~g/ml. Sometimes 6-aminopenicillanic acid (100 ~g/ml) or cloxacillin (0.2 ~g/ml) is used in place of methicillin. Benzylpenicillin, ampicillin, and phenoxymethyl penicillin are poor inducers. Production of Constitutive Mutants 4
Experience has shown that enzyme purification from cultures synthesizing the enzyme constitutively is very much easier than from those which have to be induced. This is partly because higher titers may be obtained from such mutants and partly because avoiding induction ensures that there is less cell damage and therefore less contaminating protein of bacterial origin released into the growth medium. In general constitutive mutants are easy to obtain from fl-lactamaseproducing staphylococcal cultures by mutagenesis either with ethylmethane-sulfonate (EMS) or with N-methyl-N-nitro-N-nitrosoguanidine (NMG). With the first of these mutagens an exponentially growing culture is treated with 0.02 ml of EMS per milliliter of culture and incubation continued for 2 hr at 35 °. At the end of this period the culture is centrifuged (5000 g, 10 min) and resuspended in fresh, prewarmed growth medium to a density of about 5 X 107 bacteria/ml. This culture is then grown for about 4 hr under normal growth conditions, and the bacteria are then plated to give rise to separate colonies on appropriate medium. 5M. H. Richmond, M. T. Parker, M. P. Jevons, and H. John, Lancet 1, 293 (1964).
I53C]
~-LACTAMASE (Staphylococcus aureus)
667
Mutagenesis with NMG is carried out on exponentially growing bacteria that have been removed from their growth medium by centrifugation (5000 g, 10 min) and resuspended in buffer containing NMG. The buffer is normally 0.1 M sodium citrate, pH 5.5, and the NMG added to a final concentration of 100 ~g/ml. After incubation for 15 min, without shaking at 35 °, the bacteria are separated by centrifugation (5000 g, 10 rain) and resuspended in prewarmed growth medium to a density of about 5 X 107 organisms per milliliter. This culture is then grown for 2-4 hr at 35°; the bacteria are then plated to give separate colonies on appropriate medium. The simplest method of detecting the constitutive mutant colonies is by growing the bacteria after muta.gen treatment on nutrient agar containing 1% (w/v) soluble starch and then staining the colonies after overnight growth with a solution of 20 mg of benzylpenicillin per milliliter of a solution containing 0.1 N I2 in 0.4 M KI. The reagent is poured onto the plates for about 10 sec, and the excess is poured away. The agar plates become deep purple because of the reaction between the iodine in the reagent and the starch. Constitutive fl-lactamase synthesis is shown by a rapidly spreading white halo around the colony. The basis of the method is that penicilloic acid, liberated from the penicillin by B-lactamase action, reacts with iodine in the reagent, thus decolorizing the agar plate in the vicinity. Constitutive colonies about 1 mm in diameter should show a positive reaction in about 20 sec after the reagent is added. Normally the action of the fl-lactamase protects the colony against the action both of the penicillin and the iodine used in the reagent, and it is possible to pick the desired colonies directly into liquid medium or on to agar plates for purification and isolation. The method can be made more sensitive either by decreasing the iodine content in the reagent or the duration of treatment. When constitutive mutants are used for enzyme production cultures are grown exponentially without induction until the enzyme titer in the culture medium reaches a maximum. High-yielding strains may produce supernatant enzyme titers in the range of 1500 enzyme units per milliliter of culture. Purification Procedure 6
If the enzyme titer in the growth medium is more than about 300 enzyme units/ml, the enzyme can be purified without removing the bacteria. At lower titers better yields are obtained if the organisms are first removed by centrifugation. Normally, continuous-flow centrifuges (e.g., M. H. Richmond,Biochem. I. 88, 452 (1963).
66S
A N T I B I O T I INACTIVATION C AND MODIFICATION
[53C]
Spinco, MSE, or Sharpies) provide a supernatant fluid clear enough to process satisfactorily. T a b l e I I summarizes the results obtained in a typical purification sequence with the constitutive strain PC1. The first step in the purification is to add cellulose phosphate (Grade P40, equilibrated as described by the manufacturer) to the culture (either containing bacteria or free of b a c t e r i a - - s e e above) until about 80% of the enzyme activity is adsorbed to the substituted cellulose. The treated culture is then allowed to settle, and the cellulose is collected by decantation. This material is transferred to a Biichner funnel or similar device, and the cellulose phosphate is washed with several liters of 0.1 M Na_~HPO4/KH~P04 buffer, p H 7.0, until the washings are free of bacteria and color derived from the growth medium. After washing in this way, the cellulose is transferred from the washing funnel and made into a relatively short column. A further 500 ml of 0.1 M phosphate buffer is then run through the column and discarded, and the enzyme is eluted by a strong salt solution at p H 7.5. In the original experiments 2 M Tris C1 buffer, pH. 7.5, was used, but more recently 20% (w/v) a m m o n i u m sulfate buffered to p H 7.5 has been shown to be effective. N o r m a l l y one obtains about 90% recovery of the enzyme at this stage. I t should be a water-clear solution of specific activity about 3 units per microgram of protein (see T a b l e I I ) . The purification procedure can be carried out at room temperature up to this point. Thereafter
A TYPICAL
TABLE II PURIFICATIONOF STAPHYLOCOCCAL/3-LACTAMASE
Step Adsorption to cellulose phosphate Elution from cellulose phosphate with 2 M Tris C1 pH 7.5 Dialysis agent distilled water Absorption to CM-cellulose Chromatography on CMcellulose Filtration through Sephadex G75
Recovery Specific enzyme (%) Enzyme activity activity recovered (units/ug Per Over(units) a enzyme protein) stage all
3.7 X 106
3.0
93 90.1
2.3 X 106 2.1 X 106 (by difference) 1.4 X 106
3.4 --
61.0 51.1 91 46.4
31
66
28.6
0.98 X 106
62
70
24
4 X 106
--
93 83.7
a The 4 X 106 units used in this experiment were derived from an 8.5-liter culture of S . a u r e u s PC1 grown in 1% CY medium.
[53e]
~-LACTAMASE (Staphylococcus aureus)
669
it should be done in a cold room. If the eluate from cellulose phosphate is dialyzed against distilled water, or against solutions of low ionic strength, precipitation occurs and much enzyme is lost. To help overcome this difficulty, CM-cellulose (Grade C 70) is added to the enzyme preparation in the dialysis bag, 1 g of substituted cellulose being used for every 2.5 2< 105 units of enzyme. Under these circumstances the enzyme tends to adsorb to the cellulose rather than to the precipitate as the ionic strength fails. In practice, however, this tends to be the most variable and difficult step of the purification procedure and the best results are usually obtained when tile total amount of enzyme being purified is large (greater than 2 >( l0 G units) and its concentration is kept high. After dialysis is complete (see Table II), the CM-cellulose is removed by decantation and added to the top of a colmnn of CM-cellulose which has already been poured. In a typical example fresh CM-cellulose is equilibrated as required by the manufacturer and poured as a column, about 2 g of CM-cellulose being used for every 10,3 enzyme units anticipated from the preceding purification step. The CM-eellulose with enzyme adsorbed originating in the previous purification step is then loaded on top of this column, and the whole column is washed with 10 mM sodium acetate buffer, pH 5.9, until the eluate from the column has reached that pH wdue. The enzyme is then eluted with a linear concentration gradient of sodium acetate buffer, pH 5.9. A typical example of the gradient used for a column 15 em long }( 1.5 cm in diameter is as follows: in the concentrated buffer vessel, 200 ml of 0.4 M sodium acetate buffer, pH 5.9; in the mixer 200 ml of 20 mM sodium acetate buffer, pH 5.9. The enzyme should appear at a buffer concentration of 60-80 raM. The recovery obtained at this step is somewhat variable, as implied above. The best obtained to date is 66%; and the specific enzyme activity of the product. should be about 30 units per microgram of protein (see Table II). The eluate from CM-eellulose is concentrated to about 2 ml by membrane dialysis against 0.1 M sodium acetate buffer, pH 5.9, and then loaded onto a Sephadex G-75 column (1.5 em )< 175 era) equilibrated against 0.1 M sodium acetate, pH 5.9. The column is then developed with the same sodium acetate buffer. This step gives a single peak of specific enzyme activity about 65 units per microgram of protein. Moreover, the specific activity of the enzyme should be constant across the peak--a good index of purity. This stage of the purification procedure normally gives recoveries in the region of 90% (see Table II). Purification on Sephadex has replaced tile earlier and more cumbersome procedures involving centrifugat!on and eleetrophoresis in sucrose gradients. This entire purification procedure as described here normally gives about 20% overall yield of enzyme of more than 95% purity.
670
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53C]
Spot Tests ]or fl-Lactamase. A number of simple tests, based on the decolorization'of iodine by penicilloic acid, have been used for detecting the position of fl-lactamase activity in column effluents. The simplest for those with access to nutrient agar plates containing l % ( w / v ) soluble starch (see the section on isolation of constitutive mutants) is to flood such a plate with a benzyl penicillin/iodine solution so that a deep violet plate is produced. Excess reagent is poured off, and after a minute or two the excess moisture soaks into the plate. The fractions from the column can then be tested by placing drops in sequence on the surface of the agar plate with a bacteriological loop. The presence of enzyme is denoted by the appearance of a white spot. An alternative method is to use a 100 t~g/ml solution of the cephalosporin 87/312 in 0.1 M sodium phosphate buffer, pH 7.0. This cephalosporin is orange when unhydrolyzed but bright red when destroyed by fl-lactamase.7 Both these methods may easily be adapted for use in small volumes of liquid such as are needed for standard immunological microtiter or indicator plates. Stability and Storage. Solutions of staphylococcal fl-lactamase may be stored with only slow loss of activity in 0.1 M phosphate buffer, pH 7.0, or 0.1 M sodium acetate buffer, pH 5.9. In general the higher the enzyme concentration the more stable the solution. The enzyme may also be stored in the frozen dried state and reconstituted in either phosphate or acetate buffer for use. Cell-Bound Enzyme. This method of purification can be used only for extracellular enzyme, a fraction that constitutes the majority of activity expressed by most staphylococci. Little progress has been made with the purification of the cell-bound enzyme. All that is known is that it is carried in the bacterial cytoplasmic membrane and that no technique that will liberate it from this structure in active form has yet been evolved. The enzyme in this form is relatively resistant to ultrasound, yet treatment of isolated staphylococcal cell membranes with ultrasound merely fragments the membranes with the enzyme in situ. Properties of Staphylococcal B-Lactamase 1,6 The activity of purified staphylococcal fl-laetamase A is summarized in Table III. The enzyme is most active against the naturally occurrirLg penicillins (benzylpenicillin and phenoxymethylpenicillin) because it combines a high V.... against these substrates with a high affinity. This property is associated with the physiological function of the enzyme, C. It. O'Callaghan, A. Morris, S. M. Kirby, and A. H. Shingler, Antimicrob. Ag.
Chemother. 1, 283 (1972).
[53E]
/~-LACTAMASE (Staphylococcus aureus)
671
TABLE III SUBSTRATE SPECIFICITY OF STAPHYLOCOCCAL ~-LACTAMASE TYPE A a
Substrate specificity
Benzylpenicillin Phenoxymethylpenicillin Ampicillin Cephalosporin C Cephaloridine 6-Aminopenicillanic acid Methieillin Cloxaeillin
Vmaxb
Kin
100 109 185 1.2 10 10 1.5 c 2c
5 X 10-6 7 X 10-6 1.7 X 10-5 10_4 5 X 10_4 7 X 10-4 10-2 10-2
a Data from N. Citri and M. R. Pollock, Adv. Enzymol. 28, 237 (1966); and M. H. Richmond, Biochem. J. 88, 452 (1963). b Values relative to benzylpenicillin = 100. c The Vm~x values for methicillin and cloxacillin were determined at l0 -~ M substrate. namely, to protect staphylococcal cultures which synthesize the enzyme against the killing action of naturally occurring penicillins. The high affinity of the enzyme is a particular advantage since it allows the enzyme, which is normally liberated into the growth medium, to sweep the environment clear of antibiotic. Since staphylococcal cultures t h a t lack the enzyme are so sensitive to these antibiotics, a high affinity for the enzyme is necessary for complete protection. Two classes of fl-lactam antibiotics have shown themselves to be effective against penicillinase-producing cultures of Staphylococcus aureus. T h e y are the semisynthetic penicillins (methicillin and cloxacillin), on the one hand, and some of the cephalosporins (notably cephaloridine and cephalothin~ on the other. The effectiveness of both these groups of compounds depends crucially on their interaction with staphylococcal penicillinase since both induce the enzyme and have to act in the presence of large amounts of it. T h e y solve the problem in distinct ways. The new penicillins have an extremely low affinity for the enzyme so t h a t although they are susceptible to hydrolysis in laboratory tests at high substrate concentrations, this sensitivity is unavailing at therapeutic levels. The cephalosporins, on the other hand, are more conventional in their resistance: they show a much reduced V~x for the enzyme when compared with penicillins. T a b l e I V summarizes the molecular properties of staphylococcal fl-lactamase. The enzyme consists of a single polypeptide chain of 257 resi-
672
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53d]
TABLE IV MOLECULAR PROPERTIES OF STAPHYLOCOCCAL PENICILLINASE TYPE A a
Molecular weight (approx.) 29,600 Number of residues 257 Specific enzyme activity 315 units/ug protein Amino terminal amino acid Lysine Isoelectric point pH 8.9 Amino acid composition: residues/mole Gly Ala Val Leu Ile Ser Thr Aspb Glub
12 18 16 22 19 19 13 39 18
Phe Tyr Trp Cys Met Pro Lys His Arg
7 13 0 0 3 9 43 2 4
a Data from M. H. Richmond, Biochem. J. 88, 452 (1963) and R. P. Ambler and J. Medway, Nature (London) 222, 24 (1969). b Includes both the free acid and the amide. dues commencing with an amino-terminal lysine residue, s The isoelectric point of the enzyme is about pH. 8.9. The enzyme is remarkable for its relatively high content of polar amino acids and the relatively large difference between its content of lysine (43 residues) and arginine (4 residues). 8R. P. Ambler and R. J. Meadway, Nature (London) 222, 24 (1969).
[53d] ~-Lactamase (gscherichia coil R+~) B y M. Hi RICHMOND Thc fl-lactamases of gram-negative bacteria present a sharp contrast in properties when compared with similar enzymes from gram-positive species. First, these enzymes are invariably cell-associated, and second, their synthesis is usually constitutive. 1 This means that fl-lactamase purification from enteric bacteria involves the isolation of a protein from the bacterial cells themselves without induction or the need to isolate constitutive mutantsY M. H. Richmond and R. B. Sykes, Advan. Microbial Physiol. 9, 31 (1973). 2This volume [53c].
672
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53d]
TABLE IV MOLECULAR PROPERTIES OF STAPHYLOCOCCAL PENICILLINASE TYPE A a
Molecular weight (approx.) 29,600 Number of residues 257 Specific enzyme activity 315 units/ug protein Amino terminal amino acid Lysine Isoelectric point pH 8.9 Amino acid composition: residues/mole Gly Ala Val Leu Ile Ser Thr Aspb Glub
12 18 16 22 19 19 13 39 18
Phe Tyr Trp Cys Met Pro Lys His Arg
7 13 0 0 3 9 43 2 4
a Data from M. H. Richmond, Biochem. J. 88, 452 (1963) and R. P. Ambler and J. Medway, Nature (London) 222, 24 (1969). b Includes both the free acid and the amide. dues commencing with an amino-terminal lysine residue, s The isoelectric point of the enzyme is about pH. 8.9. The enzyme is remarkable for its relatively high content of polar amino acids and the relatively large difference between its content of lysine (43 residues) and arginine (4 residues). 8R. P. Ambler and R. J. Meadway, Nature (London) 222, 24 (1969).
[53d] ~-Lactamase (gscherichia coil R+~) B y M. Hi RICHMOND Thc fl-lactamases of gram-negative bacteria present a sharp contrast in properties when compared with similar enzymes from gram-positive species. First, these enzymes are invariably cell-associated, and second, their synthesis is usually constitutive. 1 This means that fl-lactamase purification from enteric bacteria involves the isolation of a protein from the bacterial cells themselves without induction or the need to isolate constitutive mutantsY M. H. Richmond and R. B. Sykes, Advan. Microbial Physiol. 9, 31 (1973). 2This volume [53c].
[53d]
~-LACTAMASIg [Escherichia coli R+EM)
673
The location of this fl-lactamase in the cells that express it has an important influence on the purification procedure that may be used. Neu and his colleagues have shown that this particular type of fl-lactamase occupies the periplasmic space of gram-negative bacteria, that is, in the space lying between the inner and outer membrane of the bacterial cell surface. :~ Moreover, the enzyme appears to be free in solution in this space since relatively mild shock techniques liberate the enzyme into the surrounding growth medium. ~ Although not employed in the purification procedure described below, it is possible to obtain relatively pure preparations of this enzyme by "shocking" it out of the cells, by resuspending the bacteria either in small volumes of ice-cold distilled water or in a solution of E D T A in sucrose. T y p e I I I a fl-lactamase from gram-negative bacteria is very commonly R factor-mediated when found in naturally occurring strains(' and tl~is means that this particular type of enzyme may be encountered il~ a wide range of gram-negative species. It is particularly common in isolates of Escherichia coli, Aerobacter aerogenes, and, Klebsiella aerogenes isolated from urinary tract infections, but may also rarely be found ou~side the Enterobaeteriaceae, for example, in isolates of Pse~domom~s aeruginosa. '~ In fact, it is probably very misleading to think of this enzyme as associated with any particular bacterial species. Rather it should be seen as characteristic of a wide range of R factors. In practice the T y p e I I I a enzyme gene has been found on plasmids from all the comnlonly encountered plasmid incompatibility groups. In this respect, therefore, this enzyme is unlike the majority of other bacterial fl-lactamases which tend to be confined to one bacterial species. Before going on to describe the purification of T y p e I I I a enzyme in detail, it is important to stress that this type of fl-lactamase is only one of the 13 distinct types that have been detected so far in gram-negative bacteria. ~ Relatively few of the others have yet been purified (but see T y p e Ia fl-lactamase. 7)
Purification Procedure A wide range of gram-negative species of bacteria carrying an appropriate R factor can be used for this purpose although the original method was worked out with Escherichia coli K12 carrying the X-class plasmid 3H. C. Neu and L. A. Heppel, Biochem. Biophys. Res. Commun. 14, 215 (1964). H. C. Neu and J. Chou, J. Bacteriol. 94, 1934 (1967). 5 G. W. Jack and M. H. Richmond, J. Gen. Microbiol. 61, 43 (1970). " R. B. Sykes and M. H. Richmond, Nature (London) 226, 952 (1970). This volume [53e].
674
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53d]
RT~.~.8 In general the expression of the enzyme in this strain increases with the richness of the growth medium. The best growth medium for enzyme production is 1% CY medium, 2 and in this medium the enzyme is synthesized to a level of about 55 enzyme units per milligram dry weight of bacteria by this strain. Optimum growth rates are achieved at between 35 ° and 37 ° . Aeration should be as vigorous as is compatible with the control of foaming. Care must be taken to choose the right point to crop the bacteria for enzyme purification. The optimum time is in the late exponential phase when the culture density is about 1.5 mg dry weight of bacteria per milliliter. If growth is allowed to continue beyond this point, titers may be higher, but some lysis occurs and the enzyme becomes partitioned between the cells and the growth medium--with consequent difficulties in recovery. The fl-lactamases of gram-negative bacteria show a clear difference from their counterparts in gram-positive species in that their intracellular location ensures that they may not always be expressed at full level in intact bacteria. This is certainly the case with Type IIIa enzyme. If the expression of this enzyme by E. coli is compared for a given culture before and after breaking by ultrasonic disintegration (see below) a difference in apparent enzyme activity of up to 50- or 60-fold may be observed, but the extent of difference depends upon the nature of the substrate used. In general, the difference is greater with penicillins than with cephalosporins ~ (see Table I). Apart frbm its physiological interest, this crypticity of the enzyme has implications for deciding when to crop the bacteria for enzyme purification. With certain substrates any cell lysis gives the illusion that enzyme titers are still increasing whereas, in fact, all that is happening is that the fl-lactamase is being liberated from its partially cryptic state in the periplasmic space. To overcome this difficulty it is necessary either always to break the bacteria before assay or to use a substrate, such as cephalorodine, which penetrates freely to the periplasmic space in E. coll. 1 If, on the other hand, assay of the enzyme has to be carried out in Pseudomonas aeruginosa, the need to break the bacteria before assay cannot be avoided since none of the currently available substrates can reach the periplasmic space of this species unhindered, and all give erroneously low enzyme titers in P. aeruginosa. The purification procedure is summarized in Table I. 7 Stage 1. Bacteria growing in 1% CY medium are collected by centrifugation, and the culture supernatant is discarded. The bacterial pellet is resuspended to a density of about 50 mg dry weight of bacteria in 8N. Datta and M. H. Richmond,Biochem. J. 98, 204 (1966).
[53d]
~ - L A C T A M A S(Escherichia E coli R+E~)
675
TABLE I SUMMARY OF THE PURIFICATION
OF TYPE IIXa /~-LACTAMASE FROM
Escherichia coli (RT+EM)"
Stage 1
2-3 3
Procedure Ultrasonic disintegration Centrifugation and dialysis Adsorption on DEAE-cellulose Elution from DEAE-cellulose Sephadex G-75
Enzyme activity recovered (units) 1,1X 1.05 X 7.4 X 5.7 X 5.3 X
106 l0 G 105 105 10~
Specific enzyme Recovery (%) activity (units/~g Per Overof enzyme) stage all 1 0.9 15,8 50 118
100 96.0 70.5 77 92
100 96,0 67.6 51.6 48
The starting material was derived from a 7-liter culture containing 22 g dry weight of bacteria.
0.1 M KH2PO4/Na,_,HPO4 buffer, p H 7.0. The suspension is stored at 2 ° and then subjected to ultrasonic disintegration in a Mullard ultrasonic disintegrator (Measuring and Scientific Instrmnents, Ltd.) at 25 kc/sec. Two procedures have been used. I f the total volume for disruption is small, the bacterial suspension is treated batchwise. About 5 ml of suspension is treated for about 2 min and rapidly returned to storage at 2 ° until all the preparation has been broken. On the other hand, large volumes of suspension m a y be treated in the continuous flow cell provided by the manufacturers of the ultrasonic disintegrator. In this ease the flow cell is cooled with a flow of water at 2 °, and the suspension is treated so t h a t it has a mean residence time of 2 rain in the cell. After irradiation the preparation is rapidly transferred to a reservoir cooled in ice water. Stage 2. After ultrasonic t r e a t m e n t is complete, the bacterial suspension is centrifuged at about 5000 g for 10 min at 2 ° to remove any remaining intact bacteria and much of the cell debris. The supernatant is decanted off and then centrifuged again, first at 30,000 g for 4 hr at 2 ° and then at 105,000 g for 2 hr at 2 ° . Again the supernatant is conserved in each ease, and the two pellets are discarded. These eentrifugation steps give a clear, deep amber solution. Stage 3. The solution obtained by Step 2 is then dialyzed to equilibrium against 10 m M Na~HPO4/KH2PO~ buffer, p H 7,0. The enzyme solution after dialysis is loaded onto a DEAE-eellulose column prepared as follows. DEAE-eellulose ( W h a t m a n Grade D E l l ) is suspended in 0.1 M Na~HPO~/KH~PO~ buffer p H 7.0 and the fines are removed. The c o l umn is then poured in 0.1 M phosphate buffer, p H 7.0, and washed with
676
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53d]
this buffer until the effluent reaches pH 7.0. The column is then prepared for use by washing with about 1 liter of distilled water. After loading the enzyme, the column is first washed with 500 ml of 10 mM phosphate buffer, pH 7.0. A great deal of protein and pigmented material passes straight through the column at this stage. The enzyme is then eluted with a concentration gradient of phosphate buffer, pH 7.0. In an experiment in which 600,000 units of enzyme was loaded on to a column (1.2 }( 20 cm) the gradient was constructed with 300 ml each of 3 mM and 5 mM phosphate buffer. The fl-lactamase activity was eluted at about 35 mM phosphate buffer. After testing the effluent fractions for activity, those containing the enzyme are pooled, concentrated to about 3 ml by pressure dialysis against 10 mM phosphate buffer, pH 7.0. Stage 4. The enzyme solution obtained from stage 3 is loaded onto a G-75 Sephadex column (1.2 X 150 cm) equilibrated against 0.1 M Na2HPO4/KH2P04, pH 7.0, and the enzyme eluted with a similar buffer. The activity appears as a single peak with molecular weight about 25,000. Material obtained at this stage is normally more than 95% pure and has a specific enzyme activity of about 120 enzyme units per microgram of protein. The enzyme is prepared for storage by dialysis against distilled water to remove all buffer, followed by freeze-drying. Stability and Storage. Enzyme solutions may be stored for use at 2 ° in 0.1 M Na2HPO4/KH~PO4 buffer pH 7.0 with only slow loss of activity. The enzyme may also be kept frozen-dried in ampules in a dark place. Properties of Type IIIa fl-Lactamase 1,9 The substrate profile of purified type I l i a fl-laetamase is summarized in Table II. This enzyme has a broad specificity encompassing both penicillins and cephalosporins. However, in practice the enzyme is much more effective against penicillins since, in general, the Km values for cephalosporin substrates are high. This fact probably accounts for the greater ability of this enzyme to protect gram-negative bacteria against penicillin than against cephalosporin. The exact molecular weight, amino acid composition, and specific enzyme activity of this fl-lactamase are not yet completely certain. The best data available so far is given in Table III. However, these analyses were carried out on relatively little material and the values must be regarded as approximate. The most important uncertainty concerns the molecular weight of the protein. In the original publication 8 values below 20,000 were reported on the basis of ultracentrifugation experiments, but 9 G. W. Jack and M. H. Richmond, FEBS Lett. 12, 30 (1970).
[53d]
~ - L A C T A M A S(Escherichia E coli R~EM)
677
TABLE II THE SUBSTRATE SPECIFICITY OF PURIFIED TYPE I i I a ~-LACTAMASE" Substrate specificity Vm~b Benzylpenicillin Phenoxymethylpenicillin Ampicillin Carbenicillin Cephaloridine Ceph~.lothin
100 50 180 10 140 15{)
Km 2.2 2.3 2 8 6 6
)< )< X )< X )<
10.5 10-6 10 - 6
10-G 10-4 10-~
" Data from M. H. Richmond and R. B. Sykes, Advan. Microbiol Physiol. 9, 3l (1973) and N. Datta and M. H. Richmond, Biochem. J. 98, 204 (1966). b Values in relation to benzylpenicillin = 100.
TABLE III SOME MOLECULAR PROPERTIES OF TYPE I I I a ~-LACTAMASE~
Approximate molecular weight Specific enzyme activity Isoelectric point Approximate amino acid composition: Gly Ala Val Leu Ile Ser Thr Aspb Gh?
3.2 5.0 4.8 8.7 4.0 4.3 7.2 13.8 12.7
Phe Tyr Trp Cys Met Pro Lys His Arg
25,003 214 units/t~g protein 5.7 %, by weight 8.4 5.8 ? 0.4 1.8 5.4 9.1 2.1 4.4
Data from M. H. Richmond and R. B. Sykes, Advan. Microbiol Physiol. 9, 31 (1973); G. W. Jack and M. H. Richmond, FEBS Lett. 19., 30 (1970); and R. P. Ambler, personal communication. Includes both the free acid and the amide.
these are c e r t a i n l y too low. T h e best i n f o r m a t i o n a v a i l a b l e a t the m o m e n t suggests a v a l u e closer to 25,000.1 A n o t h e r error in the earlier d a t a concerns the p r o b a b l e cysteine c o n t e n t of the enzyme. 9 A l t h o u g h o r i g i n a l l y t h o u g h t to be a b s e n t , this a m i n o acid is p r o b a b l y p r e s e n t in t y p e I I I a f l - l a c t a m a s e a t a level of 1 r e s i d u e / m o l e . TM 1, R. P, Ambler, personal communication.
678
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53e] B - L a c t a m a s e ( E n t e r o b a c t e r By GORDONW. Ross
[53e]
Species)
The cephalosporinases produced by Enterobacter species form a more closely related family of fl-lactamases than is found in most other gramnegative species. They have little activity against penicillins, but hydrolyze many of the cephalosporins; they are readily inhibited by cloxacillin, methicillin, and the cephalosporins with the acyl substituents of cloxacillin and methicillin, and they have isoelectric points between pH 7.5 and 8.7. fl-Lactamase activity can be detected in all strains of Enterobacter examined to date if a sufficiently sensitive technique is used. 1 Enzyme production is inducible in many strains and appears to be chromosomally mediated. R factor-mediated cephalosporinase has not been confirmed in Enterobacter species, although the widely occurring RT~M-mediated fl-lactamase has been located in at least one strain of E. cloacae in addition to the usual cephalosporinase. 1,2 Antiserum against the cephalosporinase from E. cloacae P99 cross-reacts with cephalosporinases from other strains of E. cloacae, but not with the cephalosporinases from strains of E. aerogenes examined to dateY Enterobacter cephalosporinase was first reported by Fleming and coworkers, 3 who isolated a strain of E. cloacae (P99) which produced relatively large amounts of a fl-lactamase capable of rapidly inactivating cephalosporin C, but having little activity against benzylpenicillin. This strain was included in Hennessey's study on induction of fl-lactamase in Enterobacter strains 4 and was found to produce more enzyme without induction than the other strains of Enterobacter produced after induction. Partially purified P99 fl-lactamase was subsequently used in inhibition studies 5,6 and compared to the wide spectrum fl-lactamase from Klebsiella aerogenes 1082EY More recently Ross and Boulton purified the P99 enzyme8 and prepared an antiserum for immunological studies2 The only 1 M. Matthew, A. M. Harris, M. J. Marshall, and G. W. Ross, J. Gen. Microbiol., in press. 1975 2 M. G. Boulton and G. W. Ross unpublished results, 1973. 3 p. C. Fleming, M. Goldner, and D. G. Glass, Lancet 1, 1399 (1963). 4 T. D. Hennessey, J. Gen. Microbiol. 49, 277 (1967). 5 C. H. O'Callaghan, P. W. Muggleton, and G. W. Ross, Antimicrob. Ag. Chemother. 1968, 57 (1968). e p. C. Fleming, M. Goldner, and D. G. Glass, J. Bacteriol. 98, 394 (1969). 7 M. J. Marshall, G. W. Ross, K. V. Chanter, and A. M. Harris, Appl. Microbiol. 23, 765 (1972). 8 G. W. Ross and M. G. Boulton, Biochim. Biophys. Acta 309, 439 (1973). G. W. Ross and M. G. Boulton, J. Bacteriol. 112, 1435 (1972).
[Sael
~-LACTAMASE (Enterobacter sPEcies)
679
other Enterobacter cephalosporinase studied in any detail is the enzyme purified from E. cloacae 214 by Hennessey and Richmond TM ; this cephalosporinase had very similar characteristics to the P99 enzyme but was shown to have a different isoelectrie focusing pattern in a recent study in which good correlation was demonstrated between the isoelectric focusing patterns of fl-lactamases and the bacterial characteristics of Enterobacteria. ~ The Enterobacter cephalosporinases are examples of one of the eight distinct types of fl-lactamases described by J a c k and Richmond 1' and have been classified as t y p e I a in the later and more exhaustive review of gram-negative fl-lactamases by Richmond and Sykes. 12
Assay Method Full details of B-lactamase assay are given elsewhere. 13 The spectrophotometric method ~ is particularly convenient for assay of these cephalosporinases and for inhibition studies. The iodometric assay TM can be used for higher substrate concentrations and is especially useful for comparison of substrate specificities of enzymes, when a wide range of substrates has to be used. The sensitive microiodometric methods have not been applied successfully to cephalosporin substrates, but microbiological assays are extremely sensitive when substrates of high antibiotic activity are used. 15 Cephaloridine or cephalothin are suitable substrates. One unit is defined as the amount of fl-lactamase required to hydrolyze 1 t~mole of cephaloridine per minute under the conditions of the assay (usually 37 °, p H 7). Specific activity is expressed as units per milligram of protein as measured by the method of L o w r y et al. TM with crystalline bovine serum albumin as a standard. T h e assay methods can be applied to culture supernatants and to broken cell suspensions as well as to purified enzyme. An indicator cephalosporin which changes color when hydrolyzed by 1oT. D. Hennessey and M. H. Richmond, Biochem. J. 1{}9, 469 (1968). 11G. W. Jack and M. H. Richmond, J. Gen. Microbiol. 61, 43 ('1970). 12M. H. Richmond and R. B. Sykes, in "Advances in Microbial Physiology" (A. H. Rose, ed), p. 31. Academic Press, New York, 1973. 1~This volume [5]. 1, C. J. Perret, Nature (London) 174, 1012 (1954). 15G. W. Ross, K. V. Chanter, A. M. Harris, S. M. Kirby, M. J. Marshall and C. H. O'Callaghan, Anal. Biochem. 54, 9 (197;]). 1, O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).
6S0
[53e]
ANTIBIOTIC INACTIVATION AND MODIFICATION
TABLE I VARIATION IN THE YIELD OF f~-LACTMASE FROM Enterobacter cloacae P99 UNDER VARIOUS CULTURAL CONDITIONS
Enzyme units/liter of initial culture Cultural conditions Static
Medium Nutrient broth Brain-heart infusion
Shaken
Microinoculum broth Nutrient broth Brain-heart infusion Microinoculum broth
Culture volume (ml) 50 250 50 250 50 50 250 50 250 50
IntraSupercellular natant preparation 1085 723 3615 1085 0 360 540 720 360 0
2890 700 4340 1160 400 4920 1450 9830 2435 0
B-lactamase 17 is a useful and sensitive aid to detection of enzyme/antibody arcs in electrophoresis and immunoelectrophoresis.
Enzyme Purification The procedure described below has been used several times for the purification of the fl-lactamase from Enterobacter cloacae P99 with reproducible results2 I t has also been applied successfully to the purification of the very similar enzyme from Enterobacter cloacae 214, which was originally .purified on CM-Sephadex and Sephadex G-75 by Hennessey and Richmond. 1° Enterobacter cloacae P99 produces larger amounts of fl-lactamase (10,000 units/liter of culture) than other strains of Enterobacter studied. When fermentation conditions were examined briefly, it was found t h a t most fl-lactamase was produced when the organism was grown in 50-ml amounts of brain-heart infusion and shaken for 18 hr at 37 ° (see T a b l e I). Production of intracellular enzyme reached a m a x i m u m at 18 hr and then fell rapidly to about 5% of the m a x i m u m by 42 hr. 7 The enzyme from five 50-ml cultures is purified routinely in each " C. H. O'Callaghan, A. Morris, S. M. Kirby, and A. H. Shingler, Antimicrob. Ag. Chemother. 1, 283 (1972).
[53e]
~-LACTAMASE (Enterobacter SPECIES)
681
batch procedure although the method has been used to purify enzyme from ten times this culture volume and can easily be applied to still larger volumes. All steps are carried out at 4 °. Although some fl-lactamase activity is released into the culture supernatant, only intracellular enzyme is purified. Step I. Ultrasonic Disruption. The cells are harvested by centrifugation of the culture at 5400 g for 45 min, washed in 50 ml of 0.1 M phosphate buffer, pH 7.0, and resuspended in the buffer (35 ml). Intracellular enzyme is released by ultrasound treatment for 3 rain at 20 kHz with icewater cooling (500 W generator). Step 2. High-Speed Centri]ugation. A clear cell-free supernatant is obtained after centrifugation at 105,000 g for 90 rain. Step 3. Sephadex G-50 Chromatography. The supernatant (34 ml) is layered directly onto a Sephadex G-50 column (25 X 6 cm) previously equilibrated in the 0.1 M phosphate buffer, pH 7.0. Descending elution with the same buffer at 55 ml/hr is monitored at 254 nm and 8 ml fractions are collected. The first sharp absorbance peak (fractions 30-50) contains all the detectable fl-lactamase activity, and these fractions are pooled (160 ml). Step 4. Dicllysis and Concentration. The pooled material from step 3 is dialyzed overnight against demineralized water, concentrated by freeze drying and then equilibrated against 25 mM TES IN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, BDH Chemicals Ltd.] buffer, pH 7.5. The considerable loss of activity which occurs at this stage can lie reduced by addition of 0.5% gelatin. Ultrafiltration or pervaporation can be used for concentration with similar results. Step 5. QAE-Sephadex Chromatography. Enzyme from step 4 (55 nil) is Nyercd onto a QAE-Sephadex A-50 column (23 X 5.5 cml equilibrated in the 25 mM TES buffer. The column is eluted with the same buffer at 55 ml/hr and fractions (5.5 ml) are monitored for protein and fl-lactamase "tctivity. The E. cloacae enzyme, which is positively charged at pH 7.5 passes through the column and is collected in fractions 60-100 (232 ml). Other proteins in the step 4 material are absorbed onto the column and are eluted with 0.1 M TES, 0.5 M NaC1, and 2 M NaC1. Slight fl-lactamase activity can be detected in the 0.5 M NaC1 eluate (see Fig. 1), Step 6. Dialysis and Concentration. The purified enzyme is dialyzed against distilled water and concentrated by freeze drying; it can be stored at --20 ° for at least two years without appreciable loss of activity. A summary of a typical purification is given in Table II. The preparations are examined by polyacrylamide disc electrophoresis at various stages of purification (Fig. 2).
682
[53e]
ANTIBIOTIC INACTIVATION AND MODIFICATION
12.0
0.025M Tes ~ : 01M Tes*--~-OSM N o C I ~ , (928 ml) ~580ml) (755 ml)
2M NaCi (1910 ml)
E 0.8
"
~ •-9c
E c o N
2 .~
0.6 "5 E
o
~
6.c
,
c~
rl
3.C
x
0.2
_
N'x"-.r-, ×~
i
°o
~oo
200
3o0
i
,~o
s~o
~o o
600
Fraction number
FIG. 1. Chromatography of the fl-lactamase from Enterobacter cloacae P99 on QAE--Sephadex. Reproduced from G. W. Ross and M. G. Boulton, Biochim. Biophys. Acta 309, 430 (1973).
TABLE II PURIFICATION OF THE Enterobacter cloacae P99
~-L.~-CTAMASE
% Yield activity Purification step 1. Ultrasonic disruption 2. High speed centrifugation 3. Sephadcx G-50 chromatography 4. Concentration and equilibration 5. QAE-Sephadex chromatography 6. Final concentration step
Step
Overall
Volume Total (ml) units
Specific Total activity protein (units/rag (rag) protein)
100
100
35
1390
420
3.3
98
98
34
1360
280
4.6
98
96.3
160
1340
120
11.2
52.0
50.0
48
696
96.8
49.1
232
673
97.4
47.4
71
646
Protein value too low for accurate measurement.
105.6 --a
4.97
6.6 __a
134.3
[53e]
~-LACTAMASE (Enterobacter SPECIES)
A
B
C
683
D
FI~. 2. Polyacrylamide disc electrophoresis of Entcrobacter cloacae P99 t~-lactamase preparations at various stages of purification. (A) Cell-free supernatant after step 2; (B) after chromatography on Sephadex G-50, step 3; (C) purified enzyme after step 6; (D) enzyme stain (cephaloridine-starch-iodine) of C. Reproduced from G. W. Ross and M. G. Boulton, Biochim. Biophys. Acta 309~ 430 (1973).
Enzyme Properties Enterobacter cephalosporinases all have very similar properties. The enzymes from 74 strains of Enterobacter studied 18 have the same inhibitor profile and all hydrolyze cephalosporins much more effectively than penicillins. Isoelectric focusing in thin layers of polyacrylamide is a valuable technique for classifying fl-lactamases, and correlation has been shown between the different isoelectric focusing patterns, differences in substrate specificity and inhibitor profiles of the enzymes, and the taxonomic differences of the strains of bacteria producing the enzymes. 1 Cephalosporinases which give the same isoelectric focusing patterns as the P99 enzyme
'~S. W. B. Newsam, M. J. Marshall, and A. M. Harris, I. Med. Microbiol. 7, 473 (1974).
684
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53e]
form the largest isoelectric focusing class at:present, although, as only 34 Enterobacter strains have been examined by this technique so far, this predominance of the P99 type may not be maintained when further strains are studied. The inducible cephalosporinase from E. cloacae 214 is an example of another group of enzymes which have properties very similar to those of the P99 group but give a different isoelectric focusing pattern. The isoelectric focusing pattern of the penicillinase mediated by the TEM R factor is clearly distinguishable from the Entezobacter cephalosporinase pattern when both fl-lactamases are present in an Enterobacter strain. All the E. cloacae fl-lactamases react with antisera against the P99 enzyme, Antiserum was originally prepared 9 in rabbits which were given two intramuscular and two subcutaneous 0.5-ml injections of a mixture of enzyme and adjuvant at four sites once a week for 4 weeks. The rabbits were bled a week after the last injection. The partially purified P99 enzyme (corresponding to the product of step 3 in the above purification) in 0.1 M phosphate buffer (2 mg of protein in 1 ml), pH 7.0, was mixed with a n equal amount of Freund's complete adjuvant for the first dose and incomplete adjuvant for subsequent doses. The specificity of the resulting antiserum was improved by absorption with a preparation from a mutant strain which produced only low levels of fl-lactamase,s The absorbed antiserum gives only one precipitin arc with P99 enzyme, and this enzyme/antibody precipitate still has fl-lactamase activity. This can be demonstrated either with the starch-iodide=cephalosporin enzyme stain 9 or with the chromogenic cephalosporinY Cross-reaction has been obtained only with other E. cloacae enzymes to date. E. aerogenes fl-lactamases have not given a cross-reaction, nor have fl-lactamases from other species (e.g., Escherichia colt, Klebsiella, Staphylococcus, Pseudomonas). More recently a highly specific antiserum has also been obtained after two injections of purified P99 enzyme (0.6 mg protein) given 1 week apart29 The specificity of this antiserum seems to be identical to that of the absorbed P99 antiserum. The constitutive cephalosporinase from E. cloacae P99 has been selected as an example of Enterobacter fl-lactamase, and its properties are listed below. Enzyme production by the other Enterobacter strains studied by Hennessey can be increased more than 100-fold by induction.4 Hennessey obtained maximum induction with 500 ~g of benzylpenicillin per milliliter. After induction E. cloacae 214 produces nearly as much ~-lactamase as the uninduced P99 strain. The other Enterobacter strains studied can 1DM. Matthew, unpublished results, 1973.
[53e]
fl-LACTAMASE (Enterobacter SPECIES)
685
produce at most 25% (induced) or 2.2% (uninduced) of the enzyme level in the P99 strain. 4 Methicillin is also a very good inducer of these B-lactamases.
Enterabacter cloacae P99 Cephalosporinase Homogeneity, Molecular Size. One major and several minor fl-lactamase bands can be detected on heavily loaded polyacrylamide disc gels (200 ~g of protein) or by the more sensitive isoelectric focusing method, and the pattern of bands is reproducible. The bands each give a precipitin arc with P99 fl-lactamase antiserum and the arcs link, showing immunological identity. The reason for the occurrence of these minor bands is not yet known, but they are possibly slight structural variants of the same enzyme protein. 1 The molecular weights of fl-lactamases quoted in the literature by different workers do not always agree, and several early values have been later modified. These discrepancies may be due to the several different methods used to measure the molecular weights of enzymes purified by very different procedures. The several fl-lactamase bands which can be detected when purified fl-lactamase is examined on heavily loaded polyacrylamide disc gels or by the isoelectric focusing technique may also give rise to errors when molecular weights are measured on polyacrylamide gels. Sargent and Lampen -~° have shown that penicillinase from Bacillus licheniformis 749/C can be isolated in forms with apparent molecular weights ranging from 24,000 to 600,000. Some binding of sodium deoxycholate to this penicillinase appears to occur. Aggregation and disaggregation may occur to some extent with other fl-lactamases, either during purification or when they are pretreated before molecular weight measurement. The molecular weight of the E. cloacae 214 cephalosporinase was originally estimated as 14,000TM but was later reported to be 29,000 in common with other type Ia fl-lactamases when examined by Sephadex column chromatography. 12 The molecular weight of the cephalosporinase from E. cloacae P99 purified on QAE Sephadex was originally given as 49,000 s as measured by sodium dodecyl sulfate (SDS) disc gel electrophoresis by the method of Weber and Osborn. 21 More recently three preparations of the purified enzyme were examined on 18 disc gel runs and a mean value of 39,000 was obtainedd 2 Eleven of the 18 values obtained were ± 1000 of the mean value. 2o M. G. Sargent and J. O. Lampen, Arch. Biochem. Biophys. 136, 167 (1970). 2~K. Weber and M. Osborn, J. Biol. Chem. 244, 4406 (1969). ~2M. G. Boulton, unpublished results, 1973.
686
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53e]
pH and Temperature E~ect. The p H optimum for the enzyme is p H 8.2 (spectrophotometric assay), and the isoelectric point of the major band is p H 7.9. If 0.2-ml aliquots of a solution of the enzyme in 0.1 M phosphate buffer, p H 7, (6.3 units/ml) are heated at different temperatures for 30 min, the temperature effect on enzyme activity can be assessed by spectrophotometric assay (20-~1 samples of the heated enzyme solutions added to 3 ml of 0.1 m M cephaloridine solution in phosphate buffer at 37°). After 30 rain at 50 °, 20% of the initial activity is lost. Heating at 55 ° for 30 rain destroys 40% of the initial activity. Activity is completely destroyed by heating at 60 ° for 30 min. Kinetic Properties. The Km and V,~x values are 0.26 m M and 313 ~moles/min per milligram of protein, respectively (spectrophotometric assay with cephaloridine as subs~rate). Substrate Specificity. Relative rates of hydrolysis of 6 cephalosporin and 4 penicillin substrates are shown in Table III. Cephalosporins 5/1 and 291/1 are 3-acetoxymethyl-7fl-(2r,6'-dimethoxybenzamido)ceph-3em-4-carboxylic acid and 3-acetoxymethyl-7fl-[3-(0-chlorophenyl)-5methylisoxazole-4-carboxamido] ceph-3-em-4-carboxylic acid, respectively, and have the same acyl substituents as methicillin and cloxacillin. The results clearly demonstrate the cephalosporinase character of this Enterobacter enzyme. Some hydrolysis of ampicillin and methicillin can be obtained when a membrane-filter microbiological technique is used to determine substrate specificity. 1 Inhibition. This type of cephalosporinase is competitively inhibited by methicillin, cloxacillin, and the cephalosporins with the same acyl substiTABLE III SUBSTRATESPECIFICITYOF THE fl-LAcTAMASEFROM Enterobacter cloacae P99 Relative activity of P99 /~-lactamase (cephaloridine = 100) Substrate
Iodometric assay
Cephaloridine Cephalothin Cephalosporin C Cephalexin 5/1 291/1 Penicillin G Ampicillin Methicillin Cloxacillin
100 20 60 10 0.5 0 1.5 0 0.5 0
UV Microbiological assay assay 100 25 100 10 2 2 -----
100 40 25 10 0 0.5 5 0 0 0
[53f]
Streptomyces ~-LACTAMASES
687
tuents as methicillin and cloxacillin (5/1 and 291/1). The value of Ki for P99 enzyme, 5/1 inhibitor and cephaloridine substrate is 0.128 gM. The structure of the acyl substituent of the inhibitor is the main determining factor; the nucleus has little effect and analogous penicillins and cephalosporins have similar inhibitory activity. For cephalosporins, the structure of the group in the 3 position can affect fl-lactamase inhibition, and it has been suggested that a stable group which does not readily accept electrons gives the cephalosporin a low affinity for the enzyme and thus reduces its effect as a competitive inhibitor2 Some substrate inhibition of P99 enzyme by cephaloridine has been detected for substrate concentrations greater than 0.5 mM. The hydrolysis of cephaloridine by the P99 enzyme is 5% and 30% inhibited by 10 gM and 0.1 mM p-chloromercuribenzoate, respectively, when inhibitor is added to enzyme immediately before substrate (37 °, spectrophotometric assay). It is completely inhibited by 0.1 InM mercuric chloride and by 0.01 mM iodine. Iodoacetate or divalent cations other than mercuric (Mg z÷, Ca ~+, Zn :+, Ni 2÷) have no inhibitor effect at concentrations between 1 gM and 1 mM, even when incubated with the enzyme at 37 ° for 1 hr before assay. Mechanical shaking (2 min) inactivates the enzyme, but this can be prevented by addition of 0.5% gelatin.
[53f] ¢~-Lactamases ( A c t i n o m y c e t e s Species)
By KENNETH JOHNSON, COLETTE DUEZ, JEAN-MARIE FRERE, and JEAN-MARIE GHUYSEN
Strains and Culture
Strains. The aerobic euactinomycetes are true gram-positive bacteria which form a characteristic mycelium and multiply by means of special spores like the fungi. Based on the structure of their wall peptidoglycan, the various genera are divided into several groups.' The two strains used are soil isolates ~,3 and are assigned arbitrary designations. Streptomyces albus G (in fact probably a Streptomyces griseus) ~ has a peptidoglycan of the general chemotype II with LL-diaminopimelic acid and glycine. 4,'~ Although strain R39 (origin: Ruwenzori; soil sample No. 1 K. H. Schleifer and 0. Kandler, Bacteriol. Rev. 36, 407 (1972). 2 M. Welsch, Rev. Belge Pathol. Med. Exp. 18, Suppl. 2, 1 (1947). ~M. Welsch and A. Rutten-Pinckaers, Bull. Soc. Roy. Sci. Liege 3-4, 374 (1963). 4 j. M. Ghuysen, BacterioI. Rev. 32, 425 (1968). 5 M. Leyh-Bouille, R. Bonaly, J. M. Ghuysen, R. Tinelli, and D. J. Tipper, Biochemistry 9, 2944 (1970).
[53f]
Streptomyces ~-LACTAMASES
687
tuents as methicillin and cloxacillin (5/1 and 291/1). The value of Ki for P99 enzyme, 5/1 inhibitor and cephaloridine substrate is 0.128 gM. The structure of the acyl substituent of the inhibitor is the main determining factor; the nucleus has little effect and analogous penicillins and cephalosporins have similar inhibitory activity. For cephalosporins, the structure of the group in the 3 position can affect fl-lactamase inhibition, and it has been suggested that a stable group which does not readily accept electrons gives the cephalosporin a low affinity for the enzyme and thus reduces its effect as a competitive inhibitor2 Some substrate inhibition of P99 enzyme by cephaloridine has been detected for substrate concentrations greater than 0.5 mM. The hydrolysis of cephaloridine by the P99 enzyme is 5% and 30% inhibited by 10 gM and 0.1 mM p-chloromercuribenzoate, respectively, when inhibitor is added to enzyme immediately before substrate (37 °, spectrophotometric assay). It is completely inhibited by 0.1 InM mercuric chloride and by 0.01 mM iodine. Iodoacetate or divalent cations other than mercuric (Mg z÷, Ca ~+, Zn :+, Ni 2÷) have no inhibitor effect at concentrations between 1 gM and 1 mM, even when incubated with the enzyme at 37 ° for 1 hr before assay. Mechanical shaking (2 min) inactivates the enzyme, but this can be prevented by addition of 0.5% gelatin.
[53f] ¢~-Lactamases ( A c t i n o m y c e t e s Species)
By KENNETH JOHNSON, COLETTE DUEZ, JEAN-MARIE FRERE, and JEAN-MARIE GHUYSEN
Strains and Culture
Strains. The aerobic euactinomycetes are true gram-positive bacteria which form a characteristic mycelium and multiply by means of special spores like the fungi. Based on the structure of their wall peptidoglycan, the various genera are divided into several groups.' The two strains used are soil isolates ~,3 and are assigned arbitrary designations. Streptomyces albus G (in fact probably a Streptomyces griseus) ~ has a peptidoglycan of the general chemotype II with LL-diaminopimelic acid and glycine. 4,'~ Although strain R39 (origin: Ruwenzori; soil sample No. 1 K. H. Schleifer and 0. Kandler, Bacteriol. Rev. 36, 407 (1972). 2 M. Welsch, Rev. Belge Pathol. Med. Exp. 18, Suppl. 2, 1 (1947). ~M. Welsch and A. Rutten-Pinckaers, Bull. Soc. Roy. Sci. Liege 3-4, 374 (1963). 4 j. M. Ghuysen, BacterioI. Rev. 32, 425 (1968). 5 M. Leyh-Bouille, R. Bonaly, J. M. Ghuysen, R. Tinelli, and D. J. Tipper, Biochemistry 9, 2944 (1970).
688
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53f]
XVI) 3 also has the appearance of a Streptomyces, its peptidoglycan is of the general chemotype I, with meso-diaminopimelic acid but no glycine. 4,6 The selected strains produce both an exocellular fl-lactamaseT, 8 and an exocellular DD-carboxypeptidase2 The DD-carboxypeptidase from strain R39 liberates the C-terminal D-Ala residue from R-D-Ala-D-Ala peptides (R-D-AIa-D-Ala + H 2 0 - ~ D-Ala + R-D-Ala) and catalyzes bimolecular transfer reactions in which R-D-Ala-D-Ala peptides act as acyl donors (R-D-Ala-D-AIa + NH2-R'-~ D-Ala + R-D-Ala-R').9 BLactam antibiotics react with the R39 DD-carboxypeptidase-transpeptidase to form equimolar and inactive antibiotic-enzyme complexes, lo In Tris.HC1 buffer pH 7.7 containing 0.2 M NaC1 and 50 mM MgCI~ and at 37 °, the half-life of the benzyl penicillin-enzyme complex is 70 hr. During dissociation, the enzyme undergoes reactivation whereas the released antibiotic molecule is neither benzylpenicillin nor benzylpenicilloie acid. 1° While DD-carboxypeptidase from strain albus G also hydrolyzes the R-D-AIa-D-Ala peptides, it does not catalyze transpeptidation reactions and is virtually unaffected by/~-lactam antibiotics. 9 Culture Media. Peptone oxoid medium contains 1% peptone oxoid, 0.1% K2HP04, 0.1% MgSO4.7H20, 0.2% NaNO3, and 0.05% KC1. Agar-APG medimn contains per liter of final volume: agar 20 g; asparagine, 0.5 g; peptone oxoid 0.5 g; glucose 10 g; and K~HPO4, 0.5 g. Agar KC medium contains per liter of final volume: agar, 20 g; partially hydrolyzed keratin from white hens' feathers, 2.5 g; partially hydrolyzed casein, 2.5 g; NaC1, 0.5 g; CaCO.% 0.1 g; MgSO4"7H20 0.1 g; K~HPO~, 1 g; and FeSO~.7H~O, 0.1 g. Final pH is 7.5. Hydrolyzed keratin and casein are prepared as follows: 100 g of dried, white hens' feathers are treated for 1 hr at 100 ° with 1 liter of N/8 KOH. After centrifugation, casein (100 g) is added to the supernatant fraction and dissolved by heating at 70 °. The pH of the mixture (final volume: 1 liter) is adjusted to 7.5-8.0; 25 ml contain 2.5 g of both partially hydrolyzed keratin and casein. Maintenance of Strains. Strain R39 is grown at 28 ° on slants of agar KC and strain albus G on slants of agar APG. Abundant sporulation occurs after 4-5 days. The strains are then maintained at 4 ° . ~J. M. Ghuysen, M. Leyh-Bouille, J. N. Campbell, R. Moreno, J. M. Fr~re, C. Duez, M. Nieto, and H. R. Perkins, Biochemistry 12, 1243 (1973). M. Welsch, Proc. ~th Int. Congr. Microbiol., Copenhagen, 19~7, Section 1, p. 144. K. Johnson, J. Dusart, J. N. Campbell, and J. M. Ghuysen, Antimicrob. Ag. Chemother. 3, 289 (1973). 9j. M. Ghuysen, M. Leyh-Bouille, J. M. Fr~re, J. Dusart, A. Marquet, H. R. Perkins, and M. Nieto, Ann. N.Y. Acad. Sci. 235, 236 (1974). ~oj. M. Fr~re, J. M. Ghuysen, P. E. Reynolds, R. Moreno, and H. R. Perkins, Biochem. J. 143, 233 (1974).
Streptomyces ~-LACTAMASES
[53f]
6S9
Assay Method for fl-Lactamases 11
Unit. One unit of fl-lactamase catalyzes the hydrolysis of 1 ~mole of benzylpenicillin per minute at 30 °. Reagents Sodium acetate buffer, 1 M, pH 3.6 Color reagent: equal volumes of a water-soluble starch solution (0.8%, w/v) and of a 240 ~M I.. + 4.8 m M K I solution.
Procedure. The following is a microscale adaptation of the technique of Novick and Dubnau. 1~ Benzylpenicillin (0.3 tLmoles) is incubated with the enzyme preparation in 30 ~l (final volume) of 25 m M sodium phosphate buffer, pH 7.0. The benzylpenicillin concentration in the mixture is 10 m M and, hence, any fl-lactamase with a Km value for this antibiotic equal to or lower than 1 m M is saturated. After 10-30 min, to the reaction mixture are added in sequence 200 ~l of 1 M acetate buffer, and then 200 ~l of color reagent. After 10 min at 25 °, the optical density of the solution is measured at 620 ran. Control consisting of the same mixture lacking enzyme is incubated as above. Acetate buffer, color reagent, and finally the same amount of enzyme as used in the test are added, and the optical density at 620 nm is determined. A decrease of the optical density of 0.1 corresponds to about 0.37 nmole of hydrolyzed benzylpenicillin. Determination of Km and Vm~x Values for Various fl-Lactam Antibiotics
Reagents Sodium acetate buffer, 5 M pH 3.6 Color reagent: the same as above
Procedure ]or Benzylpenicillin. Enzyme and benzylpenicillin are incubated together at 30 ° in 190 td, final volume, of 25 m M sodium phosphate buffer pH 7.0. After 10-30 rain, to the reaction mixtures are added 40 t~l of 5 M acetate buffer and then 200 ~l of color reagent. The remainder of the procedure is as described above. This technique allows the determination of a Km value as low as 30 ~M. With initial concentrations of benzylpenicillin ranging from 9 to 90 tLM (i.e., from 0.3 to 3 X a Km value of 30 t~M), a 10% utilization of the substrate corresponds to decreases of the absorbance of the final solutions ranging from 0.046 to 0.460. "This volume [5]. 12R. P. Novick, J. Gen. Microbiol. 33, 121 (1963).
690
ANTIBIOTIC INACTIVATION AND MODIFICATION
[53f]
Procedure ]or Other fl-Lactam Antibiotics. The same procedure as for benzylpenicillin is used. However, the products of fl-lactamase action upon 6-aminopenicillanic acid and its derivatives and those obtained from 7-aminocephalosporanic acid and its derivatives have not the same iodine uptake. A decrease of the absorbance at 620 nm of 0.1 corresponds to more hydrolyzed cephalothin (about 0.58 nmole) than to hydrolyzed benzylpenicillin. Standard curves [hydrolyzed fl-lactam antibiotic vs iodine uptake] must be used as references.
Assay Methods for DD-Carboxypeptidases Reaction Ac2-L-Lys-D-Ala-D-Ala + H20 --~ D-Ala + Ac~-L-Lys-D-Ala
Unit. One unit of DD-carboxypeptidase catalyses the hydrolysis of 1 ~mole of N%N~-diacetyl-L-lysyl-D-alanyl-D-alanine into D-alanine and N",N~-diacetyl-L-lysyl-D-alanine, per minute at 37 °. Substrates. Nonradioactive tripeptide Ac~-L-Lys-D-Ala-D-Ala is prepared as described by Nieto and Perkins. 13 The same peptide radioactively labeled with 1~C in both acetyl groups (specific activity 10,000 dpm/nmole) is prepared as described by Perkins et al. 14 Standard Incubation Conditions. Ac2-L-Lys-D-Ala-D-Ala (0.25 ~mole) is incubated with the relevant enzyme (Km values: 0.8 and 0.33 mM for R39 and albus G enzymes, respectively) at 37 ° in 30 ~l (final volumes) of 30 mM Tris.HC1 buffer, pH 7.5, supplemented with 3 mM MgCL_. Hydrolysis products (D-Ala or Ac2-L-Lys-D-Ala dipeptide) are then estimated by using one of the following procedures. Chemical Estimation o] Free Alanine
The following is a modification of the techn;~que of Ghuysen et al. 15 F D N B Reagent. Fluorodinitrobenzene, 130 ~l in 10 ml 100% ethanol. Procedure. Samples containing 10-50 nmoles of alanine are mixed with 10% K~B~O7 and water to give a total volume of 100 ~l of 1% K2B407. F N D B reagent (10 ~l) is added. The solutions are mixed and incubated at 60 ° for 30 min. After acidification with 50 ~l of 12 N HC1, the DNPalanine is extracted three times with 200 ~l of ether. The ether extracts are evaporated in a stream of hot air and dried in vacuo. The residues are dissolved in methanol and chromatographed at room temperature on thin-layer plates of silica gel G in chloroform:methanol:acetic acid " M. Nieto and H. R. Perkins, Biochem. J. 123, 789 (1971). 14H. R. Perkins, M. Nieto, J. M. Fr~re, M. Leyh-Bouille, and J. M. Ghuysen, Biochem. J. 131, 707 (1973). ~ J. M. Ghuysen, D. J. Tipper, and J. L. Strominger, this series, Vol. 8, p. 685.
Streptomyces ~-LACTAMASES
[53f]
691
(220:25:5, v / v / v ) . DNP-alanine moves faster than DNP-Tris. After drying, the DNP-alanine spots are transferred to 1-ml tubes and eluted by vigorous mixing with 500 t~l of water:ethanol:25% (0.91) ammonia (100:100:054, v / v / v ) . After centrifugation, the optical density of the supernatant fractions is measured at 360 nm. The molar extinction coefficient for DNP-alanine is about 15,000.
Enzymic Estimation o] D-Alanine The following modifications of the technique of Ghuysen et al. 15 permit one to carry out many simultaneous tests in a very short time.
Reagents o-Dianisidine (Merck, pro analysis): 10 mg/ml in methanol (freshly prepared) K pyrophosphate buffer, 0.1 M, pH 8.3 FAD (monosodium; Boehringer): 0.3 mg/ml in pyrophosphate buffer Peroxidase (Boehringer. Reinheitsgrad 1. Fiir analytische Zwecke; suspension 10 mg/ml) to be diluted to 10 t~g/ml in H20 D-Amino acid oxidase (Boehringer. Fiir analytisehe Zwecke; Kristallsuspension: 5 mg/ml) Enzymes and coenzyme mixture (freshly prepared): pyrophosphate buffer:FAD solution:diluted peroxidase solution:D-amino acid oxidase suspension (20:10: 5: 1, v / v / v / v ) .
Procedure. Samples (30 t~l) containing 5-40 nmoles of D-alanine are mixed with 5 ~l of o-dianisidine solution and 70 td of the enzymes-coenzyme mixture. To such solutions, incubated 5 min at 37 °, is added 400 ~l of methanol:water (v/v). Following an additional 2-min incubation at 37 °, the absorbance at 460 nm is immediately measured. (Coloration of the solution is slightly labilized after addition of the methanol-water solution.) Blanks consist of the same mixtures as above lacking AC2-LLys-D-Ala-D-Ala tripeptide. Controls are blanks containing known amounts of D-alanine. Estimation o] Radioactive [14C]Ac.,.-L-Lys-D-Ala Dipeptide Reagents Collidine-acetic acid-water (9.1:2.65: 1000, v / v / v ) , buffer pH 6.4 Liquid scintillation: 2,2-p-phenylenebis (5-phenyloxazole) (POPOP), 100 mg; 2,5-diphenyloxazole, (PPO), 4 g; toluene, 1 liter
692
ANTIBIOTIC
INACTIVATION
AND
MODIFICATION
[53f] /.
Procedure. 14 Samples (30 ~I) containing 10,000-20,000 dpm are diluted with 40 t~l of water. These are spotted as bands, 30 cm from the cathode on 4 cm X 1.5 m strips of Whatman 3MM paper, and subjected to electrophoresis at pH 6.4 for 4 hr at 60 V/cm, under a Sol T Shell. A Gilson high voltage, 10,000 V, Electrophoretor Model DW equipped with a cooling device is used as power source. Residual [~4CJAc2-L-Lys-D-Ala-D-AIa and the reaction product [~4C]Ac.,-L-Lys-D-Ala move 65 and 75 cm, respectively, toward the anode. The radioactive compounds are located on the dried strips with a Packard Radiochromatogram Scanner Model 7201. Cuts of the radioactive spots (10 mm section) are placed in vials, to each of which is added 0.75 ml of the scintillation liquid. Counting is performed in a Packard Tri-Carb liquid scintillation spectrometer. Excretion of B-Lactamase and DD-Carboxypeptidase by Streptomyces Strains
~Streptomyces can be grown aerobically in l-liter flasks containing 500 ml of peptone oxoid medium on a New Brunswick shaker at 28 °. Inoculation is made with a 20-ml suspension of actively growing Streptomyces. Maximal fl-lactamase activity (about 3 to 7 X 10--0 units/ml) and maximal DD-carboxypeptidase activity (about 2.5 to 10 X 10-3 units/ml) occur after 48-72 hr of culture. Both activities then disappear progressively and are negligible after 6 days, at which time mycelium production is maximal (1.5-2 g dry weight per liter). With time, the two enzyme activities increase and decrease independently of each other. Streptomyces can also be grown in 500-liter tanks. After two successire subcultures of increasing size, 100 liters of culture of actively growing Streptomyces are used to inoculate 400 liters of medium contained in a 500-liter tank. This culture is grown at 28 ° for 72-96 hr with mechanical stirring (120 rpm) and an air flow rate of 100 liter/min at an air pressure of 1.5 kg/cm 2. Silicone A emulsien (Dow Coming, 20 ml) is used as antifoam. Maximal fl-lactamase activity and maximal DD-carboxypeptidase activity are comparable to those obtained with cultures carried out in l-liter flasks. Adsorption and Storage of Crude Enzymes. Both B-lactamase and DDcarboxypeptidase in 500 liters of culture fluid are adsorbed either on 5 kg of moist diethylaminoethyl (DEAE)-cellulose previously equilibrated against 50 mM sodium phosphate buffer pH 7.0 (Streptomyces R39), or on 10 kg of Amberlite CG50 or XE64 H ÷ by adiusting the pH to 5 with acetic acid (Streptomyces albus G). The enzyme-resin complexes are collected by centrifugation with a Sharpless type MU865P33 (17,000 g). They can be stored for months at 0 ° without loss of activity.
[53f]
Streptomyces ~-LACTAMASES
693
Isolation of Streptomyces ~-Lactamases
Concentration o/Enzyme Preparations Unless otherwise stated, concentration is carried out by ultrafiltration through UMIO membranes with an Amicon apparatus (Amicon Corporation, Lexington, Massachusetts).
Preparative Polyacrylamide Gel Electrophoresis It is carried out with a Shandon preparative apparatus No. SAE-2782. The gel, polymerized in the presence of 0.14~ ammonium persulfate, is prepared in Tris-glycine buffer pH 8.3, 42 mM with respect to Tris. Height of the gel colmnn: 3 era. Electrophoresis: 2 hr at 320 V (40 mA/tube) at 10 °. Fractions reaching the bottom of tile gel are collected by means of a flow of 0.5 M Tris-acetate buffer pH 8.3.
Phosphate Buf]er Unless otherwise stated, 50 mM sodium phosphate buffer, pH 7.0, is used.
General Remarks During fractionation, it is essential to monitor the collected fractions for fl-lactamase and DD-carboxypeptidase activity and to keep for further processing those fractions preferentially enriched in fl-lactamase. The fl-lactamasc and DD-carboxypeptidase from Streptomyces R39 are anionic proteins at pH 8.3. At this pH, the fl-lactamase of Streptomyces albus G is anionic whereas its DD-earboxypeptidase is cationic. The procedures described below give rise to preparations devoid of any detectable DDcarboxypeptidase activity after periods up to 24 hr of incubation with Ac~-L-Lys-D-Ala-D-Ala.
Procedures Unless otherwise stated, all the operations described below are carried out at 4 °. Recoveries and specific activities with benzylpenicillin as substrate are given in Table I.
fl-Lactamase ]rom Strain R39 Step 1. The enzyme-DEAE-cellulose complex (640 g; corresponding to about 60 liters of culture fluid) is extracted twice with 1200 ml of
694
ANTIBIOTIC INACTIVATION AND MODIFICATION
i
09
c~
~
i~
o o c~
¢9
>
O
.<
~v O
0
O
09
v
•~ c~
~D
o ~
[53f]
[53fl
Streptomyces ~-LACTAMASES
695
phosphate buffer containing 1 M NaC1. The combined extracts (1 volume) at 0 ° are slowly added with 1.60 volume of acetone previously cooled at --20 °, the precipitate collected by centrifugation and dissolved in 200 ml of phosphate buffer containing 0.2 M NaC1. The solution is centrifuged and concentrated to 80 ml. Step 2. After step 1, the enzyme is adsorbed onto a 180 ml colmnn of DEAE-cellulose equilibrated against phosphate buffer containing 0.2 M NaC1. After washing with the same buffer until only traces of material absorbing at 280 nm remain, most of the DD-carboxypeptidase is eluted with phosphate buffer containing 0.33 M NaC1, fl-Lactamase is eluted in its turn with an increasing convex gradient of NaC1 (mixing flask at constant volume: 750 ml phosphate buffer containing 0.33. M NaC1 ; adding solution: phosphate buffer containing 0.45 M NaC1). Active fractions (from 1550 to 2000 ml) are pooled and concentrated by ultrafiltration to 25 ml. The concentrated solution is filtered through a 700-ml column of Sephadex G-100 in phosphate buffer containing 0.2 M NaC1. fl-Lactamase is eluted at a KD value of 0.21 and is at least partially separated from two yellow and brown pigments of lower and higher KI) values, respectively. Active fractions (from 340 to 390 ml) are pooled. Step 3. After step 2, the enzyme solution is submitted to additional chromatography on DEAE-cellulose and filtration on Sephadex G-100 as described in step 2. Active fractions are pooled, concentrated by ultra filtration to 5 ml, and the concentrated solution is filtered through a 100 ml column of Sephadex G-75 in phosphate buffer containing 0.2 M NaC1. All the active fractions exhibit the same high specific activity. They are pooled and concentrated by ultrafiltration. The final enzyme sohltion (3 mg of protein/ml) is stored at 0 ° in the presence of thymol.
fl-Lactamase ]rorn Strain Albus G Step 1. The enzyme-Amberlite complex (200 g; corresponding to about 10 liter of culture fluid) is suspended in 0.15 M K=,HPO4 and the pH is adjusted to 8.0 with concentrated ammonia. The extract (1 liter) is adjusted to 40% saturation by the addition of 226 g of solid ( N H j 2SO4, and after removal of the precipitate, is adjusted to 75% saturation by the addition of 222 g of solid (NHj:SO~. The precipitate is dissolved in phosphate buffer (final volume: 50 ml), dialyzed, and filtered on a 2 liter column of Sephadex G-75 in phosphate buffer. The fractions containing fl-lactamase activity (elution volume: 800-1300 ml) are pooled and concentrated to a final volume of 30 ml. Step 2. After step 1, the enzyme (3.0 ml) is adsorbed onto a 200-ml column of DEAE-cellulose equilibrated against phosphate buffer and,
696
ANTIBIOTIC
INACTIVATION
AND MODIFICATION
[53f]
after washing with the same buffer, is eluted with an increasing NaC1 gradient from 0 to 0.25 M in phosphate buffer (total elution volume: 500 ml). Most of the fl-lactamase (usually 75% of the total activity) is eluted between 0 and 0.1 M NaC1 and fractions containing maximal activity are about 50 mM with respect to NaC1. They are pooled, concentrated, dialyzed, and subjected to an additional chromatography on the 200 ml column of DEAE-cellulose as above except that the elution is carried out with an increasing NaC1 gradient from 0 to 0.1 M in phosphate buffer (total elution volume: 1 liter). The active fractions (elution volume: 300-500 ml) are pooled and concentrated. Step 3. After step 2, the enzyme is transferred into 20 mM Tris.HC1 buffer pH 7.0 by filtration through Sephadex G-75, and is dialyzed against this buffer. Residual pigment is then removed by preparative polyacrylamide gel electrophoresis at pH 8.3. The fl-lactamase fractions are pooled, concentrated, dialyzed against phosphate buffer containing 10% glycerol (v/v) and stored in 100-t~l aliquots at --20%
Properties of Streptomyces ~-Lactamases Stability. They are completely inactivated by heating at 60 ° for 5 rain. The albus G B-lactamase is sensitive to dilution; activity is rerained and assays are performed in the presence of 10% glycerol (w/v; final concentration). pH and Salt Optimum. pH optimum is between 6 and 8 in 30 mM phosphate buffer. At pH 7, rate of hydrolysis is maximal between 10 and 30 mM phosphate buffer. At pH 8, activity is higher in phosphate buffer than in Tris. HC1 buffer. Metal Ion Requirement. Sodium EDTA, Mg -°+ Mn 2÷, Ca 2÷, and Zn 2÷, each cation being used as its chloride at 2 raM, have no effect. Cu ~÷ (2 mM) inhibited both R39 and albus G enzymes by 50 and 90%, respectively. Sensitivity to -SH Group Reagents. p-Chloromercuribenzoate, iodoacetate, and p-aminobenzoate (1 mM) have no effect on the enzymes. Sensitivity to Iodine. Unlike most B-lactamases, neither the R39 nor the albus G enzymes are inactivated by 2.5 mM iodine/KI solution, for 30 min at 30% Physical Properties. Both R39 and albus G fl-lactamases are anionic at pH 8.3. Polyacrylamide gel eleetrophoresis at this pH, in 25 mM Trisglyeine bufferTM with respect to Tris, discloses only one band of protein 1~M. Leyh-Bouille, M. Nakel, J. M. Fr~re, K. Johnson, J. M. Ghuysen, M. Nieto, and H. R. Perkins, Biochemistry 11, 1290 (1972).
Streptomyces /~-LACTAMASES
[53f]
697
(detected with Coomassie blue) which migrates toward the anode with the buffer discontinuity, fl-Lactamase activity is associated with this band. Isoelectric points and molecular weights are not known. Specificity. Typical Michaelis-Menten kinetics are observed over a wide range of antibiotic concentrations. K .... V,...... and efficiency values (Table II) are determined in 30 m M sodium phosphate pH 7.0 and 30 °. The K,,~ values are well within the norm of other fl-lactamases. The V,..... values of the enzyme preparations, as they are obtained, and therefore. their physiological efficiencies, are low comi)ared with other fl-lactamases. Based on their substrate profiles, the two Streptomgces fl-laetamases (lifter from one another and from other fl-lactamases of bacterial origin. Effect of DD-Carboxgpeptidase Substrates and Inhibitors. 3,'",N~-Dia cetyl-L-lysyl-D-alanyl-D-alanine, N"-acetyl-L-lysyl-D-alanyl-D-alanine and the disaceharide pcntapeptide-pentaglycine [N"-(fl-l,4-N-acetylglucosaminyl - N - acetylmuramyl-L-alanyl-D-isoglutaminyl ) -N ~- (pentaglycyl)-L-lysyl-D-alanyl-D-alanine] arc substrates of both R39 and albus G Dn-carboxypeptidases. Acetyl-D-alanyl-D-glutamic acid, X%N~-disuc cinyl-L-lysyl-D-alanyl-D-glutamic acid, and L-lysyl-D-glutamyl-D-atanine inhibit the albus G DD-carboxypeptidase but have no effect on the R39
T A B L E II HYDROLYSIS OF ~-LACTAM ANTIBIOTICS BY ~-LACTAMASES 1/39 AND albus (~
albus G
R39
[rnlax
Wmax K.,
(t~moles/mg protein/inin)
Km
Efficiency ~
(raM)
(ulnoles/ms Eftiprotein/nlin) eiency ~
Substrate
(raM)
Benzylpenicillin 9-Aminopenicillanic acid Penicillin V Ampieillin Carbenicillin Oxacillin Cloxacillin Methicillin Cephalosporin C Cephaloglycin Cephalexin Cephalothin
0.07
198
2835
0.74
33
45
0.07 0.28 0.57 0.26 0.54 0.42 0.29 1.05 2.86 0.91 0.12
152 452 1038 78 646 60 55 12 429 35 111
2175 1615 1820 300 1200 140 190 10 150 40 920
0.6() 0,62 0.90 1,00 0.33 0.25 0.77 3,33 3,84 4,53 1.33
25 27 70 3.3 6.2 0.6 0.5 2 2.5 0,4 0,8
42 44 78 3,3 19 2.5 0.7 (I, 7 0,7 0.1 0,6
r, Ffficiency: V ..... divided by Kin.
698
ANTIBIOTIC INACTIVATION AND MODIFICATION
[54a]
DD-carboxypeptidase-transpeptidase. 17 The above peptides (1.1-1.7 mM, final concentrations) neither inhibit nor activate the S t r e p t o m y c e s fl-lactamases (using benzylpenicillin at concentrations near the relevant Km values). " M . Nieto, H. R. Perkins, M. Leyh-Bouille, J. M. Fr~re, and J. M. Ghuysen, Biochem. ]. 131, 163 (1973).
[54a] Penicillin Acylase (Assay) B y M. COLE, T. SAVlDGE, and H. VANDERHAr~IE RCONH
.S
+
H~N ~----~N_~
H20
O~--N----~CO,~
6-Aminopenicillanic acid
Penicillin
6-APA)
R
=
R
=
R
=
~ ~ ~
CH 2-
+ RCO~H Carboxylic acid
in benzylpenicillin
_OCH2__ in phenoxymethylpenicillin -CH-NI-I2
in ampicillin
The enzyme catalyzing the above reversible reaction is usually called penicillin acylase and is produced by m a n y types of microorganisms in intracellular and extracellular forms. I t was called penicillin amidase in early literature and has been given the E n z y m e Commission number EC 3.5.1.11. For a discussion of enzyme nomenclature and early work, the reviews by H a m i l t o n - M i l l e r 1 and Cole 2 can be consulted. The enzyme is not specific for penicillins but can also hydrolyze certain cephalosporins ~,4 and a variety of acylamino acids, amides, and esters. ~ The 1j. M. Hamilton-Miller, Bacteriol. Rev. 30, 761 (1966). M. Cole, Process. Biochem. 2, 35 (1967). 3 M. Cole, Biochem. J. 115, 733 (1969). 4B. Sjoberg, L. Nathorst-Westfelt, and B. Ortengren, Acta Chem. Scand. 21, 547 (1967). 5 M. Cole, Biochem. J. 115, 741 (1969).
698
ANTIBIOTIC INACTIVATION AND MODIFICATION
[54a]
DD-carboxypeptidase-transpeptidase. 17 The above peptides (1.1-1.7 mM, final concentrations) neither inhibit nor activate the S t r e p t o m y c e s fl-lactamases (using benzylpenicillin at concentrations near the relevant Km values). " M . Nieto, H. R. Perkins, M. Leyh-Bouille, J. M. Fr~re, and J. M. Ghuysen, Biochem. ]. 131, 163 (1973).
[54a] Penicillin Acylase (Assay) B y M. COLE, T. SAVlDGE, and H. VANDERHAr~IE RCONH
.S
+
H~N ~----~N_~
H20
O~--N----~CO,~
6-Aminopenicillanic acid
Penicillin
6-APA)
R
=
R
=
R
=
~ ~ ~
CH 2-
+ RCO~H Carboxylic acid
in benzylpenicillin
_OCH2__ in phenoxymethylpenicillin -CH-NI-I2
in ampicillin
The enzyme catalyzing the above reversible reaction is usually called penicillin acylase and is produced by m a n y types of microorganisms in intracellular and extracellular forms. I t was called penicillin amidase in early literature and has been given the E n z y m e Commission number EC 3.5.1.11. For a discussion of enzyme nomenclature and early work, the reviews by H a m i l t o n - M i l l e r 1 and Cole 2 can be consulted. The enzyme is not specific for penicillins but can also hydrolyze certain cephalosporins ~,4 and a variety of acylamino acids, amides, and esters. ~ The 1j. M. Hamilton-Miller, Bacteriol. Rev. 30, 761 (1966). M. Cole, Process. Biochem. 2, 35 (1967). 3 M. Cole, Biochem. J. 115, 733 (1969). 4B. Sjoberg, L. Nathorst-Westfelt, and B. Ortengren, Acta Chem. Scand. 21, 547 (1967). 5 M. Cole, Biochem. J. 115, 741 (1969).
[54a1
PENICILLIN ACVLASE (ASSAY)
699
specificity resides mainly in the structure of the acyl group, and this m a y be used to distinguish different enzymes. 6 Thus the fungal penicillin acylases more readily deacylate phenoxymethylpenicillin t h a n benzylpenicillin whereas for m a n y bacterial penicillin acylases the reverse is the case. The reaction in the synthetic direction proceeds more readily at slightly acid p H values (e.g., 5.0) whereas the deacylation reaction is usually faster at slightly alkaline p H values. :,~ Reactions in the synthetic direction often proceed more readily at neutral p H values when the carboxylic acid is in an activated form, such as amide or ester. 8 The penicillin acylase of E s c h e r i c h i a coli is specific for acyl-L-amino acids 5 and can be used for amino acid resolution. The use of acylases in the preparation of 6-aminopenicillanic acid (6-APA) and 7-aminocephalosporanic acid has been recently reviewed2
Assay Methods Principle. The most specific method of assaying these enzymes involves the determination of the rate of formation of 6-APA from a readily available penicillin, such as benzylpenicillin or phenoxymethylpenicillin. The method of choice involves the hydroxylamine assay of 6-APA 1° after removal of residual penicillin substrate by solvent extraction at p H 2 (Assay 1 below). Alternatively the 6-APA m a y be assayed in the presence of residual substrate by the p-dimethylaminobenzaldehyde method. The method lends itself to automation, 11 but the fl-lactam ringopened form of 6-APA also reacts. A n o n a u t o m a t e d version has also been described."-' A recently published method for assaying 6-APA in the presence of penicillins involves reaction with glucosamine. TM This method shows promise but has not yet been used for penicillin acylase assays by the present authors. A less specific but readily performed method involves
6 G. N. Rolinson et al., Nature (London) 187, 236 (1960). 7 W. Kaufmann, K. Bauer, and H. A. Offe. Antimicrobial. Ag. Annu. 1960, p. 1 (1961). s M. Cole, Biochem. J. 115, 747 (1969). 9 F. M. Huber, R. R. Chauvette, and B. G. Jackson, in "Cephalosporins and Penicillins" (E. H. Flynn, ed.), Chapter 2, p. 27. Academic Press, New York, 1972. 1oF. R. Batchelor, E. B. Chain, T. L. Hardy, K. R. Mansford, and G. N. Rolinson, Proc. Roy. Soc. Set. B 154, 498 (1961). 11j. Bomstein and W. G. Evans, Anal. Chem. 37, 576 (1965). i.~K. BMasingham, D. Warburton, P. Dunnill, and M. D. Lilly. Biochim. Biophys. Acta 276, 250 (1972). 13K. Shaikh, P. G. Talati, and D. M. Gang. Antimicrob. Ag. Chemother. 3, 194 (1973).
700
ANTIBIOTIC INACTIVATION AND MODIFICATION
[54a1
the pH-stat titration of carboxylic acid side chain liberated from the substrate penicillin. This method is suitable for following large-scale reactions or obtaining kinetic data provided that no fl-lactamase enzyme is present to liberate penicilloic acid. A microbiological assay for 6-APA in the presence of benzylpenicillin has been described using S e r r a t i a m a r c e s c e n s ATCC 27117. TM In the examination of new strains of microorganisms for the presence of the enzyme, the chromatographic detection of liberated 6-APA is probably the best method. After separation of the 6-APA from residual penicillin by paper chromatography, t h e 6-APA is converted to antibacterially active benzylpenicillin by phenylacetylation, and the zones of activity are detected and assayed by the bioautographic procedure (Assay 2 below). As penicillin acylase enzymes are reversible, they may also be assayed by determining the rate of formation of penicillin by bioassay ~5 or after solvent extraction by the hydroxylamine assay. 15 P r e c a u t i o n s . Crude microbial acylase enzyme preparations may be contaminated with fl-lactamase, an enzyme that catalyzes the opening of the fl-lactam ring of penicillins and 6-APA to give antibacterially inactive penicilloic acids. In some bacteria and actinomycetes this fl-lactamase is coproduced with penicillin acylase under certain cultural conditions. This subject is reviewed by Hamilton-Miller? Alternatively the /%lactamase may arise from bacterial contamination of enzyme preparations during storage. Enzyme preparations should be filtered through bacteria-retaining membranes (e.g., Millipore 0.22 ~m pore size) or shaken with n-butylacetate (1% v / v final concentration) to prevent bacterial contamination. It is essential to ensure that no fl-lactamase activity is present in penicillin acylase preparations, or to make due allowance for it if it is unavoidable. Enzyme preparations can be checked for the presence of fl-lactamase by incubating at 3 7 ° a 2 mg/ml solution of benzylpenicillin at pH 7 and examining the reaction mixture after 1 hr for the presence of benzylpenicilloic acid by paper TM or thin-layer chromatography. 17 Alternatively a 2 mg/ml solution of 6-APA in pH 8.0 buffer is incubated with the enzyme preparation for 1 hr at 37 °, and the loss of 6-APA is measured by hydroxylamine assay (procedure below). A control in which the enzyme is replaced by water is also assayed after 1 hr. ~'ff. G. Oostendorp, Antonie van Leeuwenhoek, J. Microbiol. Serol. 38, 201 (1972). 15M. Cole, Biochem. J. 115, 757 (1969). 1, M. Cole and R. Sutherland, J. Gen. Microbiol. 42, 345 (1966). 1~M. Cole, M. D. Kenig, and V. A. Hewitt. Antimicrob. Ag. Chemother. 3, 463 (1973).
[54a]
PENICILLIN ACYLASE (ASSAY)
701
The effect of the presence of fl-lactamase on the assays for penicillin acylase based on 6-APA liberation is to give an underestimate of reaction rate because of destruction of substrate and product, although 6-APA is often more stable to fl-lactamases than is benzylpenicillin. In the titrimetric or pH-stat method, a serious overestimate of rate of reaction is made because of the formation of a second acid group from the opened fi-lactam ring. Thus this method will record activity even in the absence of penicillin acylase if fl-lactamase is present. Penicilloic acids may also be generated from penicillin or 6-APA by extremes of pH, particularly if temperature is also elevated. Values of I)H above 8.5 or below 5.0 should be avoided. The hydroxylamine assay procedure described below is not suitable when using ampicillin (D-a-aminobenzylpenicillin), penicillin N, or other amphoteric penicillins as substrate because these penicillins are not solvent extractable. Instead the chromatographic separation and bioassay of 6-APA may be used iG (see Assay 2 below). This latter method is also more suitable when assaying low enzyme activities. Care should be taken to set up and assay a non-substrate control when examining cultures which may make 6-APA biosyntheticly5~ In preliminary experiments a substrate control (no enzyme) should be used to check for nonenzymic deacylation, which can occur for example with penicillins ~:) and cephalosporins 4 at high pH values. These two precautions particularly apply when using the sensitive biochromatographic method.
Assay 1 Reagents
Potassium or sodium benzylpenicillin or potassium phenoxymethyl penicillin substrate, (Glaxo Laboratories Ltd., Greenford, Middlesex, England), freshly prepared in 50 mM potassium phosphate buffer, pH 8.0 6-Aminopenicillanic acid dissolved in water by pH adjustment to 7.0 with 0.1 M NaOH, 1 mg/ml (standard) (Beecham Research Laboratories, Worthing, Sussex) Hydroxylamine hydrochloride solution, 347.6 g/liter Alkaline buffer solution, 173 g NaOH + 20.6 g sodium acetate/liter '~ M. Cole, Appl. Microbiol. 14, 98 (1966). 19F. R. Batehelor and J. Cameron-Wood, Nature (London) 195, 1000 (1962).
702
ANTIBIOTIC INACTIVATION AND MODIFICATION
[54a]
Hydroxylamine reagent: alkaline buffer + hydroxylamine solution ethanol mixed in the ratio 1:1:4 v/v. The final pH of the reagent must be 7.0 Ferric ammonium sulfate solution, 200 g of ferric ammonium sulfate, and 93.4 ml of sulfuric acid/liter
Procedure (Hydroxylamine Assay). The enzyme preparation is mixed with substrate penicillin to give a final concentration of 20 mg of penicillin per milliliter in 50 mM phosphate buffer, pH 8.0. Choice of substrate depends on the type of acylase (see later sections) but benzylpenicillin or phenoxymethylpenicillin are most frequently used. When working with bacterial cells as source of enzyme, they should be thoroughly resuspended after collection by centrifugation and prior to dilution with substrafe solution. Failure to disperse the cells can give lowered assays. Duplicate reaction mixtures are incubated for 1 hr at 37% If they contain cells or mycelium, they should be shaken continuously. The yield of 6-APA is then determined as follows: If present, microbial cells are centrifuged from each reaction mixture (4000 g, 12 rain), and a suitable volume of supernatant is adjusted to pH 2.0 with 5 M HC1. The unconverted penicillin and liberated side-chain acid are then removed by shaking twice with 1 volume of n-butylacetate and rejecting the acetate phase. Diethylether may be used here if it is necessary to diminish the small acetate interference in the hydroxylamine assay. The aqueous phase is returned to pH 7.0, and after dilution to a known final volume the 6-APA content is assayed by the hydroxylamine method as follows: Into each of two tubes pipette 2.0 ml of sample. Into one of the tubes (blank) pipette 0.25 ml of 5 M NaOH followed, after 10 min, by 0.25 ml of 2.5 M H._,SO~. Into the other tube pipette 0.5 ml of water. Into both tubes pipette 6.0 ml of hydroxylamine reagent followed, after 10 min at 25 °, by 2.0 ml of ferric ammonium sulfate solution. Mix the contents of the tubes, leave for 3 min, and read absorption in an E.E.L. Co]orimeter using a blue-green filter No. 623 or a spectrophotometer at 490 nm. 6-APA solutions in the range 0.1-1.0 mg/ml, pH 7, are put through the same procedure, and the results are used to prepare a standard line. Using as enzyme the cells of E. coli NCIB 8743, 3 approximately 30% and 50% hydrolysis of benzylpenicillin to 6-APA is obtained at 0.5 hr and 1.0 hr, respectively. The cells for this reaction are grown in a yeast extract/phenylacetic acid medium and resuspended in the reaction mixture to give the same concentration as in the growth medium. One unit of enzyme is the amount required to liberate 1 ~mole of 6-APA per minute at pH 8 and 37 °.
[54a]
PENICILLIN ACYLASE (ASSAY)
703
Assay 2 Reagents Substrate solutions of benzylpenicillin (Glaxo), phenoxymethylpenicillin (Glaxo), or ampicillin (Beecham), in 50 raM, pH 8, phosphate buffer Standard solutions of 6-APA (Beecham) approximately in the range 12.5-400 t~g/ml, doubling concentrations, in 50 mM, pH 7, phosphate buffer Potassium phosphate buffer; stock 0.5 M, 24.3 g KH~P04 plus 56.0 g K~HPO4 in 1 liter of deionized water plus 1 ml of n-butylacetate. Dilute to 50 mM for use Whatman No. 1 paper strips 1 cm X 45 cm (100 meter reels manufactured by W. and R. Balston Ltd., Maidstone, Kent, England) Chromatography solvent: butan-l-ol/ethanol/water; 4:1:5, v/v, top phase after equilibration at 4 ° NaHCO3 solution, 5% in water Phenylacetyl chloride (BDH Ltd., Poole, England), 5% in acetone, freshly prepared Nutrient broth No. 2, 2.5%, + Agar 2% (Oxoid Ltd., London, SE1 9HF, England.) Blood agar base 3.5% (CM55) (Oxoid Ltd.) Spores of Bacillus subtilis ATCC 6633
Procedure (Biochromatographic Assay). Enzyme solution, culture illtrate, or suspension of microbial cells is mixed with substrate penicillin solution to give a final concentration of 5 mg/ml. The pH is adjusted to 8.0 with 0.1 M NaOH if necessary, and duplicate reaction mixtures are incubated for 1-4 hr at 30 or 37 ° with shaking if cells are present. Choice of substrate concentration, temperature, pH, and duration of reaction will depend on the type of enzyme preparation being examined. If this procedure is used for preliminary testing of cultures grown in shaken flasks, it is convenient to add the penicillin substrate so as to give I mg/ml final concentration. The pH may be left unadjusted if it is between 7 and 8. The flasks are then shaken at 30-37 ° and assayed for 6-APA content after intervals of up to 4 hr. When testing cultures that may produce 6-APA biosynthetically a nonsubstrate control should be set up. A further control of substrate alone should be used if pH values higher than 8.0 are used. Cell-free samples from the reaction mixtures are examined for 6-APA content by the following method.
704
ANTIBIOTIC INACTIVATION AND MODIFICATION
[54a]
Duplicate Whatman No. 1 paper chromatography strips, 1 cm X 45 cm, are spotted with 5 ~l of cell-free reaction mixture diluted up to 1 in 4 if necessary. Other strips are spotted with the substrate and culture controls, and 6-APA standards (400-12.5 ug/ml in doubling dilutions). After air drying, the strips are placed in a chromatography tank at 4 ° and developed overnight by descending chromatography using the butan1-ol/ethanol/water solvent. Whatman No. 4 strips may be used when chromatograms are run during the day. The solvent fronts are marked, then the strips are dried in a stream of antibiotic-free air at no more than 40 ° . One of the pair of strips for each reaction mixture and control is then sprayed consecutively with 5% aqueous NaHC03, 5% phenylacetyl chloride in acetone, and 5% aqueous NaHC03 so that the paper strip is damp but not running with liquid. All the 6-APA standard chromatograms are similarly sprayed. The paper strips are left to dry in a fume cupboard supplied with air free from antibiotic dust. This phenylacetylation converts 6-APA into antibacterially active benzylpenicillin. Without such treatment 6-APA shows no antibacterial activity unless loaded at very high concentration (2%). The sprayed and unsprayed strips are then laid on a sheet of B. subtilis-seeded agar prepared as described below. This bioautogram plate is incubated for 6 hr at 40 ° or overnight at 30 ° to reveal the antibiotic zones as areas of inhibited bacterial growth. Most substrate penicillins (and cephalosporins), except penicillin N, have higher Rs values in the butan-l-ol/ethanol/water system than 6-APA, which, if it has been formed in the reaction mixture, is seen as a slow-moving zone at about RI 0.1-0.2, occurring only on the phenylacetylated chromatogram. For a photograph of a bioautogram prepared using the above solvent see Cole. 2° Other solvent systems that may be used for separating 6-APA (slow moving) from substrate (fast) are butan-l-ol/glacial acetic acid/water, 12:3:5 v/v, or butan-l-ol/pyridine/water, 1 : 1 : 1, v/v. The yield of 6-APA in the reaction mixture can be obtained by measuring the width of the 6-APA zone and reading off the corresponding concentration of 6-APA from a graph of log concentration against zone width for the 6-APA standards. Alternatively the surface area of the zones may be plotted against log concentration. The method is capable of detecting down to 1 ~g of 6-APA per milliliter. The accuracy of the method can be increased by replicating the chromatograms for the reaction mixtures and standards, only phenylacetylated chromatograms being necessary if the presence of 6-APA has already been confirmed and c o n ~oM. Cole, Process. Biochem. 1, 334 (1966).
[54b]
PENICILLIN ACYLASE (BACTERIAL)
705
trols carried out. As 6-APA runs slowly in the butan-l-ol/ethanol/water system, the top half of the chromatogram strip can be cut off after spraying and only the lower half laid on the B. subtilis plate. M a n y more strips can thus be handled allowing for replication and randomization of standards and unknowns. The above biochromatographic method is applicable also to measuring the rate of formation of 7-aminocephalosI)oranic acid from cephalosporins such as cephalothin which is also a substrate for the penicillin acylase of Escherichia coli. The l)rep,aration of the B. subtilis-seeded agar plate is e'~rrie(t out as follows : The spores of B. subtilis ATCC 6633 are prei)ared by inoculating Roux bottles containing 350 ml of Oxoid nutrient t)roth plus 2%, agar. The bottles are incubated for 5 days a t 3 7 ° before draihing off surphls liquid and incubating for a further 9 days. Sterile water ~50 mlt is then added to each bottle, and the spores are dislodged by shaking. The suspension so obtained is filtered through sterile glass wool to remove debris, and the filtrate is pasteurized at 70 ° for 1 hr before dispensing into small sterile glass bottles with screw caps for storage at 4 °. The stock sust)ension can be stored for a t least 6 months. A 300-ml volume of Oxoid blood agar base, 3.5% (CM55) is melted, cooled to 60% and inoculated with about 0.5 ml of a suitable dilution (about 1 in 40) of the stock B. subtilis spore suspension. The agar is then poured into a previously leveled tray or plate having a fiat glass base 33 X 40 cm to give a layer about 2 mm thick. The amount of spore inoculum should be such that the individual colonies of B. subtilis are only just touching one another and a 10 ~,g/ml solution of benzylpenicillin when placed in a 8-mm hole cut in the agar gives a zone of inhibition of about 30 mm after incubation. Too heavy an inoculum reduces the sensitivity of the assay.
[54b] Penicillin Acylase (Bacterial) By T. A. SAVIDGE and M. COLt: Penicillin acylases are widely distributed among bacteria and actinomycetes. 1,'-' Most of the published work is on the intracellular enzyme 1M. Cole, Process Biochem. 2, 35 (1967). : M. Cole, Biochem. J. 115, 733 (1969).
[54b]
PENICILLIN ACYLASE (BACTERIAL)
705
trols carried out. As 6-APA runs slowly in the butan-l-ol/ethanol/water system, the top half of the chromatogram strip can be cut off after spraying and only the lower half laid on the B. subtilis plate. M a n y more strips can thus be handled allowing for replication and randomization of standards and unknowns. The above biochromatographic method is applicable also to measuring the rate of formation of 7-aminocephalosI)oranic acid from cephalosporins such as cephalothin which is also a substrate for the penicillin acylase of Escherichia coli. The l)rep,aration of the B. subtilis-seeded agar plate is e'~rrie(t out as follows : The spores of B. subtilis ATCC 6633 are prei)ared by inoculating Roux bottles containing 350 ml of Oxoid nutrient t)roth plus 2%, agar. The bottles are incubated for 5 days a t 3 7 ° before draihing off surphls liquid and incubating for a further 9 days. Sterile water ~50 mlt is then added to each bottle, and the spores are dislodged by shaking. The suspension so obtained is filtered through sterile glass wool to remove debris, and the filtrate is pasteurized at 70 ° for 1 hr before dispensing into small sterile glass bottles with screw caps for storage at 4 °. The stock sust)ension can be stored for a t least 6 months. A 300-ml volume of Oxoid blood agar base, 3.5% (CM55) is melted, cooled to 60% and inoculated with about 0.5 ml of a suitable dilution (about 1 in 40) of the stock B. subtilis spore suspension. The agar is then poured into a previously leveled tray or plate having a fiat glass base 33 X 40 cm to give a layer about 2 mm thick. The amount of spore inoculum should be such that the individual colonies of B. subtilis are only just touching one another and a 10 ~,g/ml solution of benzylpenicillin when placed in a 8-mm hole cut in the agar gives a zone of inhibition of about 30 mm after incubation. Too heavy an inoculum reduces the sensitivity of the assay.
[54b] Penicillin Acylase (Bacterial) By T. A. SAVIDGE and M. COLt: Penicillin acylases are widely distributed among bacteria and actinomycetes. 1,'-' Most of the published work is on the intracellular enzyme 1M. Cole, Process Biochem. 2, 35 (1967). : M. Cole, Biochem. J. 115, 733 (1969).
706
ANTIBIOTIC INACTIVATION AND MODIFICATION
[54b]
produced by Escherichia coli, and the preparation and properties of this enzyme are described in detail below. In addition and for comparison, we describe the published work 3-6 on the preparation and properties of the extracellular enzyme produced by the gram-positive organism Bacillus megaterium. A selection of other penicillin acylase-producing bacteria is listed in Table VI together with the summary of their properties.
Assay Assay procedures for penicillin acylase are described in a previous article. ~
T h e Penicillin Acylase of E s c h e r i c h i a c o i l Preparation Culture. The enzyme is produced by various strains, e.g., ATCC 9637, ATCC 11105, N C I B 8743,1 or N C I B 8743, selection A, a natural variant capable of growing well on a corr-steep liquor based medium. 2 For industrial use, various higher-yielding derivatives have been obtained by mutation and selection. Stock cultures are prepared from freeze-dried cultures by inoculating nutrient agar slopes and incubating at 27 ° for 24 hr. These slopes m a y be stored at 4 ° for up to 1 month. Workir~g slopes are prepared by subculturing the stock slopes onto the same medium and incubating for 24-48 hr at 270. 2 Fermentation. A most important constituent of the fermentation medium is phenylacetic acid, which is metabolized during the course of bacterial growth, increasing enzyme production by 5- to 10-fold. The nitrogen content of the medium m a y be provided by complex organic sources, such as corn-steep liquor (CSL)S yeast extract, ~ Casamino acids, ~ or by simple chemically defined sources, such as sodium glutamate. 1° Enzyme 3C. Chiang and R. E. Bennett, J. Bacteriol. 93, 302 (1967). U.S. Patent 3,144,395 (1964). 5U.S. Patent 3,446,705 (1969). 6D. Y. Ryu, C. F. Bruno, B. K. Lee, and K. Venkatasubramanian, in "Fermentation Technology Today" (G. Terui, ed.), p. 307. Society of Fermentation Technology, Japan, 1972. This volume [54a]. s British Patent 1,015,554 (1966). ' A. Szentirmai, Acta Microbiol. Acad. Sci. Hung. 12, 395 (1966). loD. A. Self, G. Kay, M. D. Lilly, and P. Dunnill, Biotechnol. Bioeng. 11, 337 (1969).
[54b]
PENICILLIN ACYLASE (BACTERIAL)
707
production is greatest at incubation temperatures of between 24 ° and 30 ° and at rather low aeration rates. A particularly suitable fermentation medium is one based on CSL, the procedure being as follows. A working slope is flooded with saline solution, scraped, and 0.5-ml aliquots of the resulting cell suspension are used to inoculate 500-ml conical flasks closed with lint caps and containing 100 ml of medium consisting of CSL (usually supplied as a concentrated solution), 1.2% w/v on a dry solids basis, adjusted to pH 7.0 with NaOH and sterilized by autoclaving at 121 ° for 10 rain. The flasks are incubated on a rotary shaker (250 rpm) at 24 ° for 24 hr and then used to inoculate fermentation medium consisting of CSL 1.2% w/v dry solids; ammonium sulfate, 0.1% w/v; and phenylaeetic acid added as the ammoniun~, sodium, or potassium salt, 0.1% w/v. The pH is adjusted to 6.5 with NaOH, and aliquots (100 inl) are dispensed into 500-ml conical flasks closed with lint caps and sterilized at 121 ° for 10 rain. The flasks are inoculated with 1 ml of seed culture and incubated on a rotary shaker 1240 rpm, 1 inch radius circle) at 24 ° for 24 hr. For larger volumes a stirred fermentor is inoculated with seed culture (0.5% v/v) developed either in shaken flasks or, depending on the volume of the fermentor, from a second seed stage using the same medium in a stirred fermentor. The aeration and agitation rates during fermentation must be optimized for individual fermentor vessels, aeration rates of 0.4 to 0.9 v/v per minute having been reported. '-',11 Incubation at 24 ° is continued until the pH of the medium increases to 8.0, generally after around 20 hr; typical enzyme levels are around 0.3 units/ml2 ~ Higher enzyme levels have been reported to occur, '~ when the phenylacetate is added gradually from 8 to 21 hr after inoculation as a solution of the ammonium salt at pH 7.0. For a fermentation medimn volume of 600 liters, 600-ml aliquots of ammonium phenylacetate solution ,~re added hourly from 8 to 21 hr after inoculation to give a total addition equivalent to 0.35% w/v phenylacetic acid. The ammonium phcnylacetare solution is prepared by treating 25% w/v aqueous phenylacetic acid with concentrated aqueous ammonium hydroxide to pH 7.0 and sterilizing at 121 ° for 30 rain. The fermentation is terminated when the pH value rises to 8.0 after around 28 hr incubation. In circumstances where it is difficult to obtain corn-steep liquor, yeast extract (e.g., The English Grain Co. Ltd., Burton-on-Trent, Staffordshire) is a suitable alternative. Yeast extract (generally supplied at a solids content of 70%) is added to give a concentration in the medium of 2.5% w/v. With this medium, enzyme titers of 0.03 and 0.07 unit per ~'British Patent 1,250,069 (1971). ~2British Patent 1,261,711 (1972).
708
ANTIBIOTIC INACTIVATION AND MODIFICATION
[54b]
milligram of dry cells were obtained with NCIB 8743 and NCIB 8743 selection A, respectively. ~ Cell Harvest. For small volumes of fermentation broth, the cells may be collected as a paste by centrifugation at 13,000 g. Disc bowl centrifuges are preferable for larger volumesl:~; thus for ca. 500 liters, a Westfalia Disc Bowl Separator, Model SAOOH 205, may be used to produce a slurry at a cell concentration around 10 times that of the fermentor broth. Since the strain of E. coli can be of fecal origin, it may be desirable to kill the cells before harvesting by adding n-butyl acetate to the fermentor broth to a concentration of 1% (v/v), and stirring for 20 rain. Cell Disruption. Chemical methods for releasing enzyme from cells have been reported, TM but the following two mechanical methods are preferred. For laboratory-scale extractions, ultrasonic disintegration is carried out as follows. After resuspension of centrifuged cells in 0.1 M phosphate buffer, pH 7.8 and 4 °, a 25-ml aliquot is transferred to a chilled vessel, and the probe of an M.S.E. ultrasonicator (M.S.E., Crawley, Sussex, England) Model M158 is inserted to a depth of approximately 1 cm. The suspension is sonicated at 20 kc/sec and 100 W for 30 sec. After cooling for 30 sec, sonication is repeated in this fashion for a total of 3-5 rain depending upon the cell concentration. Larger volumes of cell slurry are best disrupted by passing the suspension through an industrial high pressure homogenizer. An example of a machine in common use for releasing intracellular enzymes is the Manton-Gaulin homogenizer (A.P.V. Co. Ltd., Crawley, Sussex, England), in which the suspended microorganisms are forced through a narrow slit between two steel faces. 13 It has been shown, 1~ that the release of fl-galactosidase by disruption of E. coli is dependent on pressure to the power 2.2. To release quantitatively the enzyme from 50 liters of concentrated cell suspension, the slurry is cooled to 4 ° and passed 4 times through a Manton-Gaulin Model 15M/SBA operated at a pressure of 500 kg/cm 2. After each pass the slurry is cooled to below 5 ° . On a small scale, cell debris is removed by centrifugation (e.g., Sharples Centrifuge Model 6P, Sharples Centrifuges Ltd., Camberley, Surrey, England), at. a speed of 15,000 rpm. The use of air-driven laboratory models should be avoided because of the danger of forming aerosols containing endotoxins. Tile large-scale removal of debris may prove difficult ~3S. E. C h a r m and C. C. Matteo, this series Vol. 22, p. 476. ~4Canadian P a t e n t 975,769 (1970). ~ P. P. Gray, P. Dunnill, and M. D. Lilly, in " F e r m e n t a t i o n Technology Today" (G. Terui, ed.), p. 347. Society of F e r m e n t a t i o n Technology, Japan, 1972.
[54bl
PENICILLIN ACYLASE (BACTERIAL)
709
because of the very small size of the solid particles having a small density differential with respect to the aqueous phase, but rotary vacuum filtration has been found to be satisfactory. ~5 Purification
A number of purification procedures have been reported but the following two methods I~','T are recommended. Crystalline enzyme has been obtained by the second method. M e t h o d A. TM After disruption of the E. coli cell slurry in tile MantonGaulin homogenizer and precipitation of nucleic acids with streptomycin sulfate (0.7% w/v), the enzyme is precipitated from the clarified solution by the slow addition of ammonium sulfate to give a 60% saturated solution. After leaving overnight at --5 ° the precipitate is centrifuged off in a Sharplcs 6P centrifuge, and the supernatant is discarded. The enzyme-containing precipitate is stored at 2 ° and processed further when required by redissolving it in 10 mM phosphate buffer, pH 7.0 and fraet.ionally precipitating with polyethylencglycol molecular weight 6000 (Shell Chenficals Ltd.). Tile fraction collected between 10 and 20% w/v polyethyleneglycol is redissolved in 10 mM phosphate buffer, pH 8.0, dialyzed against more of this buffer, and then passed down a column (45 }( 7.5 cm diameter) of DEAE-cellulose (Whatman grade DE-52~. The purified enzyme apl)ears in the first protein-containing fractions (the ~pecific activity of the most active fraction was 12.1 units/rag) which are pooled, dialyzed against 5 mM phosphate l)uffer, I)H 7.5, and freezedried. The powder is stored at --20°; all the isolation steps are carried out at .iround 5 °. The results obtained with this isolation procedure are shown in Table I. The apparent increase in total enzyme activity at the start of the polyethyleneglycol precipitation stage is because the hydroxylamine assay r which was used in the initial stages was replaced by the p-dimethylaminobenzaldehyde assay, ~ which, although less specific, is sufficiently sensitive to permit determination of the initial reaction rate. M e t h o d B. ~ After disruption of the cells, the 1)H of a 4-liter aliquot of ceil homogenate is adjusted to 5.0 with dilute sulfuric acid, and ttlc cell debris is removed by centrifugation. The pH of the supernatant is adjusted to 6.0 and fractionated by the addition of solid ammonium sulfate. The fraction precit)itating between 40 and 6 0 ~ saturation is collected and dissolved in 400 ml of 50 mM sodium acetate pH 5.0 and dialyzed against 20 volumes of the same buffer. The dialyzed solution ~'~K. Balasingham, D. Warburton, P. Dunnill, and M. D. Lilly, Biochim. Biopl~gs. Act(~ 276, 250 (1972). '~ C. Kutzbach and E. Rauenbusch, Hoppe Seyler's Z. Pliysiol. Chem. 354, 45 (1974).
710
ANTIBIOTIC INACTIVATION A N D MODIFICATION
•-3 .~.
6666
09
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[54b]
[54b]
PENICILLIN ACYLASE (BACTERIAL)
711
is applied to a column (90 X 5 cm) of SE-Sephadex C-50 and equilibrated with the same buffer (chromatography is carried out in a constant temperature room at 20% and the fractions are cooled to 5-10°). Elution is carried out with a linear gradient composed of equal volume {5 liters) of 0.07 and 0.25 M sodium acetate, pH 5.0, added at a rate of 150 ml/hr. After a breakthrough peak, the acylase is eluted between 4.2 and 5.0 liters at a buffer concentration of approximately 0.15 M. The enzymecontaining fractions are concentrated to 130 ml under vacuum, slight amounts of precipitate being removed by centrifugation. The solution is then dialyzed against 10 mM potassium phosphate pH 7.0 and applied to a column (40 X 2.5 cm) of DEAE-Sephadex A-50 in the same buffer. The enzyme is eluted with a linear gradient composed of equal volumes (1 liter) of 10 mM and 0.1 M phosphate, pH 7.0, the acylase occurring in the first protein peak. The enzyme-containing fractions are combined and brought to 1 mM EDTA; the enzyme is precipitated with ammonium sulfate, which is added to give 70% saturation. The precipitate is recovered by centrifugation and dissolved in a minimum volume of ice-cold 45% saturated ammonium sulfate, pH 6.0. A slight turbidity is centrifuged off, and the enzyme is induced to crystallize by storage at room temperature for a few days. The crystals are recovered by centrifugation and suspended in 50% saturated ammonium sulfate. The overall yield of crystalline enzyme is 25%, and the total purification is close to 200fold, the most important step being gradient elution from the cation exchanger (purification factor: 13) The Penicillin Acylase of Bacillus rnega teriurn Preparation Culture. Bacillus megaterium, ATCC 14945 is maintained as lyophilized cell suspensions or on meat extract agar slopes. ~ Fermentation. ~ A seed medium consisting of enzyme-hydrolyzed casein (Amber Lab. Inc., Milwaukee, Wisconsin), 4%; glucose 0.5%, and Ucon antifoam LB 625 (Dow Chemical Co., Midland, Michigan), 0.05% is adjusted to pH 7.0 before and after sterilization at 121 ° for 30 rain. It is then inoculated and incubated with aeration and agitation at 30 ° for 24 hr. This seed is used to inoculate at a concentration of 3% (v/v), fermentation medium having the same composition as the seed medium with the exception of enzyme-hydrolyzed casein at 3% instead of 4%. After incubation with aeration and agitation (0.2 HP/100 gal) at 30 ° for 8 hr, 0.15% (w/v) phenylacetic acid is added and incubation is continued for about 70 hr. An enzyme titer of 0.22 unit per milliliter of cul-
712
ANTIBIOTIC INACTIVATION AND MODIFICATION
[54b]
ture fluid has been reported. 3 The whole broth is then treated with a floceulant, 0.5% (v/v) Primafloc C-3 (Rohm & Haas Co. Philadelphia, Pennsylvania) and 0.2% v/v toluene. The pH is adjusted to 7.0-7.5, the cells are removed by centrifugation as described for the E. coli enzyme and the enzyme-containing supernatant is retained. Extraction and Purification 3 The supernatant liquid is acidified with dilute acetic acid to pH 6.4, and the enzyme is adsorbed onto acid-washed Celite (No. 545, JohnsManville Co., New York) added at a ratio of 15 g/liter. The suspension is stirred for 2 hr, during which time the pH is maintained at 6.4 by further addition of acetic acid; the Celite-enzyme complex is collected by filtration and then slurried in 24% (w/v) ammonium sulfate in 0.1 M Tris buffer adjusted to pH 8.4 with NH40H and transferred to a glass column. Enzyme is eluted from the Celite column by further addition of the ammonium sulfate solution, and the eluate is concentrated under vacuum at 40 ° . The protein gradually precipitates from the solution, concentration being stopped as soon as ammonium sulfate begins to crystallize. The precipitated protein is separated by filtration and then resuspended in 50 mM phosphate buffer, pH 7.0 and dialyzed for 24 hr against a 50-fold volume of 25 mM phosphate buffer, pH 6.4. Further purification is achieved by fractionation on a column (47 X 4.7 cm) containing carboxymethylcellulose, impurities being eluted at a flow rate of 100 ml/hr with 25 mM phosphate buffer, pH 6.4, and the enzyme with 0.1 M phosphate buffer, pH 6.5. By repeating the Celite adsorption at a ratio of 75 g/liter and ammonium sulfate precipitation, the enzyme is purified still more, as indicated in Table II. Properties o] the Enzymes The properties of the enzymes produced by E. coli and B. megaterium are given below. Those for other bacteria and actinomycetes are given in Table VI. Physical Characteristics. The crystalline E. coli enzyme forms regular rectangular plates (ca. 150 X 80 ~m) having a specific activity of 48 units/mg, a molecular weight of 71,000 ± 2000 by sedimentation equilibrium (cf. the B. megaterium enzyme, 120,000 determined by the same method~), and 70,000 ± 5000 by thin-layer gel filtration. The isoelectric point determined by gel electrofocusing is 6.8 ± 0.2. For partially purified enzyme preparations, enzyme is visualized by staining the gel with a
[54b]
PENICILLIN ACYLASE (BACTERIAL)
713
TABLE II PURIFICATION OF PFNICILLIN ACYbASE FROM Bacillus megaterium
Fraction Crude broth supernatant fluid First-cycle Celite adsorption and ammonium sulfate precipitation Carboxymethylcellulose column fractionation Second-cycle Celite adsorption and ammonimn sulfate precipitation
Total Total Specific Volume activity ~ protein activity Yield (ml) (kilounits) ( m g ) (units/rag) (%) 34,000 58
7.6 5.25
23,000 340
0.33 15.5
101) 69
565
3.9
160
24.6
52
10
2.55
81
31.5
33
" Unit of enzyme activity is defined as the amount of enzyme required to produce i tmmle 6-aminopenicillanic acid from benzylpenicillin per minute in 0.1 M borate buffer (pH 8.7) at 37°.
novel substrate, 6-nitro-3-phenylacetamidobenzoic acid which is cleaved to form a yellow product, 6-nitro-3-aminobenzoic acidY pH and Temperature Optima. The optimum temperature for the initial rates of reaction using benzylpenicillin as substrate is around 55 ° for tile E. coli'-' and 45 ° for the B. megaterium 3 enzymes. Both enzymes are unstable at these temperatures however and 37 ° is generally chosen. Tilt- ot)timmn pH values for the two enzymes are about 8.2 for E. coli and about 8.5 for B. megaterium, but at these values, benzylpenicillin can be rather unstable and slightly lower values, e.g., 7.8, are used. Kinetics and Inhibitors. Both of the enzymes described above are inhibited by the reaction products produced by the hydrolysis of benzylpenicillin. ~,'" Phenylacetie acid competitively inhibits tile two enzymes, although as the kinetic data in Table I I I show, the inhibitory effect on the B. megaterium enzyme is very much less than on E. coli acylase. Both enzymes are noncompetitively inhibited by 6-APA and again, the effect is greater with the E. coli enzyme. A further difference between tile two enzymes is that the E. coli acylase only is inhibited by substrate. The K,, values of the two enzymes are also shown in Table I I I ; this constant has been determined for the enzymes obtained from various sources, and these are smmnarized in Table IV. Although the conditions of temperature and pH under which these values were determined m a y differ, the very high value for E. coli cells suggests considerable diffusional restrictions across the cell wall. Nevertheless the use of the cell-
714
ANTIBIOTIC INACTIVATION AND MODIFICATION
[54b]
TABLE I l l COMPARISON OF KINETIC CONSTANTS OF PENICILLIN ACYLASES PRODUC~'D BY Escherichia coli AND Bacillus megaterium Parameter
E. coli NCIB 8743A~
B. megaterium ATCC 14945b
Vm,x (~moles 6-APAd/minute/mg) Km (mM) Ki, 6-APA, (mM) Ki, phenylaeetic acid (mM) K., benzylpenicillin (mM)
7.1 0.67 7.1 4.8 270
1.1 c 4.5 26 450 Not inhibited
a Kinetic constants obtained at pH 8.0 and 37 °. a Kinetic constants obtained at pH 8.7 and 37 °. Calculated from graphical data of C. Chiang and R. E. Bennett [J. Bacteriol. 93, 302 (1967)]. a 6-APA, 6-aminopenicillanicacid. TABLE IV COMPARISON OF Era VALUES OF PENICILLIN ACYLASE FROM DIFFERENT SOURCES Organism Escherichia coli ATCC 9637 E. coli NCIB 8743A Crystalline enzyme of Escherichia coli ATCC 11105 Whole cells of E. coli NCIB 8743A Bacillus megaterium Streptomyces lavendulae Fusarium semitectum
Km Value (mM)
Reference
7.7 0.67 0.02
a b c
30.0 4.5 10.3 2.5
/
a D. A. Self, G. Kay, M. D. Lilly, and P. Dunnill, Biotechnol. Bioeng. 11, 337 (1969). a K. Balasingham, D. Warburton, P. Dunnill, and M. D. Lilly, Biochim. Biophys. Acta 276, 250 (1972). c C. Kutzbach and E. Rauenbusch, Hoppe Seyler's Z. Physiol. Chem. $54, 45 (1974). d M. Cole, Biochem. J. 116, 733 (1969). e C. Chiang and R. E. Bennett, J. Bacteriol. 93, 302 (1967). / F . R. Batchelor, E. B. Chain, M. Richards, and G. N. Rolinson, Proc. Roy. Soc. Ser. B 154~, 522 (1961). g E. Brandl, Hoppe-Seyler's, Z. Physiol. Chem. 342, 86 (1965). b o u n d e n z y m e for the c o m m e r c i a l p r o d u c t i o n of 6 - A P A is still widely practiced. T h e k i n e t i c d a t a for the E . coli 18 a n d t h e B. m e g a t e r i u m ~ e n z y m e s h a v e been used i n derived r a t e e q u a t i o n s d e s c r i b i a g the h y d r o l y s i s of lSD. Warburton, P. Dunnill, and M. D. Lilly, Biotechnol. Bioeng. 15, 13 (1973).
[54b]
PENICILLIN ACYLASE (BACTERIAL)
715
benzylpenicillin to 6-APA. These equations have been used to predict satisfactorily, the progress of the reaction in batch and continuous stirred tank reactors. Substrate Profile--Hydrolytic Direction. The relative rates of deacylation of various penicillins by the cell bound acylase of E. coli 2 and by the extracellular acylase of B. megaterium ~ are shown in Table V. Both TABLE V SUBSTRATE PROFILES FOR THE ACYLASES OF Escherichia coil AND
Bacillus megaterium Relative Penicillins a n d other substrates
r a t e s of hydrolysis
Cell-bound acylase of Cell-free acylase of E. coli N C I B 8743 B. megaterium A T C C 14945
P enicillins Benzylp-HydroxybenzylDL-a-HydroxybenzylD-a-Aminobenzyla-Carboxybenzyl2-Furyhnethyl2-ThienylmethylPhenoxymethyl~-Phenoxyethyln-PropoxymethylIsobutoxymethyln - H e p t ylA2-Pentenyl AllylmercaptomethylEthylthiomethylPhenyl2,6-Dimethoxyphenyl5- M e t hyl-3-phenyl-4-isox azolyl-
100 (6.3 ~:moles/hr) 150 88 50 < 5 91 80 5.5 < 5 40 29 < 5 ---< 5 < 5 --
100 (38 ~ m o l e s / h r ) --
---3.4 4.0
0 16.3 10.5 10. 5 .... 0 0
Other substrates Phenylacetamide N-Methylphenylacetamide N-Phenylacetylglycine N-Phenylacetyl-DL-leucine N-Phenylacetyl-DL-alanine N-Pheiiylacetyl-D-~-aminop h e n y l a c e t i c acid N- Phenylacetyt-L-c~-aminop h e n y l a c e t i e acid Benzylpenicilloic acid
109 -182 73 -0
23 !0 -15 --
139
---
22
--
716
ANTIBIOTIC INACTIVATION AND MODIFICATION
[54b]
enzymes readily hydrolyze benzylpenicillin and more slowly hydrolyze alkylpenieillins. The substrate profiles for the penicillin acylases of other organisms are shown in Table VI. For the E. coli enzyme it would seem that aeyl groups of the size of phenylacetyl are optimal and a-substitution with amino or hydroxy is tolerated, but not carboxy. The structure of the acyl side chain is the main factor controlling enzyme activity. Derivatives at the 3-earboxy group of benzylpenicillin such as amides and esters are good substrates giving 6-APA amides and 6-APA esters. 2 Cephalosporins with suitable acyl side chains, such as cephalothin (2thienylmethylcephalosporin), are good substrates, yielding the nucleus 7-aminocephalosporanic acid (7-ACA2), however the naturally occurring substance cephalosporin C (~-aminoadipylcephalosporin) is not a substrate29 Both enzymes are able to hydrolyze acyl groups from compounds other than penicillins and cephalosporins as shown in Table V. The B. megaterium enzyme carries out this reaction less readily than the E. coli enzyme. The enzyme from E. coli is stereospecific, only phenylacetyl-Lamino acids being hydrolyzed at a significant rate. Details of this reaction and others involving nonpenicillin substrates have been published. 2°-22 The substrate profile of the crystalline enzyme1~ confirms that a single enzyme is responsible for the deacylation of both penicillins and nonpenicillins. Substrate P r o f i l e - S y n t h e t i c Direction. The penicillin acylase of E. coli is reversible. 2~,24 At optimum pH values using the enzyme from E. coli NCIB 8743, the rate of synthesis of benzylpenicillin from 6-APA and phenylacetic acid (pH 5.0) is about 12% of the rate of hydrolysis of benzylpenicillin (pH 8.5). The best carboxylic acids for reactions in the synthetic direction are phenylacetic acids, with or without hydroxy and amino substituents in the ring. However a-hydroxyphenylacetic acid is a poor substrate while phenylacetic acids substituted with a-amino, a-carboxy, or a-methoxy are not substrates. The best alkyl carboxylic acid is 3-hexenoic acid. The use of derivatives of a-aminophenylacetic acid such as amide, N-glyeyl, or methyl ester, enables the D-a aminophenylacetyl group to be readily lgB. Sjoberg, L. Nathorst-Westfelt, and B. Ortengren, Acta Chem. Scand. 21, 547 (1967). 20M. Cole, Biochem. J. 115, 747 (1969). 21G. Lucente, A. Romeo, and D. Rossi, Experie~tia 21, 317 (1965). 22A. Romeo, G. Lucente, D. Rossi, and G. Zanotti, Tetrahedon Lett. 21, 1799 (1971). 23W. Kaufmann, K. Bauer, H. A. Offe, in Antimicrob. Ag. Ann. 1960, 1 (1961). 24G. N. Rolinson, F. R. Batchelor, D. Butterworth, J. Cameron-Wood, M. Cole, G. C. Eustace, M. V. Hart, M. Richards, and E. B. Chain, Nature (London) 187, 236 (1960).
[54b]
PENICILLIN ACYLASE (BACTERIAL)
717
transferred to 6-APA to give ampicillin. 23,-~5 The rate of synthesis of benzylpenicillin and a-hydroxybenzylpenicillin is also increased by such means, but not a-carboxybenzylpenicillin. The accumulation of penicillin in the above synthetic reactions has an inhibitory effect on the reaction which thus comes to equilibrium. Use of a molar excess of carboxylic acid or its derivative shifts the equilibrium in favor of a greater conversion of 6-APA into penicillin. 25 If the pH is allowed to drift upward during synthetic reactions, product penicillin hydrolyzes to 6-APA. The optimum pH for synthesis is between 5.0 and 7.0, depending on substrates and duration of reaction. 2~ By the use of derivatives of carboxylic acids, the substrate range of penicillin acylase can be much extended; for example, phenoxyacetic acid does not support the synthesis of phenoxymethylpenicillin but N-phenoxyacetylglycine does? ° The acyl transferase reaction is stereospecific, penicillins being synthesized at a much greater rate by transfer of an acyl group from an L-amino acid than from a D-amino acid. x° Immobilized Enzyme. The technique of immobilizing enzymes in general with either soluble, or more usually insoluble inert carriers has been reported extensively.2s The main advantage of immobilized enzyme is greater stability, enabling the enzyme to be re-used many times. It has been reported in the literature (',~; that some of these immobilization techniques have been applied to the penicillin acylases produced by E. coli and B. megaterium, but a particularly useful method employing a commercially available carrier is that based on the attachment of proteins to water-insoluble polysaccharide polymers or derivatives thereof activated by reaction with cyanogen halides. 2s To immobilize E. coli enzyme, the following procedure may be used. Cyanogen bromide-activated Sepharose 4B (Pharmacia Co.) (15 g) i,~ suspended in 10 mM HC1 (3 liters) and stirred for 15 min. The gel is recovered on a glass filter and resuspended in a solution of partially purified penicillin acylase in 0.1 M borate buffer, pH 8.75, containing 0.5 M NaC1 (200 ml). The specific activity of the enzyme solution is 6.33 units of protein per milligram, and the total activity is 7407 units. The enzyme and carrier are gently stirred at room temperature for 18 hr. The gel is recovered and resuspended in 1.0 M ethanolamine pH 8.0 (500 ml) and stirred for 2 hr to block any remaining active groups. Ionically bound enzyme is removed by washing the gel three times alternately with 0.1 M acetate buffer pH M. Cole, Biochem. J. 115, 757 (1969). :s O. Zaborsky, "Immobilized Enzymes." Chem. Rubber Publ. Co., Cleveland, Ohio, 1973. ~ D. Warburton, K. Balasingham, P. Dunnill, and M. D. Lilly Biochirn. Biophys. Acta 284, 278 (1972). 2~R. Axen, J. Porath, and S. Ernb~ck, Nature (London) 214, 1302 (1967).
718
ANTIBIOTIC INACTIVATION AND MODIFICATION
[54b]
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[54b1
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ANTIBIOTIC
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AND
[54b]
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[54C]
PENICILLIN ACYLASE (FUNGAL)
721
4.0 containing 1 M NaC1 (200 ml) and 0.1 M borate buffer pH 8.0 containing 1 M NaC1 (200 ml). The final product, 36 g at an activity of 171.2 units/g, is stored damp at 4 ° and used as required; the enzyme activity is quite stable under these storage conditions. The properties of the enzyme may be altered by immobilization, depending upon the carrier and the method of attachment. For example, diffusional limitations may affect the apparent K,~ value. The binding of an enzyme to a charged support may alter the pH-activity profile especially at low ionic strengths; a shift in the pH optimum from 8.2 to 7.65 was reported for E. coli enzyme attached to triazinyl cellulose. -~; Attachment may alter the inhibition constants due to occlusion of allosteric centers or to repulsion effects in the case of charged supports. For example, with E. coli enzyme attached to triazinyl cellulose, the change in the K~ value for 6-APA from 7.1 mM for the free enzyme to 9.0 m M for the immobilized enzyme enabled a 90% conversion of 50 mM benzylpenicillin by the immobilized enzyrae to be achieved in about 80c~ of the time required using free enzyme with the same activity. ~s Immobilization of the B. megaterium enzyme onto bentonite increased the K~ for 6-APA from 26 to 250 mM and the K~ for phenylacetic acid from 450 to 620 mM. ~
[54c] Penicillin Acylase (Fungal) By
HL'BE~T VANDERHAEGHE
The "fungal penicillin acylase," which efficiently hydrolyzes phenoxymethylpenicillin, and also some other penicillins, is produced not only by filamentous fungi, but also by some actinomycetes and bacteria. The production by Penicillium chrysogenum and by F~sarium will be described below. Other producing microorganisms are: Aspergillus ochraceus, 1 Cephalosporium CMI 49137,1--3 Emericellopsis minima, 3 Epidermophyton floccosum, ~,~ Trichophyton mentagrophyta? ,~ Calonectria. ~ Nectria, ~ Pleurotus ostreatus(' Streptomyces lavendulae. ~ Strepto~Tyces M. Cole, Appl. Microbiol. 14, 98 (1966). : C. A. Claridge, J. R. Luttinger, and J. Lein, Proc. Soc. Exp. Biol. Med. 113, 1008 (1963). 3 M. Cole and G. N. Rolinson, Proc. Roy. Soc. Ser. B 154, 490 (1961). 4 j. Uri, G. Valu, and I. Bekesi, Naturu~issenscha]ten 51, 298 (1964). 5 I. C. I., British Patent 924,455 (1963), Chem. Abstr. 59, 2133 (1963). ~Biochemie Ges., Belg. Patent 615,659 (1962), Chem. Abstr. 58, 2819 (1963).
F. R. Batchelor, E. B. Chain, M. Richards, and G. N. Rolinson, Proc. Roy. Soc. Ser. B. 154, 522 (1961).
[54C]
PENICILLIN ACYLASE (FUNGAL)
721
4.0 containing 1 M NaC1 (200 ml) and 0.1 M borate buffer pH 8.0 containing 1 M NaC1 (200 ml). The final product, 36 g at an activity of 171.2 units/g, is stored damp at 4 ° and used as required; the enzyme activity is quite stable under these storage conditions. The properties of the enzyme may be altered by immobilization, depending upon the carrier and the method of attachment. For example, diffusional limitations may affect the apparent K,~ value. The binding of an enzyme to a charged support may alter the pH-activity profile especially at low ionic strengths; a shift in the pH optimum from 8.2 to 7.65 was reported for E. coli enzyme attached to triazinyl cellulose. -~; Attachment may alter the inhibition constants due to occlusion of allosteric centers or to repulsion effects in the case of charged supports. For example, with E. coli enzyme attached to triazinyl cellulose, the change in the K~ value for 6-APA from 7.1 mM for the free enzyme to 9.0 m M for the immobilized enzyme enabled a 90% conversion of 50 mM benzylpenicillin by the immobilized enzyrae to be achieved in about 80c~ of the time required using free enzyme with the same activity. ~s Immobilization of the B. megaterium enzyme onto bentonite increased the K~ for 6-APA from 26 to 250 mM and the K~ for phenylacetic acid from 450 to 620 mM. ~
[54c] Penicillin Acylase (Fungal) By
HL'BE~T VANDERHAEGHE
The "fungal penicillin acylase," which efficiently hydrolyzes phenoxymethylpenicillin, and also some other penicillins, is produced not only by filamentous fungi, but also by some actinomycetes and bacteria. The production by Penicillium chrysogenum and by F~sarium will be described below. Other producing microorganisms are: Aspergillus ochraceus, 1 Cephalosporium CMI 49137,1--3 Emericellopsis minima, 3 Epidermophyton floccosum, ~,~ Trichophyton mentagrophyta? ,~ Calonectria. ~ Nectria, ~ Pleurotus ostreatus(' Streptomyces lavendulae. ~ Strepto~Tyces M. Cole, Appl. Microbiol. 14, 98 (1966). : C. A. Claridge, J. R. Luttinger, and J. Lein, Proc. Soc. Exp. Biol. Med. 113, 1008 (1963). 3 M. Cole and G. N. Rolinson, Proc. Roy. Soc. Ser. B 154, 490 (1961). 4 j. Uri, G. Valu, and I. Bekesi, Naturu~issenscha]ten 51, 298 (1964). 5 I. C. I., British Patent 924,455 (1963), Chem. Abstr. 59, 2133 (1963). ~Biochemie Ges., Belg. Patent 615,659 (1962), Chem. Abstr. 58, 2819 (1963).
F. R. Batchelor, E. B. Chain, M. Richards, and G. N. Rolinson, Proc. Roy. Soc. Ser. B. 154, 522 (1961).
722
ANTIBIOTIC INACTIVATION AND MODIFICATION
[54c]
noursei, 8 Streptomyces erythreus, s Streptomyces netropsis, s and also Achromobacter sp. N C I B 94249 and Erwinia aroideae. ~° For other microorganisms, see the review by Cole. 1~ I t is likely that there exist differences between penicillin acylases produced by these microorganisms. Although phenoxymethylpenicillin is a good substrate, in several cases marked differences in pH optimum and substrate specificity are described.
Assay Assay procedures for penicillin acylase are described in a previous article. TM P r o d u c t i o n and Purification PeniciUium chrysogenum Acylase
The penicillin acylase may be obtained from a penicillin-producing strain or from a mutant which has lost this property. 13 With the latter strain, the interpretation of the results is easier because no corrections must be made for endogenous {i-APA. The fermentation media described here TM differ somewhat from those used by other authors 13 for the same strain. It is, however, impossible t o tell which media give the highest yield, because the conditions of assay were different. Culture. ~ Penicillium chrysogenum mutant Wis. 49.408 (obtainable from the University of Wisconsin) is grown for 5 days at 26 ° on potato dextrose agar slants , containing (in g/liter): potato infusion, 200; dextrose, 20; and agar, 15. A spore suspension obtained from the agar slant is used to inoculate four 250-ml Erlenmeyer flasks, each containing 50 ml of the following medium (in g/liter): corn-steep powder, 25.0; sucrose, 20.0; CaC03 5.0; Na~S203.5H.,O, 0.2 adjusted to p H 5.5 with 10% N a O H before autoclaving. The flasks are incubated at 26 ° for 3 days on a rotary shaker operating at 150 rpm. These precultures are added to twenty 250-mi Erlenmeyer flasks (5 ml per flask) containing 50 ml of a synthetic medium 15 containing, additionally, 0.2% phenoxyacetic acid. The composition is (in g/liter): lactose, s I. Haupt and H. Thrum, Z. Allgem. Mikrobiol. 7, 343 (1967). o M. Cole, Nature (London) 203, 519 (1964). 1, E. J. Vandamme, J. P. Voets, and A. Dhaese, Ann. Inst. Pasteur 121, 435 (1971). 11M. Cole, Process Bioehem. 2, 35 (1967). 12This volume [54a]. is R. C. Erlckson and R. E. Bennett, Appl. Microbiol. 13, 738 ('1965). 14H. Vanderhaeghe, M. Claesen, A. Vlietinck, and G. Parmentier, Appl. Microbiol. 16, 1557 (1968). ~ F. G. Jarvis and M. J. Johnson, J. Bacteriol. 59, 51 (1950).
[54c]
PENICILLIN ACYLASE (FUNGAL)
723
30.0; dextrose, 10.0; ammonium lactate, 6.0; ammonium acetate, 3.5; KH2PO4, 3.0; Na2SO4, 0.5; MgSO4.TH._,O, 0.25; ZnSO~.7H_~O, 0.02; M n S Q . H 2 0 , 0.02; Fe(NH4)2(SO4)._,'6H20, 0.10; CuSO4"5H20, 0.005; CaCl.,-2H20, 0.05; and phenoxyacetic acid, 2.0. The sugars are sterilized separately and added aseptically to the sterile solution of the mineral components. The flasks are shaken at 26 ° for 2 days. The mycelium is collected on a Biichner funnel, washed with water, and then four times with 0.5 liter of cold (--30 to --40 °) acetone. The mycelium is kept in the air until the odor of acetone has disappeared. Either 60 g (46-82 g) of wet mycelium or 17 g (15-20 g) of dry mycelium are obtained from 20 Erlenmeyer flasks. The dry mycelimn may be kept for several months in a deep-freeze. Extraction. 1~ To remove the enzyme from the cells it is necessary to use salt solutions of sufficiently high concentration. Higher yields are obtained with 0.2 M sodium chloride than with 0.2 M sodium acetate, but there is no significant difference between wet and acetone-dried mycelium. Sonic treatment or disruption of the cells with mechanical methods does not improve the yield. The mycelium is shaken with 0.2 M sodium chloride (60 ml for 1.8 g of dried mycelium in a 250-ml flask) for 16 hr at 26 °. The contents of 10 flasks are filtered on a Biichner filter, and the filtrate (600 ml) is put in dialysis bags. After dialysis for 24 hr at 0-5 ° against l0 liters of distilled water, the operation is repeated for another 24 hr. The dialyzed filtrate is concentrated by submerging the dialysis bags in Carbowax G25000. When the volume is reduced to one-tenth, the dialysis bags are put in distilled water, and the solution is freeze-dried. The mycelium that has been extracted can be used for a second and eventually a third extraction. The activity of these extracts is sometimes similar to the first one, sometimes much lower. Only extracts of sufficiently high activity should be used. The activity of a freeze-dried extract is 0.0005 unit/mg. The unit is defined as the amount of enzyme which hydrolyzes 1 /~mole of phenoxymethylpenicillin per minute in 0.15 M phosphate buffer pH 7.5 at 37 °. The amount of 6-aminopenicillanic acid (6-APA) formed is determined by the t)iochromatographic assay, 12 but using Staphylococcus aureus instead of B. subtilis. Fusari~m Acylase
Penicillin acylase has been found in the mycelia of Fusarium semirectum, ~,~ F. avenaceum TM and in the spores of Fusarium monili'~E. Waldschmidt-Leitz and G. Bretzel, Hoppe-Seyler's Z. Physiol. Chem. 337, 222 (1964), ~7E. Brandl, Hoppe-Seyler's Z. Physiol. Chem. 342, 86 (1965).
724
ANTIBIOTIC INACTIVATION AND MODIFICATION
[54C]
]orme. ls,1~ With the last microorganism, no acylase activity is detected
in the mycelium nor in the fermentation broth. 2° The purification of the enzyme from F. s e m i t e c t u m is described below. 1~ Another purification of the enzyme from the same microorganism has also been described. 21 Although the specific activity of the latter preparation was lower, it was homogeneous when chromatographed on Sephadex G-150. This could be due to the fact that extraneous protein was tightly bound to the enzyme. No direct comparison of the two preparations has been made. Culture. F u s a r i u m a v e n a c e u m 14 and F. s e m i t e c t u m IG are grown on Jarvis-Johnson medium ~ containing 0.2% phenoxyacetic acid for 3 days at 26 or 28 °. For the first species, the preparation of the spore inoculum aDd the preculture is the same as described above for P e n i c i l l i u m chrysogenum, for the second no details are given. The different steps for growing the mycelium of F. s e m i t e c t u m in slightly different media have been given in another publication. 2~ The yield of acylase is apparently very similar. E x t r a c t i o n . ~6 The mycelium of F u s a r i u m s e m i t e c t u m BC805 is suspended in 0.2 M sodium acetate buffer at pH 6.0 (600 ml for 18 g of dry mycelium containing 18 units/g, and which is obtained from 1 liter of fermentation culture) and shaken for 15 hr. After filtration, the solution is alkalinized to pH 11.0 with NaOH, and the inactive precipitate is removed by centrifugation. The supernatant is immediately adjusted to p H 8.0 with acetic acid. The solution is treated three times with D E A E cellulose (10 g per 500 ml of solution). The clear solution, which is obtained by passing the liquid through a Sephadex G-25 column, is freezedried. From 190 g of mycelium containing 1905 units ~ (specific activity 0.01 unit/rag), 9.5 g of freeze-dried extract with a specific activity of 0.035 unit/mg (330 units, 17% yield) is obtained. F r a c t i o n a t i o n . ~ The extrac~ (9.5 g) is dissolved in 5 m M sodium citrate solution (100 ml/3 g) and while cooling to --10 °, acetone is added until a concentration of 44% is reached. After 20 min, the precipitate containing the enzyme is removed by centrifugation. After the precipitate is dry, 1.92 g of extract with a specific activity of 0.160 unit/mg 1~K. Singh, S. N. Seghal, and C. V~zina, Appl. Microbiol. 17, 643 (1969). 1~E. J. Vandamme, J. P. Voets, and C. Beyaert, Meded. Rijks]ac. Landbouwwetensch. Gent 36, 577 (1971). 2, E. J. Vandamme and J, P. Voets, Meded. Rijks]ac. Landbouwwetensch. Gent 37, 1185 (1972). 2~F. Baumann, R. Brunner, and M. RShr, Hoppe-Seyler's Z. Physiol. Chem. 352, 853 (1971). 22The unit is defined as the amount of enzyme that hydrolyzes 1 #mole of phenoxymethylpenicillin per minute at pH 7.5 and 32°. The amount of 6-APA was determined by iodometric assay of the aqueous phase after extraction of the residual penicillin in butylacetate from the acidified solution. See also footnote 17.
[54(::]
PENICILLIN ACYLASE (FUNGAL)
725
is obtained. The operation is repeated, but the concentration of acetone is raised only to 37%. This yields 254 mg of extract with a specific activity of 1.10 unit/mg (280 units, 15%). Final Purification. TM The enzyme preparation (180 rag) is dissolved in 10 ml of 0.2 M sodium citrate buffer pH 4.8 and adsorbed on an Amberlite 1RC-50 column and eluted with the same buffer, the pH gradually being increased to 5.1. The elution is monitored by measuring the extinction at 280 nm and assaying the enzyme. The column chromatography is repeated. The volume of the eluate is reduced to 10 ml by freeze-drying, and salt is removed by passing this solution through a column of Sephadex G-25. After elution with water and lyophilization, 17 mg of a product with a specific activity 3.02 units/rag is obtained.
Properties Stability. A solution of the enzyme preparation from Fusarium semitectum is stable for at least 24 hr at 32 ° and pH 6.0 to 10.0. Loss of activity is observed at pH 4.2.16 The extracellular enzyme of Streptomyces lavendulae seems to be less stable. Only 67% of the original activity is present after storing the sterile culture filtrate at 35 ° and pH 8.0 for 24 hrY Chemical and Physical Properties. Examination of the purified preparation from Fusarium semitectum in the ultracentrifuge indicates that the product is homogeneous, with a sedimentation constant of 7.76 S. The molecular weight is estimated to be in the range of 65,000, but a precise determination of the diffusion constant has not been performed because of the limited amount of product. This acylase contains two atoms of zinc per molecule. TM A molecular weight of 62,000 is inferred for the acylase of Erwinia aroideae from thin-layer chromatography on Sephadex G-100 gelY° Activators and Inhibitors. No activators have been described, but the Fusarium enzyme is inactivated when zinc is complexed with 8-hydroxyquinoline. The activity is fully restored by addition of ZnSO4, and partially with MnSO,, MgS04, COSO4, and FeS0t. TM In this connection, it should be noted that Szentirmai ~ reported the inhibition of acylase production in Escherichia coli by 130 ~g/ml of 8-hydroxyquinoline. Optimum pH and Temperature. It is difficult to determine optimum pH and temperature for this enzyme because the substrate (phenoxymethylpenicillin) is rapidly destroyed at alkaline pH and high temperature. The optimum pH for Penicillium acylase has not been determined, but there is a marked increase of the rate of hydrolysis when the pH ='A. Szentirmai, Appl. Microbiol. 12, 185 (1964).
726
ANTIBIOTIC INACTIVATION AND MODIFICATION
[54C]
is raised from 6.5 to 8.5.13,~4 The reaction rate at 35 ° is 1.5 times that observed at 300.13 For the Fusarium enzyme, the rate of hydrolysis of phenoxymethylpenicillin increases from 25 ° to 370,14,1' but the effect levels off from 40 ° to 500.17 The optimum pH is between 7.0 and 8.0, with a broad maximum at 7.5 for the acylase of F. semitectum, 1°,1~ F. avenaceum 14 and F. monili]orme. ~8,19 The optimum pH of acylase of Streptomyces lavendulae is 10.0, and the highest rate is observed at 50 ° when measured for a short period of time (30 rain).8 For Erwinia aroideae a pH optimum of 4.5-5.5 is observed with phenoxymethylpenicillin as substrate, and a pH optimum of 8.0 for cloxacillin and methicillin. 11 It may be concluded from these data that the acylases produced by these microorganisms are not identical, although all hydrolyze phenoxymethylpenicillin much better than benzylpenicillin. This conclusion is supported by the observations on the substrate specificity (see below). Kinetic Properties. All Km values are high, indicating a low affinity of penicillin acylase for the substrate. For Fusarium enzyme the following Km values have been reported for the cleavage of phenoxymethylpenicillin: 2.5-2.8 mM (without indication of pH or temperature),17 4.75 mM at pH 7.5 and 370, 21 and 5.75 mM at pH 8 and 28 ° using Fusarium moniliforme spores. 19 No Km has been published for the hydrolysis of penicillin by Penicillium chrysogenum acylase, but Km values are 1.1 to 1.4 mM for the deacylation of some phenoxyacetylamino acids. 2~ In this publication, ~4 however, there are indications that this enzyme is different from that responsible for the cleavage of penicillin. The extracellular acylase of Streptomyces lavendulae has a Km of 10.3 mM for the hydrolysis of phenoxymethylpenicillin at pH 8.7 and 340. 8 Substrate Specificity. Only semiquantitative data are available concerning the rate of hydrolysis of different penicillins by "fungal acylase." They are given in the table for the acylases of Penicillium chrysogenum and Fusarium aveneceum. Penicillin N, T M isopenicillin N, 14 methicillin, 2 and cephalosporin 2 are not hydrolyzed by Penicillium chrysagenum acylase. The enzyme of Streptomyces lavendulae rapidly deacylates penicillin V, penicillin H2F, and penicillin K, but very slowly deacylates penicillin G 7 (for Erwinia aroideae, see under "Optimum pH"). It has been reported that several phenoxyacetylamino acids are hydrolyzed by Penicillium chrysogenum, but the enzyme seems to be different from that responsible for the deacylation of penicillins. 24 An enzyme 24R. Brunner, M. RShr, and M. Zinner, Monatsh. Chem. 97, 952 (1966).
[54c1
PENICILLIN ACYLASE (FUNGAL)
727
~C ¢D
¢D '-~
,,-1
--I+-FI -F
I
I
I
+ ÷ l J l ÷ + + ÷
©
c~
.< m¢*
U
• ~
~'~
o°~-~-~j~l~j
~"~
728
A N T I B I O T IINACTIVATION C AND MODIFICATION
[55a]
preparation obtained by extraction of Fusarium semitectum with 0.2 M sodium chloride deacylates phenoxymethylpenicillin but not phenoxyacetylamino acids. 2l The converse was observed for an extract prepared in a different way from the same microorganism. ~5 ~F. Baumann, M. RShr, and R. Brunner, Zentr. Bakteriol. Abt. I, Re]. 229, 351 (1972).
[55a] Cephalosporin Acetylesterase (Citrus) By E. P. ABRAHAMand PATRIClA FAWCETT +
This enzyme hydrolyzcs cephalosporin C [(I), R = ()~C.CH(NHa) (CH2) 3C0] to deacetylcephalosporin C.X It also hydrolyzes other cephalosporins containing an O-acetyl group to the corresponding deacetyleephalosporins (II) 2 and 7-aminocephalosporanic acid (I, R = H) to 7-aminodeacetylcephalosporanic acid (7-aminocephalosporadesic acid), a H H RNH [ ~
H H R N t t ~ + HOAe
+ H20
O
CHzOAe CO2- M+
(I)
0 ~/
~ "CH:~OH CO~- M+
(n)
Assay Methods The assay is based on measurement of the acid liberated during hydrolysis of an acetyl ester. Cephalosporin C or triacetin may be used as a substrate. Manometric. The rate of hydrolysis at 30 ° is determined in a Warburg respirometer at pH 7.0 by measurement of the liberation of CO2 from NaHCO3. With cephalosporin C as substrate, the main compartment of. each vessel contains 1.7 ml of 0.15 M NaC1, 0.3 ml of esterase solution, and 0.5 ml of 43 mM N a H C Q ; the side bulb contains 0.1 ml of 43 mM NaHCO~ and 0.4 ml of a solution of cephalosporin C Na salt (40 mg). With triacetin as substrate the main compartment contains 85 mg of triacetin in 2 ml of 0.15 M NaC1 and 0.5 ml of 43 mM NaHCO~; the side 1j. D'A. Jeffery, E. P. Abraham, and G. G. F. Newton, Biochem. J. 81, 591 (1961). "~U.S. Patent 3,459,746 (1969). 3U.K. Patent 1,066,347 (1967).
728
A N T I B I O T IINACTIVATION C AND MODIFICATION
[55a]
preparation obtained by extraction of Fusarium semitectum with 0.2 M sodium chloride deacylates phenoxymethylpenicillin but not phenoxyacetylamino acids. 2l The converse was observed for an extract prepared in a different way from the same microorganism. ~5 ~F. Baumann, M. RShr, and R. Brunner, Zentr. Bakteriol. Abt. I, Re]. 229, 351 (1972).
[55a] Cephalosporin Acetylesterase (Citrus) By E. P. ABRAHAMand PATRIClA FAWCETT +
This enzyme hydrolyzcs cephalosporin C [(I), R = ()~C.CH(NHa) (CH2) 3C0] to deacetylcephalosporin C.X It also hydrolyzes other cephalosporins containing an O-acetyl group to the corresponding deacetyleephalosporins (II) 2 and 7-aminocephalosporanic acid (I, R = H) to 7-aminodeacetylcephalosporanic acid (7-aminocephalosporadesic acid), a H H RNH [ ~
H H R N t t ~ + HOAe
+ H20
O
CHzOAe CO2- M+
(I)
0 ~/
~ "CH:~OH CO~- M+
(n)
Assay Methods The assay is based on measurement of the acid liberated during hydrolysis of an acetyl ester. Cephalosporin C or triacetin may be used as a substrate. Manometric. The rate of hydrolysis at 30 ° is determined in a Warburg respirometer at pH 7.0 by measurement of the liberation of CO2 from NaHCO3. With cephalosporin C as substrate, the main compartment of. each vessel contains 1.7 ml of 0.15 M NaC1, 0.3 ml of esterase solution, and 0.5 ml of 43 mM N a H C Q ; the side bulb contains 0.1 ml of 43 mM NaHCO~ and 0.4 ml of a solution of cephalosporin C Na salt (40 mg). With triacetin as substrate the main compartment contains 85 mg of triacetin in 2 ml of 0.15 M NaC1 and 0.5 ml of 43 mM NaHCO~; the side 1j. D'A. Jeffery, E. P. Abraham, and G. G. F. Newton, Biochem. J. 81, 591 (1961). "~U.S. Patent 3,459,746 (1969). 3U.K. Patent 1,066,347 (1967).
[55a]
CEPHALOSPORIN ACETYLESTERASE (CITRUS)
729
bulb contains 0.4 ml of esterase solution and 0.1 ml of 43 mM NaHC0:,. The vessels are gassed with CO., + N=, (5:95, v/v). Hydrolysis is allowed to proceed for 20 rain, and the initial rate of evolution of CO~ is determined. Potentiometric. A solution of cephalosporin C sodimn salt (27 rag), or of triacetin (57 rag), in 1.6 ml of 0.15 M NaC1 are placed in a vessel maintained at 30 ° and carrying a magnetic stirrer. A glass electrode and a reference electrode, coupled to a pH stat, are immersed in the solution. A solution of the esterase (0.4 ml), whose pH has been adjusted to 6.5, is added. The pH is maintained at 6.5 by automatic addition of 40 mM NaOH, and the initial rate of hydrolysis is determined. Unit o] Activity. Jansen et al. 4 defined a unit of citrus acetylesterase activity as the amount of enzyme that liberates 1 mmole of acetic acid per minute at 30 ° and pH 6.5 from a solution of triacetin (5%, w/v) in 0.15 M NaC1. The unit is defined here as the amount of enzyme that liberates 1 ~mole of acid per minute from a 2.85% (w/v) solution of triacetin (130 mM) under these conditions. Detection o] Activity. A solution may be tested for acetylesterase activity by the method of Byrde and Fielding2 A sample (10 ~l) of the solution is spotted on chromatography paper, and the paper is sprayed with diacetylfluorescein (0.01%) in 50% (v/v) acetone. The presence of the enzyme is revealed after 15 min by a spot which shows a bright green fluorescence in ultraviolet light.
Preparation The esterase was found by Jansen et al. ~ in the rind of California citrus fruits. Similar preparations of the enzyme have been obtained from oranges from California, Israel, and South Africa. ~,~ It is present in highest concentration in the colored "flavedo" of the peel. It may be extracted and partially purified as follows1,3.4: Crude Extract. The peel from 30 oranges (about 1500 g) is minced at 2 ° in a domestic food mincer, mixed with sodimn chloride (22.6 g), and minced again. The minced peel is mixed with sand or Celite (155.6 g) and pressed at + 2 ° in a laboratory press, pressing cloth being used for filtration. The press juice is cooled to + 2 ° and saturated with sodium oxalate to protect the enzyme from oxidative deactivation. ~ The pH of the stabilized juice is raised to 5.2 by addition of Na~CO..~ solution, and the juice is then centrifuged at 1700 g at 0% Oil and pith are removed as completely as possible, and the solution is filtered through a thin layer 4 E. F. Jansen, R. Jang, and L. 1R. MacDonnell, Arch. Biochem. 15, 415 (1947). R. J. W. Byrde and A. H. Fielding, Biochem. J. 61,337 (1955).
730
ANTIBIOTIC INACTIVATION AND MODIFICATION
[55a]
of Hyflo-Supcrcel to give about 800 ml of clear fluid with an activity of about 5 units/ml. To this solution sodium dithionite (250 mg) is added, 1 and the solution is allowed to stand for 2 or 3 min. Without this treatment the precipitate obtained at the next stage may be sticky and dark. Precipitation. Sufficient ammonium sulfate is stirred into the above solution at 0 ° to give 70% saturation (349 g), and the mixture is allowed to stand at 2 ° overnight. The precipitate is removed and taken up in 100 ml of 0.1 M Na2C20~. The mixture is dialyzed at 2 ° for 24 hr against 0.1 M Na2C204 (5 liters), and dialysis is continued for two further 24-hr periods against fresh Na2C20~ solution. A small precipitate is removed by centrifugation, and the clear supernatant (200 ml), which contains about 90% of the activity of the press juice, is stored at ~ 2 ° . 1,3,4 In a modification of the above procedure 17.5 g of Darco G-60 activated carbon is added to 340 ml of the crude extract, and the mixture is stirred slowly at 0 ° for 15 min. ~ The carbon is removed by centrifugation and to the resulting fluid (285 ml) at 0 ° sufficient ammonium sulfate (49.9 g) to give 30% saturation is added slowly with stirring. The mixture is centrifuged and the solid discarded. To the cold supernatant fluid (305 ml) is added 60 g of ammonium sulfate, with stirring, to give a solution which is 60% saturated with ammonium sulfate. The mixture is centrifuged and the supernatant liquid is discarded. The solid is dissolved in cold water to give 95 ml of enzyme solution. Solutions of citrus acetylesterase obtained in this manner have been used for the preparation of 7-aminodeacetylcephalosporanic acid, 8 deacetylcephalosporin C, 1 deacetylcephalothin (7-fl-thienylacetamidocephalosporadesic acid) and a series of other acylamido derivatives of deacetylcephalosporanic acid. ",3
Properties Specificity. The enzyme is an esterase which shows highest activity with esters of acetic acid as substrates. Good substrates include 7-aminocephalosporanic acid and a variety of cephalosporins. Butyryl esters are hydrolyzed much less rapidly than acetyl esters, and the rate of hydrolysis of glycerides decreases as the chain length of the fatty acid increases. 4 N-Acetyl compounds are not hydrolyzed.4 Activators. The activity of a solution of the enzyme which had been dialyzed against water increased by about 20% in the presence of 0.15 M NaC12 Stability. Oxidation and pigment formation occur in crude extracts of the enzyme and are accompanied by loss of activity. This loss may be
[55b]
CEPHALOSPORINACETYLESTERASE (Bacillus subt~lis)
731
minimized by addition of reducing agents, including 0.1 M sodium oxalate 4 and Na.~S.,04 (0.5 mg/ml).l After precipitation with (NH,)2S0~ and dialysis against 0.1 M Na~C_~O, the enzyme retains its activity for several months in solution at pH 7.0 and at 2 °. At 35 ° it is relatively unstable in this solution, and at 50 ° about 90% of its activity is lost in 30 rain. At 25 ° it is relatively stable between pH 5.0 and pH 8.25, but is rapidly inactivated at pH values of 4.0 or below. 4 Dialysis of the enzyme against water is accompanied by about 50% loss of activity.4 pH. The pH-activity curve with monoacetin as a substrate shows a maximum between pH 6.0 and 6.5. The activities at pH 5.5 and 7.0 are about 10% lower than that at the optimum pH. Kinetic Constants. Km values of 30 mM and 32 mM have been reported with triacetin as substrate, TM and values of 4.7 mM and 8.6 mM with cephalosporin C 1 and cephalothin, 6 respectively, as substrates. The value for V,,,~x with triacetin as substrate is 3.4 times that with cephalosporin C as substrate in the presence of the same amount of enzyme. 1 With one preparation of enzyme, the maximum rate of hydrolysis of cephalothin was 0.36 mg cephalothin per milligram of esterase per minute2 6B. J. Abbott, personal communication, 1969.
[55b] Cephalosporin Acetylesterase (Bacillus subtilis) By BERNARDJ. ABBOTTand DAVID S. FUKUDA H
H ,
+
O , / ~ N ~ 9 , ~ CH2--O-- ~ -- CHa
~
COOH
CH2OH COOH
7-Aminocephalosporanic acid, R~H O Cephalothin, R = t S ~ _ C H 2 ~ NH~
'
O
Lk
Cephalosporin C, R z H O O C - - C H - - ( C H 2 ) 3 - - C -
Enzymes that deacetylate cephalosporins are widespread in nature. Jeffery et al. 1 reported that citrus peel contains an enzyme that deacety1j. D'A. Jeffery, E. P. Abraham, and G. G. F. Newton, Biochem. J. 81, 591 (1961).
[55b]
CEPHALOSPORINACETYLESTERASE (Bacillus subt~lis)
731
minimized by addition of reducing agents, including 0.1 M sodium oxalate 4 and Na.~S.,04 (0.5 mg/ml).l After precipitation with (NH,)2S0~ and dialysis against 0.1 M Na~C_~O, the enzyme retains its activity for several months in solution at pH 7.0 and at 2 °. At 35 ° it is relatively unstable in this solution, and at 50 ° about 90% of its activity is lost in 30 rain. At 25 ° it is relatively stable between pH 5.0 and pH 8.25, but is rapidly inactivated at pH values of 4.0 or below. 4 Dialysis of the enzyme against water is accompanied by about 50% loss of activity.4 pH. The pH-activity curve with monoacetin as a substrate shows a maximum between pH 6.0 and 6.5. The activities at pH 5.5 and 7.0 are about 10% lower than that at the optimum pH. Kinetic Constants. Km values of 30 mM and 32 mM have been reported with triacetin as substrate, TM and values of 4.7 mM and 8.6 mM with cephalosporin C 1 and cephalothin, 6 respectively, as substrates. The value for V,,,~x with triacetin as substrate is 3.4 times that with cephalosporin C as substrate in the presence of the same amount of enzyme. 1 With one preparation of enzyme, the maximum rate of hydrolysis of cephalothin was 0.36 mg cephalothin per milligram of esterase per minute2 6B. J. Abbott, personal communication, 1969.
[55b] Cephalosporin Acetylesterase (Bacillus subtilis) By BERNARDJ. ABBOTTand DAVID S. FUKUDA H
H ,
+
O , / ~ N ~ 9 , ~ CH2--O-- ~ -- CHa
~
COOH
CH2OH COOH
7-Aminocephalosporanic acid, R~H O Cephalothin, R = t S ~ _ C H 2 ~ NH~
'
O
Lk
Cephalosporin C, R z H O O C - - C H - - ( C H 2 ) 3 - - C -
Enzymes that deacetylate cephalosporins are widespread in nature. Jeffery et al. 1 reported that citrus peel contains an enzyme that deacety1j. D'A. Jeffery, E. P. Abraham, and G. G. F. Newton, Biochem. J. 81, 591 (1961).
732
ANTIBIOTIC INACTIVATION AND MODIFICATION
[55b]
lates cephalosporin C and triacetin. A survey of microbial sources revealed that many bacteria and actinomycetes also are capable of deacetylating cephalosporins. 2,~ A similar enzyme activity has been found in mammalian tissues, where the enzyme appears to be most prevalent in the liver and kidney. ~ Although the enzyme is widespread, it has not been purified to homogeneity and very little is known about its physical characteristics or kinetic behavior. The data in this report, except where other documentation is cited, were derived from the authors' laboratory.
Assay Method
Principle. Cephalosporin esterase activity is readily assayed by titration with an automatic pH star. The reaction generates acetic acid which causes a drop in pH of the reaction mixture. A standardized KOH solution is automatically added to maintain the pH at a preset value (usually pH = 7.0). The amount of KOH added per unit of time is directly proportional to the reaction rate. The rate of KOH addition is automatically recorded on a moving chart, and the initial reaction rate can be determined from the slope of the line. Reagents 7-(Thiophene-2-acetamido)cephalosporanic acid (cephalothin), 5.0 mg/ml 7-Aminocephalosporanic acid (7-ACA), 2.5 mg/ml. These solutions are stable for 1 day at 4 °. Enzyme: 2.0-10.0 mg/ml of unbuffered crude esterase at pH -- 7. 0 KOH, standardized 0.004 N
Procedure. The reaction is followed using a Radiometer T T T l l automatic titrator, ABU l I T 0.25-ml burette and SBR3 recorder. The reaction vessel TTA31 contains 2.0 ml of substrate solution adjusted to pH -- 7.0 and is maintained at 25 ° with a water jacket. A stream of humidified nitrogen must be passed over the surface of the reaction mixture to exclude CO2. In the absence of N2, CO2 absorption lowers the pH and triggers the addition of the standardized KOH solution. At pH = 7.0 and 25% water attacks the fl-lactam ring causing hydrolysis 5 and release of protons. The nonenzymic hydrolysis, which is a firstorder reaction, is minimal at pH = 7.0, but it increases at both alkaline A. L. Demain, R. B. Walton, J. F. Newkirk, and I. M. Miller, Nature (London) 199, 909 (1963). 3U.S. Patent 3,304,236 (1967). 4 C. H. O'Callaghan and P. W. Muggleton, Biochem. J. 89, 304 (1963). 5 j. Konecny, E. Felber, and J. Gruner, J. Antibiotic. 36, 135 (1973).
[55b]
CEPHALOSPORINACETYLESTERASE (Bacillus subtilis)
733
and acidic pH values. Nonenzymic hydrolysis is also accelerated at higher temperatures. The rate of nonenzymic hydrolysis of the substrate is measured by determining the rate of KOH addition in the absence of enzyme. The reaction is then started by adding 100 ~l of enzyme solution, and the total rate of KOH addition is determined. The reaction is linear for at least 15 rain. A convenient chart speed for the recorder is 0.5 cm/min. The chart contains 100 divisions, and each division corresponds to the addition of 10 nmoles of titrant. The delivery of 0.25 ml from the burette causes full-scale deflection of the recorder pen and corresponds to the hydrolysis of 1 ~mole of substrate in the 2.0-ml reaction vessel. Units and Specific Activity. A unit may be defined as the amount of enzyme that hydrolyzes 10 nmoles of cephalothin per minute at 25 °, pH = 7.0, at a cephalothin concentration of 5 mg/ml. Specific activity may be expressed as units per milligram of protein.
Preparation of the Enzyme
Cultivation of the Microo,rganism. The enzyme is prevalent in strains of Bacillus subtilis. Strain WRRL-B-558 (from the Western Regional Research Laboratory) is cultivated in trypticase soy broth on a rotary shaker at 30 °. During cultivation the pH declines to 6.2 and then begins to rise. After 22 hr, when the pH is about 7.8, the cells are harvested by centrifugation. The enzyme may be recovered from either the culture supernatant or from the sedimented biomass. About 75% of the total esterase activity is associated with the biomass fraction. Partial Preparation of the Enzyme. Although most of the enzyme is fo~md with the biomass, the specific activity of a crude biomass preparation is sinfilar to that obtained from a culture supernatant. Isolation of the esterasc from the supernatant obviates the need for a sonication and nucleic acid removal step. The enzyme is recovered from the supernatant by saturation with ammonium sulfate. The ammonium sulfate is removed by dialysis against distilled water and the salt-free enzyme may be stored after lyophilization. A 2- to 3-fold purificatioll can be achieved by recovering the protein fraction that precipitates between 50% and 80% of ammonium sulfate saturation. Additional purification can be obtained by stirring 2.0 mg/ml of salt-free unbuffered esterase preparation with 4.0 mg/ml bentonite at pH -- 6.5 and 25 °. After 3 hr the bentonite is removed by centrifugation. The supernatant contains about 75% of the initial esterase activity and exhibits a 2.5-fold increase in specific activity. Further purification (~40-fold~ is possible with Sephadex G-200 chromatography. Using a calibrated Sephadex column, it was determined
734
ANTIBIOTIC INACTIVATION A N D
MODIFICATION
[55]
that the cephalosporin acetylesterase has a molecular weight of about 190,000.
Properties Stability. The enzyme is extremely stable. It may be stored in an unbuffered solution at 25 ° and pH - 7.0 for 3 weeks with little or no loss of activity. Sodium azide (0.02~) must be added to prevent the growth of microorganisms. At 80 ° all activity is lost within 5 rain; however, the activity is retained after 10 rain at 80 ° if heating occurs in the presence of 1.0 M phosphate buffer. The salt-free enzyme loses 50~ of its activity in 5 min at 70 °, but no activity is lost at 60 ° after 1 hr. pH and Temperature Optima. The enzyme has a narrow pH optimum at pH -- 7.0, and the optimum temperature is between 40 ° and 50 °. Substrates and Inhibitors. In addition to cephalosporins, the enzyme will hydrolyze mono- and triacetin, a-naphthyl acetate, and glucose pentaacetate. The enzyme does not hydrolyze casein, acetanilide, p-nitrophenylacetate, p-nitrophenylsulfate, and it is not inhibited by arsenilic acid or bis (p-nitrophenyl) phosphate. Kinetic Properties. The enzyme exhibits Michaelis-Menten kinetics with cephalosporins and triacetin. The Michaelis constants measured with these substrates are cephalothin, 8.3 raM; 7-ACA, 2.8 raM; triacetin, 0.46 raM. The products of 7-ACA hydrolysis (acetate q- deacetyl 7-ACA) are weak competitive inhibitors of the reaction. The Ki values are 5 )< 10.2 M for acetate and 3.6 X 10-2 M for deacetyl 7-ACA.
[56] Chloramphenicol Hydrolase By
CH20H I H2N-C-H I H- C-OH
CHzOH CI2CHCOHN- C-H !
H-C-OH
0
L. C. VINING
HzO
NO2
CHLORAMPHENICOL
Cl2CHCOOH +
0 NOz
p-NITROPHENYLSERINOL
734
ANTIBIOTIC INACTIVATION A N D
MODIFICATION
[55]
that the cephalosporin acetylesterase has a molecular weight of about 190,000.
Properties Stability. The enzyme is extremely stable. It may be stored in an unbuffered solution at 25 ° and pH - 7.0 for 3 weeks with little or no loss of activity. Sodium azide (0.02~) must be added to prevent the growth of microorganisms. At 80 ° all activity is lost within 5 rain; however, the activity is retained after 10 rain at 80 ° if heating occurs in the presence of 1.0 M phosphate buffer. The salt-free enzyme loses 50~ of its activity in 5 min at 70 °, but no activity is lost at 60 ° after 1 hr. pH and Temperature Optima. The enzyme has a narrow pH optimum at pH -- 7.0, and the optimum temperature is between 40 ° and 50 °. Substrates and Inhibitors. In addition to cephalosporins, the enzyme will hydrolyze mono- and triacetin, a-naphthyl acetate, and glucose pentaacetate. The enzyme does not hydrolyze casein, acetanilide, p-nitrophenylacetate, p-nitrophenylsulfate, and it is not inhibited by arsenilic acid or bis (p-nitrophenyl) phosphate. Kinetic Properties. The enzyme exhibits Michaelis-Menten kinetics with cephalosporins and triacetin. The Michaelis constants measured with these substrates are cephalothin, 8.3 raM; 7-ACA, 2.8 raM; triacetin, 0.46 raM. The products of 7-ACA hydrolysis (acetate q- deacetyl 7-ACA) are weak competitive inhibitors of the reaction. The Ki values are 5 )< 10.2 M for acetate and 3.6 X 10-2 M for deacetyl 7-ACA.
[56] Chloramphenicol Hydrolase By
CH20H I H2N-C-H I H- C-OH
CHzOH CI2CHCOHN- C-H !
H-C-OH
0
L. C. VINING
HzO
NO2
CHLORAMPHENICOL
Cl2CHCOOH +
0 NOz
p-NITROPHENYLSERINOL
[55]
CHLORAMPHENICOL HYDROLASE
735
The enzyme which hydrolyzes the amide bond in chloramphenicol has been isolated from the mycelium of Streptomyces species 3022a, an organism which produces chloramphenicol. ~ A similar enzyme (enzyme A) has been obtained from Proteus vulgaris and Bacillus subtilis, 2 and the presence of a hydrolase in Escherichia coli, 2,~ B. mycoides, °- a Flavobacterium, 4 and humans 5 is inferred from reports that p-nitrophenylserinol can be detected after exposure to the antibiotic.
Assay Principle. Activity is measured as the amount of radioactivity retained in the aqueous phase when chloramphenicol labeled in the p-nitrophenylserinol moiety is incubated with the enzyme, acidified, and extracted with a chloroform-isopropanol (3:1) mixture. Reagents D-threo [Methylene-14C ] chloramphenicol [ 1- (p-nitrophenyl-2-dichloracetamido-l,3-propanediol-3-14C, Amersham/Searle Corporation, Arlington Heights, Illinois 60005], aqueous solution, 1 raM. Sorenson phosphate buffer, 0.1 M, pH 7.2 Hydrochloric acid, 1 M Chloroform-isopropanol (3:1) Scintillation fluid: 0.4% 2,5-diphenyloxazole in toluene-ethanol (3:2) Procedure. The assay mixture contains: D-threo-[methylene-14C]chloramphenicol solution, 0.1 ml; Sorenson buffer 0.1 ml; and enzyme, 0.5 ml. It is incubated in a 15-ml conical, centrifuge tube at 37 ° for 2 hr, then mixed with 1 N hydrochloric acid, 2 ml, and immediately centrifuged at sufficient speed to sediment the precipitate. The clarified solution is decanted and extracted 3 times with 10-ml portions of chloroform-isopropanol (3:1). Samples, 0.5 ml, of the aqueous phase are mixed with 15-ml scintillation fluid and counted for radioactivity. Each series of assays should include a blank in which the enzyme is replaced by an equivalent volume of buffer; the time between acidification of the assay mixture and the final extraction should be as short as possible to avoid high blanks caused by acid-catalyzed hydrolysis. V. 2 G. 3 L. 4 F. 5 A.
S. Malik and L. C. Vining, Can. J. Microbiol. 17, 1287 (1971). N. Smith, C. S. Worrel, and B. L. Lilligren, Science 110, 297 (1949). Molho-Lacroix and D. Molho, Bull. Soc. Chim. Biol. 34, 93 (1952). Lingens, It. Eberhardt, and O. Altmann, Biochim. Biophys. Acta 130, 345 (1966). J. Glazko, Antimicrob. Ag. Chemother. 1966, 655 (1967).
736
ANTIBIOTIC INACTIVATION AND MODIFICATION
[55]
Enzyme activity is estimated as nanomoles of p-nitrophenylserinol formed per hour.
Purification Source. Streptomyces species 3022a can be grown in 500-ml Erlenmeyer flasks, each containing 100 ml of a medium consisting of (in grams per liter): glycerol, 1%, yeast extract (Difco Laboratories, Detroit, Michigan), 0.3%; nutrient broth (Difco), 0.8%; dipotassium hydrogen phosphate, 0.5%, in distilled water. With a 4% (v/v) inoculum good mycelial growth is obtained after a 24-hr incubation on a rotary shaker (220 rpm, 1.5 inch eccentricity) at 25 °. The enzyme specific activity of the mycelium is similar throughout the growth cycle. Acetone Powder. The mycelium is collected and washed with 0.9% saline by centrifugation (10 min at 10,000 g). The packed cells are then dropped into a blender containing 20 volumes of acetone at --20 ° and operated at low speed. After the suspension has been allowed to settle for 10 min at this temperature, most of the supernatant can be decanted, and the slurry is filtered in vacuo through a sintered-glass funnel. The residue is washed 3-4 times with additional acetone, dried, and stored at --20 ° over phosphorus pentoxide. Soluble Enzyme. The mycelium, collected and washed with saline as above, is resuspended in 4 times its weight of 0.1 M Sorenson buffer, pH 7.2, and cooled in an ice-salt bath. The cells are disrupted by sonic oscillation (Branson Sonifier, Heat Systems, Inc., Great Neck, New York, at a maximum tuned current of 12 A) for 10 min, interrupted at 2-min intervals to avoid overheating. After centrifugation for 10 min at 15,000 g and 5 °, the supernatant solution is collected and ammonium sulfate is added to 40% saturation. The precipitate is removed by centrifugation and discarded. A second precipitate obtained at 60% ammonium sulfate saturation is collected and redissolved in Sorenson buffer, pH 7.2.
Properties Few properties have been reported. The crude soluble preparation from Streptomyces species 3022a gave a K.~ of 0.2 mM and a V.... of 2.86 ~nmoles per hour. In 0.1 M phosphate buffer the pH for optimum activity is 6.8.1 The value for the bacterial enzyme is given as 7.5, and the temperature optimum as 37.5-40o. 2 Most of the enzyme activity in Streptomyces species 3022a cells is not solubilized after prolonged sonic disruption or grinding with abrasives. Acetone powders are stable and retain activity after storage for
[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
737
at least 1 month. Solutions in Sorenson buffer, pH 7.2, may be stored at 5 °, but the rate of inactivation has not been measured. The E. coli enzyme is reported to have high specificity for chloramphenicol. "~
Miscellaneous As a product of the enzymic reaction within the organism, p-nitrophenylserinol does not accumulate in large amounts, but is N - a c y l a t e d or metabolized to p-nitrobenzoic acid and other products. ~,~," The Streptomyces and bacterial enzymes are intracellular, except under autolytic conditions. -° In Streptomyces species 3022a substrate access to the enzyme appears to be prevented in cultures producing chloramphenicol, or in cultures adapted to grow under nonproducing conditions in the presence of the antibiotic? 6 G. N. Smith and C. S. Worrel, Arch. Biochem. 28, 232 (1950).
[57] Chloramphenicol Acetyltransferase from Chloramphenicol-Resistant Bacteria B y W. V. SHAW Chloramphenicol q- acetyl-S-CoA-~ chloramphenicol 3-acehtte + IIS-(?oA
(1)
H~.N/R2 OH FId. 1. Structure of compounds related to chloramphenicol. The asymmetric carboa atoms at C-1 and C-2 generate d~e four possible stereoisomers of which only the D-threo isomer has significant antibiotic activity. Chloramphenicol has the following functional groups: R, = --NO:; R: = --COCHCh ; and R~ = --OH. Chloramphenicol resistance is frequently encountered among many genera of bacteria. Although in some instances the underlying mechanism may be a relative impermeability to chloramphenicol (CM), the resistance phenotype is most commonly the result of inactivation of the antibiotic by the enzyme chloramphenicol acetyltransferase (CAT)?,'-' The 1W. V. Shaw, J. Biol. Chem. 241~, 687 (1967). : Y. Suzuki and S. Okamoio, J. Biol. Chem. 242, 4722 (1967).
[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
737
at least 1 month. Solutions in Sorenson buffer, pH 7.2, may be stored at 5 °, but the rate of inactivation has not been measured. The E. coli enzyme is reported to have high specificity for chloramphenicol. "~
Miscellaneous As a product of the enzymic reaction within the organism, p-nitrophenylserinol does not accumulate in large amounts, but is N - a c y l a t e d or metabolized to p-nitrobenzoic acid and other products. ~,~," The Streptomyces and bacterial enzymes are intracellular, except under autolytic conditions. -° In Streptomyces species 3022a substrate access to the enzyme appears to be prevented in cultures producing chloramphenicol, or in cultures adapted to grow under nonproducing conditions in the presence of the antibiotic? 6 G. N. Smith and C. S. Worrel, Arch. Biochem. 28, 232 (1950).
[57] Chloramphenicol Acetyltransferase from Chloramphenicol-Resistant Bacteria B y W. V. SHAW Chloramphenicol q- acetyl-S-CoA-~ chloramphenicol 3-acehtte + IIS-(?oA
(1)
H~.N/R2 OH FId. 1. Structure of compounds related to chloramphenicol. The asymmetric carboa atoms at C-1 and C-2 generate d~e four possible stereoisomers of which only the D-threo isomer has significant antibiotic activity. Chloramphenicol has the following functional groups: R, = --NO:; R: = --COCHCh ; and R~ = --OH. Chloramphenicol resistance is frequently encountered among many genera of bacteria. Although in some instances the underlying mechanism may be a relative impermeability to chloramphenicol (CM), the resistance phenotype is most commonly the result of inactivation of the antibiotic by the enzyme chloramphenicol acetyltransferase (CAT)?,'-' The 1W. V. Shaw, J. Biol. Chem. 241~, 687 (1967). : Y. Suzuki and S. Okamoio, J. Biol. Chem. 242, 4722 (1967).
738
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
O-acetoxy derivatives of CM are devoid of antibiotic activity since they do not bind to bacterial ribosomes and therefore fail to inhibit polypeptide elongation. The structural gene for CAT is commonly extraehromosomal among the Enterobacteriaceae, where it is carried by R factors conferring resistance to CM and is similarly associated with a plasmid in resistant staphylococci. CAT has also been detected in CM-resistant isolates of Streptococcus ]aecalis, Diplococcus pneumoniae, and Agrobacterium tume]aciens. Bacteria which synthesize CAT begin the exponential phase of growth only after the concentration of unmodified CM has fallen to a level that is no longer inhibitory. The duration of such an increment in the usual lag phase is roughly proportional to the concentration of CM at the time of innoculation. This relationship holds for gram-negative species harboring R factors or for any bacteria carrying plasmids which dictate the constitutive synthesis of CAT. A more complex situation exists in staphylococci, streptococci, pneumococci, and A. tume]aciens wherein CAT is an inducible enzyme. Chloramphenicol is an effective inducer of CAT in such strains, but it is also a potent inhibitor of bacterial protein synthesis. Because of these competing properties and the fact that the acetylated product is not an inducer, the kinetics of-CAT induction in bacteria with inducible CM resistance are complex. Levels of CAT activity can be maintained at a high level only by continued challenge with CM or by induction with a "gratuitous inducer" which is neither acetylated nor serves as an inhibitor of protein synthesis. Although all known R factors carrying the CAT gene in enteric bacteria mediate constitutive synthesi s of the enzyme, the specific activity of CAT in crude extracts is subject to wide variations due to gene dosage effects and also to "catabolite repression" by glucose and certain other growth substrates. Examples in the first instance are the "relaxed" replication of certain R factors in Proteus mirabilis ~ and the vegetative growth of bacteriophage P1 CM in Escherichia coli. ~ Both situations have in common a high multiplicity of plasmid-borne CAT gene copies. A less dramatic example is the occasional occurrence of strains of E. coli (or related enteric bacteria) which harbor two compatible plasmids, both of which may carry one copy of the CAT region2 Catabolite repression of CAT synthesis in E. coli is mediated by a mechanism involving cyclic adenosine 5'-monophosphate (cAMP), in common with the streptomycin adenylylating enzyme and the more gens R. Rownd, H. Kasamatsu, and S. Mickel, Ann. N . Y . Acad. Sci. 182, 188 (1971). ' E. Kondo, D. K. Haapala, and S. Falkow, Virology 40, 431 (1970). 5 W. V. Shaw, L. C. Sands, and N. Datta, Proc. Nat. Acad. Sci. U~S. 69, 3049 (1972).
[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
739
eral cases of fl-galactosidase and gatactokinase. 6 The decrease in CAT levels seen with growth in glucose-containing media can be prevented by the addition of high concentrations of cAMP or by the substitution of glycerol or other nonrepressing energy sources for glucose. Requirements ]or CM Acetylation Techniques. More techniques for studying the enzymic acetylation of CM are described below than are likely to be of use for any given research objective, The thin-layer chromatographic method for detecting CM acetylation in cell suspensions is useful in screening presumptive CAT producing strains or for exploring other possible mechanisms of CM inactivation. The spectrophotometric methods excel for general purposes and can also be adapted for the measurement of either CM or acetyl coenzyme A (CoA). The very sensitive radioisotopic method appears cumbersome but is remarkably reliable in practice and very useful for detecting and quantitating v'ery low levels of CAT activity. It should be stressed that the availability of CAT as a reagent offers interesting possibilities for (a) the specific detection of the biologically active isomer of CM and its quantitation and (b) the instantaneous removal (by inactivation) of CM present in any in vitro biological system. A preliminary investigation of the use of CAT to measure CM in body fluids is encouraging as regards specificity, sensitivity, and speed: features of enzymic assay techniques which are usually lacking in many of the commonly used bacteriological assay methods for antibiotics. ~ An additional possible application of CAT is its use to inactivate CM in instances wherein the antibiotic has been added as a potentially reversible inhibitor of protein synthesis. Apart from the above considerations it should be noted that CAT may be of value to molecular biologists as it is a gene product which can be assayed with specificity and great sensitivity. Two temperate phages have been isolated which confer CM resistance on the host cell in the lysogenic state, and in both cases the phenotype is due to the synthesis of CAT. Phage P1 CM was isolated as a recombinant between an R factor and phage P1 and, more recently, a derivative of phage lambda (~ CM) has been constructed which also carries the CAT gene. ~ It has been possible to synthesize CAT in a cell-free system from E. coli using P1 CM template DNA, 9 and it seems likely that either or both ~J. Harwood and D. H. Smith. Biochem. Biophys. Res. Commun. 42, 57 (1971). ~P. Lietman, personal communication, 1973. 8j. R. Scott, Virology 53, 327 (1973). B. de Crombrugghe, I. Pastan, W. V. Shaw, and J. L. Rosner, Nature (Lo~don) New Biol. 241, 237 (1973).
740
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
of the CM phages will be useful for studies of the control of transcription and translation.
Assay Methods
Principles. Enzyme activity can be quantitated by either measuring (a) the CM-dependent disappearance of acetyl-S-CoA; (b) the appearance of 3-O-acetoxy derivative of CM; or (c) the formation of reduced (unesterified) CoA. In practice, the choice between these alternatives will be determined by the level of sensitivity required and by complications created by interfering substances. The 3-monoacetoxy derivative of CM is the initial product of the reaction and is devoid of significant antibiotic activity. A diacetoxy product appears at a rate two orders of magnitude slower than the monoacetylation reaction, and its formation is also catalyzed by CAT. Although the mechanism has not been studied in detail, there is circumstantial evidence favoring the following sequence of reactions: CM-3-acetate ~ CM-l-acetate CM-l-acetate + acetyl-S-CoA--~ CM-1,3-diacetate + HS-CoA
(2)
(3)
The reaction described by Eq. (2) appears to be a nonenzymic and pHdependent acyl migration. It seems likely that the slow overall rate of formation of the diacetate derivative is due both to an unfavorable equilibrium of the nonenzymic step and also to a low rate of 3-O-acetylation of CM-l-acetate by CAT. Although Eqs. (2) and (3) should be kept in mind in any kinetic or mechanistic studies of CAT, they do not in fact interfere significantly with measurements of CAT which utilize the stoichiometry of Eq. (1).
Chromatographic Detection o] Chloramphenicol Acetylation Although of historical interest as a means of assaying CAT, the thinlayer chromatographic separation and quantitation of CM and its acetoxy derivatives still has limited applications, especially for the detection of CM acetylation by bacterial cell suspensions. The chromatographic determination of the fate of CM when incubated with a culture of a resistant microorganism also can reveal (a) other mechanisms than acetylation if the latter is not involved or (b) no alteration of CM, thereby suggesting a relative impermeability to CM of the bacterium in question. The use of 14C-labeled CM is recommended because of the difficulties in detecting small amounts of products formed when the initial concentra-
[571
CHLORAMPHENICOL ACETYLTRA_NSFERASE
741
tion of nonradioactive CM may not exceed 10 uM (3.2 ~g/ml). Since no general protocol can be formulated, a typical example will be given to illustrate a possible application. A strain of E. coli which is resistant to CM (minimum inhibitory concentration in nutrient broth = 100 t,g/ml) is to be tested for the presence of CAT. An overnight culture is diluted 1:1000 into 5 ml of fresh growth medium (the same liquid medium used for the sensitivity testing) containing J'~C]chloramphenicol at a concentration of 32 /~gfml, (0.1 raM) and a specific activity of 1 mCi/mmole. At the conclusion of tile exponential phase of growth the culture tube is centrifuged and 1 ml of the culture supernatant is extracted with 1 ml of ethyl acetate by agitation on a vortex mixer followed by centrifugation. The process is repeated twice and the ethyl acetate supernatants are pooled in a conical centrifuge tube and evaporated to dryness in air stream or on a steam bath. The sample is then taken up to 0.1 ml of ethyl acetate and spotted by repeated applications with a capillary pipette at the origin of a thinlayer sheet alongside an extract of an uninoculated control culture which has been treated in similar fashion. The chromatograms are developed in ascending fashion with the appropriate solvent until the front is a few centimeters from the top of the sheet. After removal from the tank or beaker the sheets are air dried and subjected to radioautography overnight after appropriately marking the sheets with reference points made with radioactive ink. On inspection the film will reveal whether bacterial growth has been accompanied by conversion of CM to its monoand diacetyl derivatives. The choice of thin-layer supports and solvents is arbitrary, but the following have been useful~: (1) alumina in benzene-methanol (85:15, v/v}; (2) silica gel in chloroform-methanol (95:5, v/v). Commercially available thin sheets of both alumina and silica gel on inert supports are available from a number of suppliers. Those which contain a fluorescent additive (such as Ladd thin fihns, Packard) are especially useful, as CM and its products give a prominent "quench" when present as major components. The translucent Mylar sheets are useful since they can be aligned with the exposed X-ray fihn and viewed by transmitted light. The areas of the chromatogram are marked, cut out with scissors, and dropped into scintillation vials for counting. Quantitation of the percent conversion of CM to acetyl products can thus be determined directly, and if samples of the culture "tre taken at several points in time, the data can be plotted to show the kinetics of inactivation. The same approach may be utilized to follow the course of the enzyme reaction, but the methods described below have distinct advantages in convenience and precision.
742
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
Spectrophotometric Assay The most convenient technique for quantitating the rate of CM acetylation takes advantage of the generation of a free CoA sulfhydryl group coincident with transfer of the acetyl group to CM. Reaction of the reduced CoA with 5,5"-dithiobis-2-nitrobenzoic acid (DTNB) yields the mixed disulfide of CoA and thionitrobenzoic acid and a molar equivalent of free 5-thio-2-nitrobenzoate. 1° The latter has a molar extinction coefficient of 13,600 at 412 nm. The assay is best carried out with a recording spectrophotometer equipped with a temperature-controlled cuvette chamber set at 37 ° .
Reagents Tris. hydrochloride, 1.0 M, pH 7.8 Acetyl-CoA, 5 mM Chloramphenicol (D-threo) 5 nlM 5,5'-Dithiobis-2-nitrobenzoic acid (DTNB) The only reagent solution that must be stored frozen is acetyl-CoA. The reaction mixture is freshly prepared from the individual reagents by dissolving 4 mg of DTNB in 1.0 ml of Tris.HC1 buffer, after which 0.2 ml of the acetyl-CoA stock solution is added and the total volume is made up to 10 ml. The final concentrations of each component are as follows: Tris.HC1 (100 mM), acetyl-CoA (0.1 mM), and DTNB (0.4 mg/ml). After the cuvette (1 cm light path) containing enzyme and the reaction mixture has been allowed to equilibrate with the waterbath, the reaction is started by the addition of CM at a final concentration of 0.1 mM. The rate of increase in absorption at 412 nM prior to the addition of CM is subtracted from the observed rate after the start of the reaction, and net change in extinction per minute is divided by 13.6 to give the result in micromoles per minute of CM-dependent DTNB reacted. Since the latter is equal to the rate of acetylation and since 1 unit of CAT = 1 ~mole of CM acetylated per minute (37°), the calculation also yields the number of units of enzyme in the cuvette. Two factors influence the precision of the DTNB assay for CAT. First, care must be taken to avoid very high concentrations of mercaptans in the enzyme solution. In practice the addition of 2-mercaptoethanol or dithiothreitol in excess of 1 mM to crude or even partially purified preparations of CAT presents problems when more than 10 ~l of enzyme must be added to a 1-ml cuvette to obtain a significant rate of CM acetylation. A more troublesome problem occurs with crude cell extracts prepared from certain genera of bacteria that contain high levels of thiolo A. F. S. A. Habeeb, this series, Vol. 25, p. 457.
[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
743
esterase activity. In such instances the control rate of increase in 412 nm absorbance due to acetyl-CoA hydrolysis prior to the addition of CM may approach or exceed the increment seen after adding the antibiotic. In some instances it may be necessary to resort to partial purification of CAT before the spectrophotometric assay can be used with confidence. The [l+C]acetate procedure which utilizes an acetyl-CoA generating system should also be considered as a possible solution to the problem of high nonspecific thioesterase background (see below). A serious limitation to the DTNB assay for CAT concerns the intrinsic high reactivity of essential cysteine thiol groups in certain variants of CAT. In the absence of protecting reduced mercaptans, enzyme activity decreases rapidly after addition of DTNB. Since the rate of CM acetylation is substantially greater than the rate of CAT inactivation, an approximation of the initial rate for CAT activity can be obtained by taking special care to add enzyme immediately prior to addition of CM. An alternative spectrophotometric method 1 can be used when a high concentration of competing mercaptans interferes with the DTNB assay. The loss of an acyl group from thioesters such as acetyl-CoA is accompanied by a decrease in absorption in the ultraviolet. The difference in molar extinction coefficients of acetyl-CoA and reduced CoA plus acetate is 4500 at 232 nm. 11 Special care must be taken to remove interfering ultraviolet absorbing material from the enzyme preparation by gel filtration or dialysis. The contribution of the absorption due to protein added to the euvette becomes a more serious obstacle in crude extracts, especially those with low levels of CAT activity. Apart from the inconvenience of measurements in the far ultraviolet region and the fact that the method is intrinsically less sensitive than the DTNB procedure, the assay of thioester cleavage at 232 nm suffers from being a difference method. The absolute decreases in absorbance per unit time due to the presence of CM and low levels of CAT may be impossible to quantitate without recourse to the use of a dual beam recording spectrophotometer.
Assay by Direct Measurement o] ["C ]Acetyl Chloramphenicol The need for a highly sensitive, rapid, and specific means of quantitating the synthesis of CAT in a complex E. coli extract 9 led to the development of a radioactive method which, unlike that described above, does not require chromatography. The principle of the method is that the neutral [14C]acetoxy-chloramphenicol derivatives can be quantitatively extracted into benzene at alkaline pH whereas ionized and polar species such as [14C]acetate and [14C]acetyl-CoA remain in the aqueous phase. 11E. R. Stadtman, this series, Vol. 3, p. 985.
744
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
The sensitivity of the method is limited only by the specific activity of [14C]acetate used. Specificity is achieved by measuring the increment in radioactivity extracted from an assay mixture containing CM as compared to that from a control incubation without CM. Should any doubt arise that the products are, in fact, the acetoxy derivative(s) of chloramphenicol, the chromatographic methods described previously may be used for confirmation. The most convenient means of terminating the assay incubation has been found to be the decomposition of acetyl-CoA catalyzed by phosphotransacetylase in the presence of 100 mM arsenate. The arsenolysis reaction obviates the need for extremes of temperature or pH and does not interfere with the extraction of products. Although the coupled assay requires numerous reagents, including three enzymes, all the components are readily available from commercial sources. A mixture of buffer, salts, and substrates may be stored frozen for convenience, but the reagent enzymes should be stored separately under the recommended conditions and added individually to the final incubation mixture immediately prior to the assay. The most convenient means of standardizing the radioactive assay to conform with the spectrophotometric unit of CAT activity (as defined above) is to assay a preparation of crude or purified CAT by both techniques. The two methods should agree within 5-10% when: (a) care is taken to ensure adequate temperature control in both instances; (b) the amount of CAT in each case is appropriate for the measurement of initial rates, and (c) the extractions are carried out with care. The experiments in which the radioactive assay have been used thus far have required that the method be specific, sensitive, and reproducible, rather than of a high absolute accuracy with respect to reference measurements. The maintenance of a saturating concentration of [14C]acetyl-CoA for CM acetylation by CAT is achieved by means of the following coupled reactions and the addition of an excess of pyruvic kinase, acetate kinase, and phosphotransacetylase: ADP -{- phosphoenolpyruvate ~ pyruvate T ATP ATP T [~4C]acetate ~ [~4C]acetyl phosphate T ADP [14C]acetyl phosphate + HS-CoA ~ [~4C]acetyl-S-CoA T phosphate
(4) (5) (6)
The pyruvic kinase system (Eq. 4) has been employed to generate a catalytic level of ATP since adenine nucleotides are competitive inhibitors (with respect to acetyl-CoA) of CAT. Although each of the reagent enzymes must be added in excess, it is usually not necessary to assay each one independently before its addition to the assay incubation. It is actually easier and more useful to ascertain that each is, in fact, present in excess than to assign a precise value to the activity of each component.
[57]
745
CHLORAMPHENICOL ACETYLTRANSFERASE
This is best accomplished by assuming the accuracy of the supplier's nominal activity specifications as a point of departure. The radioactive assay is then run with a sample of CAT with known activity by the DTNB method. Dilutions of each reagent enzyme are made, and aliquots of several such dilutions of each enzyme are tested while the other two are added at concentrations assumed to be 10-fold higher than the 1 unit required (1 umole/min) for each under the conditions of the assay. For each enzyme reagent so tested a limiting dilution shoukt be reached where the final amount of [~4C]acetyl CM radioactivity extracted is (a) lower than that with all less dilute samples of the enzyme in question and (t)~ less than that expected from the known activity of CAT present from the DTNB procedure. The assay conditions described below are a modification of those reported previously." The present procedure has evolved in order: (a) to avoid the inhibition of CAT by adenine nucleotides (see above), (b) to ensure that acetyl-CoA and CM are present at ~aturating concentrations, (el to be certain that the reagent enzymes are in excess and art present in a favorable environment for activity, and (d) to maximize the efficiency of the extraction procedure without introducing either high blank v'dues or artifacts2'-'
Reagents
Volume (ul) per 250 ul incubation
Final concentration
1 M T r i s . HCI (pH 7.8) 100 m M MgCI: 100 m M A T P 100 m M P h o s p h o e n o l p y r u v a t e 5 m M Coenzyme A (reduced; lithium salt) 10 m M [1-~4Cl Sodium :~eetate Acetate kinase P y r u v a t e kinase Phosphot r'msacet ylase
25 15 7.5 50 20 10 See text See lext See lext
100 m.'ll 6 mM 3 mM 20 m M 0.4 m M O. 4 m M
1 Unit 1 Unit I Unit
Each incubation is carried out in a conical centrifuge tube (nominal volume approximately 12-15 ml) to simplify the subsequent extraction t)rocedure. Tile final volume of 250 ul is achieved by the addition of tile ,2 Two other techniques have been tried unsuccessfully for the separation of P4C]acetoxy chloramphenicol from radioactive acetate and acetyl-CoA. Tile tatier should be absorbable by anion exchange resins (such as Dowex 1) whereas the nonionized product is not. An alternative approach is the selective absorption of C M and its P~C]acetyl derivatives to charcoal after arsenolysis of ["C]acetyl-CoA. In practice b o t h of these approaches have given high and variable blank values.
746
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
requisite volume of deionized water and the sample containing CAT, after which the components are thoroughly mixed. The blank incubation contains only water to volume, but no enzyme. In both instances the reaction is started by the addition of 5 ~l of chloramphenicol (5 mM) immediately prior to placing each Parafilm-sealed tube into a water bath at 37 °. After 20 rain of incubation the reaction is terminated by the addition of 25 ~l of 1.1 M sodium arsenate followed by thorough mixing. The extraction and processing of the product is carried out in the following manner. Two milliliters of benzene (analytical reagent grade) is added to each incubation tube, and the latter is agitated on a Vortex mixer for 20 sec. The tube is centrifuged in a nonrefrigerated clinical centrifuge for a few minutes to separate phases, and the upper benzene layer is carefully taken with a Pasteur pipette and placed in a glass scintillation vial. The extraction is repeated with a second 2 ml of benzene. The extracts are pooled, and 100 ~l of glacial acetic acid is added to each scintillation vial. After thorough mixing of the acetic acid and benzene, the samples are dried under a heat lamp in an air stream. After the samples are thoroughly dried, an appropriate scintillation counting solution is added (any convenient type is adequate since the product is soluble in most organic solvents and there is no nonvolatile residue to cause quenching) and each vial is assayed for 14C radioactivity. Blank incubations (in which CM has been added in the absence of CAT) or controls (containing CAT but no CM) should yield extracts with no more than 100-200 dpm above background when the specific activity of sodium acetate is of the order of 5 mCi per millimole. It should be apparent that initial CAT velocities will be measured accurately with this technique only if CAT is present in amounts such that both CM and acetyl-CoA are still at saturating concentrations after 20 m~n of incubation. In practice this means the addition of no more than approximately 0.0005 unit of CAT to any given incubation, an amount of enzyme sufficient to catalyze the acetylation of 10 nmole of CM in 20 min (or 40% of the 25 nmoles present initially). It is fortunate that the DTNB method begins to give reliable data at CAT levels near 0.0005 unit when a recording spectrophotometer with scale expansion (to 0 . 1 0 D units full scale) is employed. The choice of method will therefore usually favor the radioactive assay whenever the concentration of CAT is less than 0.005 unit/ml (0.005 unit per 100-~1 sample).
Purification of Chloramphenicol Acetyltransferase General Remarks. Conventional techniques of protein fractionation have been adequate for the purification of CAT from (1) R factor-bear-
[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
747
ing strains of E. coli, (2) staphylococci harboring CM plasmids. (3) CMresistant mutants of Proteus mirabilis in which the CAT gene is probably chromosomal, and (4) selected strains of Agrobacterium tume]aciens. Although the following protocol describes a typical purification for an R factor type of CAT, the principles can be applied to any bacterial source of the enzyme. Since each variant of CAT examined thus far has been an acidic protein, the use of DEAE-cellulose absorption is common to all purifications and is an important step. The molarity of sodium chloride required to elute CAT in each instance will be found to roughly parallel the net charge as measured by the apparent isoelectric point (pK~; isoelectric focusing). The most acidic CAT purified thus far (Agrobacterium tume]aciens; pK~ = 3.93) is eluted at 0.21 M ~3 whereas all other species of CAT (pK~ = range of 4.8 to 5.7) have been recovered at lower ionic strengths (sodium chloride; 0.1(}-0.19 M). The procedure described gives a homogeneous CAT product from R factor-containing strains of E. coli and isolates of S. aureus harboring a CM plasmid. The ease with which such results can be achieved is due to the fact that fully induced S. aureus cultures and virtually all R ÷ E. coli cultures synthesize CAT to levels approximating 0.5-1% of the soluble cell protein in stationary phase (see Table I for summary of typical purification). It should be anticipated that other genera or species which synthesize CAT may do so at lower rates and that additional purification steps may be required to obtain a pure enzyme preparation. A useful maneuver in such instances is a second DEAE-eellulose step (after gel filtration) employing a different pH and salt gradient (for example, zero to 0.5 M sodium phosphate at pH 7.0).
Typical Purification o] CAT ]rom E. coli Growth o] Bacteria. A prototrophic strain of E. coli carrying an R factor for CM resistance (or a P1 CM lysogen of such a strain) is grown to the stationary phase of growth at 37 ° with vigorous shaking (or alternative means of aeration) in a well buffered nonrepressing growth mediam. The latter is conveniently and inexpensively provided by a medium containing per liter: K2HPO4, 14 g; KHP04, 6 g; (NH~).,SO~, 2 g; MgS(L, 0.2 g; easamino acids (Difco), 1 g; and glycerol, 5 g. A heavyduty orbital shaker platform capable of taking twelve 2-liter Erlenmeyer flasks will yield approximately 35-40 g (wet weight) of cells from 14 liters if each flask contains only 1200 ml of the above medium to ensure adequate aeration. The cells are harvested with a continuous flow centrifuge (Sharpies Supercentrifuge or Sorvall continuous-flow attachment for 1~L. C. Sands and W. V. Shaw, unpublished studies.
748
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
the RC-2B centrifuge) and suspended in a final volume of 200 ml of 50 mM Tris.HC1 (pH 7.8) containing 2-mercaptoethanol at a concentration of 50 ttM (TM buffer). Step 1. Crude Extract. The cells are broken by sonication or by extrusion in a French pressure cell (Aminco). If the latter method is used a few crystals of deoxyribonuclease should be added to reduce the viscosity of the suspension prior to centrifugation. The cell debris is then removed by centrifugation (30,000 g for 20 min), and the supernatant is taken for purification. Such crude preparations of CAT are stable for months at --20 ° . All subsequent steps are carried out at 0-4 ° . Step 2. Streptomycin Sul]ate Precipitation. Solid streptomycin sulfate is dissolved in the crude extract at a final concentration of 1%. The precipitate obtained after 30 min is collected by centrifugation and discarded. After this step, to remove the bulk of nucleic acids the concentration of pH 7.8 Tris. HC1 buffer is increased to 100 mM prior to precipitation of CAT with ammonium sulfate. Step 3. First Ammonium Sul]ate Step. CAT is precipitated from the buffered supernatant of the streptomycin step by the addition of finely ground ammonium sulfate (enzyme grade) to a final concentration equivalent to 50% saturation. 14 The ammonium sulfate is added slowly with stirring and the preparation is allowed to stand for 30 min in an ice bath before centrifugation at 30,000 g for 20 rain. The supernatant is decanted carefully and put aside until it has been determined that it contains less than approximately 10% of the total activity in the precipitate after the latter is dissolved in 50 ml of TM buffer containing 0.2 mM chloramphenicol (TCM buffer). In the event that CAT has not been precipitated effectively, additional ammonium sulfate should be added to the supernatant to achieve 55% saturation and the centrifugation repeated. It is imperative that the purification proceed to the heat step directly and especially that freezing of ammonium sulfate containing solutions of CAT be avoided. Step 4. Heat Step. All naturally occurring variants of R-factor CAT have been found to be sufficiently thermostable to permit the use of a heat step in the purification. 5,1~ After the precipitate from the ammonium sulfate step is dissolved in TCM buffer the enzyme preparation is allowed to reach room temperature and is then placed in a heated water bath set at 60% The extract is stirred slowly for 10 min, cooled, and centrifuged, and the precipitate is discarded. Step 5. Second Ammonium Sul]ate Step. The supernatant fluid from the heat step is brought to 50% saturation with ammonium sulfate, and ~4A. A. Green and W. L. Hughes, this series, Vol. 1, p. 67. 15W. V. Shaw and R. F. Brodsky, J. Bacteriol. 95, 28 (1968).
[571
CHLORAMPHENICOL ACETYLTRANSFERASE
749
the CAT-containing precipitate is collected as in the earlier precipitation step. The precipitate is dissolved in a small volume (15-25 ml) of TCM buffer and either (a) dialyzed against the latter to remove residual ammonium sulfate, or (b) desalted on a Sephadex G-25 column of suitable size which has been equilibrated with TCM buffer. In practice the gel filtration method is more convenient since it permits completion of all steps up to and including the loading of the DEAE column in a normal working day. Step 6. DEAE-Cellulose Chromatography. The usual precautions are taken to ensure that the chromatography medium contains particles of near uniform size and that the column is well packed and completely equilibrated with the TCM buffer. Microgranular (DE-52) Whatman DEAE-cellulose (dry or prewetted) should be satisfactory in all respects, so long as the manufacturer's preeyeling instructions are followed. A packed column bed of dimensions 2.5 X 40 em should be adequate for a preparation of the size described above and can be developed with a l-liter gradient of SaC1 (0-0.4 M) in TCM buffer after the desalted sample has been applied to the top of the eohmm and the latter washed with several column volumes of TCM buffer. CAT activity should be eluted iust before the midpoint of the NaC1 gradient (0.15-0.20 M in most instanees~ and can be easily detected by the speetrophotometrie (DTNB) method. The strategy for pooling the CAT containing tubes will depend on the goal of the overall purification. Since the enzyme is stable at 0-4 ° for at least 24 hr there is much to be said for determining the CATspecific activity for each tube in the peak and for surveying the number and types of contaminants by polyaerylamide electrophoresis before pooling tubes. When the DEAE step is to be followed by gel filtration, ttw pooled peak tubes can be concentrated by membrane ultrafiltration (Amicon or equivalent types) to a volmne suitable for the column step to follow. Step 7. Gel Filtration. In principle, any gel filtration support which will include CAT (molecular weight: 80,000t should be useful for size separations. In practice, Sephadex G-100 proved satisfactory and has been the only method used. A volume (approximately 5-10 roll of concentrated enzyme solution from the prior step is applied to the bottom of such a column (2.5)< 100 era) equilibrated with TCM buffer containing 0.2 M NaC1 and the elution is continued in upward fashion using th~ same buffer. As in the prior step the peak tubes are pooled judiciously on the basis of specific activity data and/or electrophoretic purity and concentrated if required. The activity of CAT in the elution buffer is stable at --20 ° for at least one year. Step 8. Additional or Alternate Steps. Although in most instances the
750
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
TABLE I PURIFICATION OF CHLORAMPHENICOL ACETYLTRANSFERASEa
Step 1. 2. 3. 4. 5. 6.
Crude extract Streptomycin sulfate First a m m o n i u m sulfate H e a t (60 °) Second a m m o n i u m sulfate DEAE-cellulose chromatography 7. Gel filtration (Sephadex G-100)
Protein (rag total)
Enzyme activity (units total)
Specific activity (units/ rag)
Purification (fold)
Yield (%)
5400 4150 2800 900 640 51
4900 4650 4400 4150 4050 2150
0.9 1.1 1.6 4.6 6.3 42 1
(1) 1.2 1.8 5.1 7.0 47
(100) 95 90 85 83 44
14
1750
125
139
36
CAT obtained from the peak tubes of G-100 column (Step 7) was homogeneous by disc gel electrophoresis.
above procedures will yield electrophoretically homogeneous CAT, it m a y be necessary to carry out an additional step. In the past this has taken the form of either a batchwise adsorption procedure with Alumina C~ gel immediately following the heat step 15 or a terminal step involving a second D E A E column run at a different pH a n d / o r with a different buffer system than that used in step 6. The arguments in favor of the alumina C~ gel step are that it is relatively straightforward, quite effective, and does not preclude the second D E A E procedure if the latter is required. Conversely, since homogeneous CAT can often be obtained without the alumina C~ gel step, it is convenient to "wait and see" before adding an additional procedure. A totally different strategy from the conventional one described above should be mentioned, namely, that of affinity chromatography. 16 Initial experiments with CM-substituted agarose as a specific adsorbant for CAT have been encouraging as regards specificity, but the elution behavior of the enzyme has been unsatisfactory in some respects. The approach employed has been to attach the free amine of CM (where R2 = H in Fig. 1) to a solid support via the formation of an amide bond with the free carboxyl group of CH-Sepharose (Pharmacia) using a water-soluble carbodiimide reagent. The rationale for the use of CM base as ligand is that the nature of the N-acyl (R2) substituent of the CM skeleton is a less critical determinant of substrate affinity for CAT than the 1,3~ P. Cuatrecasas and C. B. Anfinsen, this series, Vol. 22, p. 345.
[57]
CHLORAMPHENICOL ACETYLTRANSFERASE
751
propanediol side chain or p-phenyl substituent. ~7 Current studies are aimed at avoiding the extremes of ionic strength, and CM concentration which are both required to quantitatively elute CAT from the adsorbant described23
Purification o] CAT ]rom Other Bacterial Species Staphylococcus sp. Since staphylococci are more fastidious in their growth requirements and CAT does not appear to be repressed by glucose in a representative collection of strains examined,13 the preferred growth medium is commercially available complex media such as Penassay Broth (Difco). All wild-type isolates of staphylococci harbor the genes for an inducible CAT is and, hence, careful attention to the details of induction (derepression) is necessary for optimum yields of enzyme. Conditions for "gratuitous" induction have been described previously 19 for the 3deoxy analog of CM (where R3 = H), but since this compound is neither available commercially nor readily synthesized, an alternative procedure will be described. A CM-resistant isolate of Staphylococcus sp. is tested for CM resistance on nutrient agar containing 50 t~g of CM per milliliter, and a single colony is picked for overnight growth in Penassay (PA) broth containing the same concentration of CM (PA-CM). Growth on a preparative scale is begun in PA-CM medium with an inoculum from the overnight starter culture equal to approximately 5% of the total volume of the large-scale culture, and the process is repeated to yield a suitable volume of inoculum (e.g., 800 ml) for a total final preparative volmne of 14 liters. By analogy with the earlier protocol for E. coli, this can be accomplished by inoculating each of 12 Erlenmeyer flasks (nominal volume of 2 liters) containing 1200 ml of PA-CM with approximately 60 ml of starter culture. Growth is then allowed to proceed at 37 ° on a rotary shaker. After each doubling of the cell mass (as approximated by turbidity measurements) fresh sterile CM is added to bring the final concentration to 50 ~g/ml. When the cultures have reached the final stationary phase of growth a final addition of CM is made and the cells are allowed to shake for an additional period of 20-30 rain to ensure maximum induction before harvest. Cells are best collected by centrifugation in large (polypropylene or stainless steel) bottles to avoid the hazardous aerosol obtained with use of an "open" centrifuge such as the conventional Sharples instrument. The yield of cells should be of the order of 40-50 g (wet weight). 1TW. V. Shaw and R. F. Brodsky, Antimicrob. Ag. Chemother. 1967, 257 (1968). ~SL. C. Sands and W. V. Shaw, Antimicrob. Ag. Chemother. 3, 299 (1973). 19E. Winshell and W. V. Shaw, J. Bacteriol. 98, 1248 (1969).
752
ANTIBIOTIC INACTIVATION AND MODIFICATION
[57]
Cell lysis is accomplished by the use of Lysostaphin (Schwarz-Mann) as follows. 19 The pooled cell pellets are washed by suspension in 1 liter of ice cold Tris-saline (TS) buffer (50 mM Tris.HC1, pH 7.5, and 145 mM sodium chloride) and collected by centrifugation. The cells are resuspended in approximately 1 liter of TS buffer containing Lysostaphin (10 units/ml) and deoxyribonuclease (50 ~g/ml) and incubated at 37 ° with agitation using a magnetic stirrer. Cell lysis is nlonitored by observing the decrease in turbidity at 660 nm of diluted aliquots of the suspension and is usually complete after 1 hr. The supernatant fluid (crude extract) obtained after centrifugation (10,000 g for 20 min) to remove cell debris should contain approximately 0.2 unit of CAT per milligram of soluble protein. The purification of staphylococcal CAT can be achieved by a straightforward 4-step procedure which omits the preliminary streptomycin sulfate precipitation and the second ammonium sulfate step of the E. coli protocol. Since the overall purification of CAT from staphylococci has been described in detail elsewhere 1~,19 and is similar to that outlined for the R factor enzyme (see above), only the ammonium sulfate precipitation step will be described here. The crude extract obtained after treatment with Lysostaphin is buffered to 100 mM with pH 7.8 Tris.HC1 and made 50 mM for 2-mercaptoethanol prior to the addition of sufficient finely ground ammonium sulfate to achieve 70% of saturation. For most variants of staphylococcal CAT, the supernatant after centrifugation of the precipitate obtained should contain more than 90% of the total enzyme activity present in the crude extract. The supernatant is then brought up to a theoretical 90% saturation with further addition of ammonium sulfate, and the precipitate is collected by centrifugation and dissolved in TCM buffer for desalting and DEAE chromatography, as described for the E. coli variants of CAT.
Properties of Chloramphenicol Acetyltransferase General Comments. Although there is no single type of CAT which
can be chosen as representative, the variants studied to date possess the following properties: pH optimum of 7.8; native molecular weight of 80,000 (and quaternary structure, of four identical subunits of 20,000 each) ; and apparent isoelectric point between 5.4 and 4.0. All CAT variants therefore behave on polyacrylamide gel electrophoresis as typical globular acidic proteins of varying net charge and a corresponding broad range of electrophoretic mobilities when examined at alkaline pH. Each variant is specific for the D-threo stereoisomer of chloramphenicol (or con-
[57l
CHLORAMPHENICOL ACETYLTRANSFERASE
753
geners) as the acyl acceptor and a marked preference for acetyl-S-CoA as the aeyl donor as compared with other acyl-S-CoA homologs. Most of the important differences observed to date between the enzymes themselves (as opposed to their mode of synthesis and its control) have been detected by kinetic or immunological means. The most easily tabulated properties are summarized in Table II. Specificity for Acyl Aeceptor. A large number of analogs and isomers of C3I have been screened for their ability to serve as substrates for O-acylation by acetyl-S-CoA. ',~5,~,~'~,~° All compounds resulting from changes in the configuration or carbon skeleton of the substituted 1,3propanediol side chain are virtually inactive as substrates for CAT. The D-erythro isomer of C3I and the pair of L isomers fall into this category as do the analogs of D-threo-CM with R~ substitutions which lack a free primary hydroxyl group or those in which the carbon skeleton is extended beyond that of the preferred 1,3-propanediol. The "3-methyl analog" of CM which preserves the 1,3-diol configuration, but introduces the complication of a secondary 3-hydroxyl substituent is illustrative of compounds of the latter class. The effect of variations in the substituent at the 2-amino position (R2 in Fig. 1) on acyl acceptor activity has been studied in some detail for the CAT from both R + E. eoli and from S. aureus. In general, the free amine of CM (R~ = H) is virtually inactive whereas any N-acyl substitution leads to measurable activity. Replacement of the nitro group at the para position (R~ in Fig. 1) of the 1-phenyl substituent with a variety of functional groups has led to analogs with a wide range of acyl acceptor activity. ~ SpeeiI%itg ]or the Aeyl Donor. Although no systematic study of donors in the acylation of CM by CAT has been made, a few compounds have been screened for activity. Whereas the propionyl and butyryl thioesters of CoA are less active than acetyl-S-CoA, the acidic acyl thioesters such as succinyl-S-CoA and malonyl-S-CoA do not serve as acyl donors. Taking all the available data into account the requisite structure for the best acyl donor is that of acetyl-S-CoA. The importance of the presence of the complete structure of CoA is apparent both from the inactivity of acetyl-S-dephospho-CoA, acetyl-S-pantetheine, and the acetyl derivative of acyl carrier protein and from the observation that adenine imeleotides are inhibitors of the CAT reaction and are competitive with respect to acetyl-S-CoA. 2~ The apparent Km for acetyl-S-CoA from an average of measurements carried out on several independently isolated vari'ults of R factor CAT is approximately 0.05 raM. ':~ '-'°W. V. Shaw, D. W. Bentley, and L. Sands, J. B(zcteHol, 104, 10% (1970). '~ N. (~arber and J. Zipser, Biochim. Biopt~,ys. Actc~ 220, 343 (1970).
754
[57]
ANTIBIOTIC INACTIVATION AND MODIFICATION
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[58]
CLINDAMYCIN PHOSPHOTRANSFERASE
755
Activators and Inhibitors. CAT does not require any known cofactors. It is not inactivated by EDTA. The most important inhibitors are those known to be specific for essential thiol groups. Iodoacetate, p-mercuribenzoate, and N-ethylmaleimide inhibit CAT irrespective of source, 2° and at least one variant of R-factor CAT is uniquely susceptible to inactivation by DTNB. 2~ Stability. Preparations of purified CAT have been stored at --20 ° for more than two years with less than 20% loss of activity. The considerable stability of the enzyme has been an asset in purification and in attempts to obtain crystals suitable for X-ray diffraction. Crystals grown at room temperature in 25% saturated solutions of ammonium sulfate (pH 7) containing dithiothreitol (0.5 raM) and chloramphenicol (0.1 raM) were dissolved and found to yield at least 60% of the initial activity after 4 weeks? 3 Reversible Denaturation and Hybridization o/Subunits. Native tetrameric CAT can be recovered after dialysis of preparations treated with 6 M guanidinium HC1 under reducing conditions. Any two homologous variants (a and fl) of CAT will hybridize with one another to yield a heteromeric species of the general structure a~fl2, but no evidence has been obtained for the asymmetric ~fl:~ and ~3fl hybrids. ~'ls The S. aureus and R-factor variants are sufficiently different in structure to preclude detectable hybrid formation./3 :2 T. J. Foster and W. V. Shaw, Antimicrob. Ag. Chemother. 3, 99 (1973).
[58] Clindamycin Phosphotransferase By JOHN H. COATS CH5
J
/N~, 5'~C3H7 ~ '
CH5
CH3
[a H--C--Cl 17
p H--C--Cl
/N~, + ATe
.o o SCH5 OH CLINDAMYCIN
CH5
I
=, 5'~c5H 7 "~2'
[7
+ ADP
.o o CH5 OPOsH2
~"
CLINDAMYCIN 3-PHOSPHATE
Several species of streptomycetes have been found to phosphorylate clindamycin, the semisynthetic antibiotic produced by chlorination of
[58]
CLINDAMYCIN PHOSPHOTRANSFERASE
755
Activators and Inhibitors. CAT does not require any known cofactors. It is not inactivated by EDTA. The most important inhibitors are those known to be specific for essential thiol groups. Iodoacetate, p-mercuribenzoate, and N-ethylmaleimide inhibit CAT irrespective of source, 2° and at least one variant of R-factor CAT is uniquely susceptible to inactivation by DTNB. 2~ Stability. Preparations of purified CAT have been stored at --20 ° for more than two years with less than 20% loss of activity. The considerable stability of the enzyme has been an asset in purification and in attempts to obtain crystals suitable for X-ray diffraction. Crystals grown at room temperature in 25% saturated solutions of ammonium sulfate (pH 7) containing dithiothreitol (0.5 raM) and chloramphenicol (0.1 raM) were dissolved and found to yield at least 60% of the initial activity after 4 weeks? 3 Reversible Denaturation and Hybridization o/Subunits. Native tetrameric CAT can be recovered after dialysis of preparations treated with 6 M guanidinium HC1 under reducing conditions. Any two homologous variants (a and fl) of CAT will hybridize with one another to yield a heteromeric species of the general structure a~fl2, but no evidence has been obtained for the asymmetric ~fl:~ and ~3fl hybrids. ~'ls The S. aureus and R-factor variants are sufficiently different in structure to preclude detectable hybrid formation./3 :2 T. J. Foster and W. V. Shaw, Antimicrob. Ag. Chemother. 3, 99 (1973).
[58] Clindamycin Phosphotransferase By JOHN H. COATS CH5
J
/N~, 5'~C3H7 ~ '
CH5
CH3
[a H--C--Cl 17
p H--C--Cl
/N~, + ATe
.o o SCH5 OH CLINDAMYCIN
CH5
I
=, 5'~c5H 7 "~2'
[7
+ ADP
.o o CH5 OPOsH2
~"
CLINDAMYCIN 3-PHOSPHATE
Several species of streptomycetes have been found to phosphorylate clindamycin, the semisynthetic antibiotic produced by chlorination of
756
ANTIBIOTIC INACTIVATION AND MODIFICATION
[58]
lincomycin.1 Through studies carried out with whole cells, lysates, and partially purified enzyme preparations of Streptomyces coelicolor Miiller the product of the phosphorylation reaction has been identified as clindamycin 3-phosphate. ~ The reaction requires a ribonucleoside triphosphate and Mg 2+.
Assay Method Principle. The product of the clindamycin phosphotransferase reaction, clindamycin 3-phosphate, lacks in vitro antibacterial activity. Its formation from clindamycin can be followed by measuring loss of antibiotic activity. To determine the amounts of clindamycin in reaction mixtures, a standard assay is employed with Sarcina lutea ATCC 9341 as the assay organism. Samples to be assayed are applied to replicate 6.5-mm paper discs and placed on antibiotic medium No. 1 agar seeded with 10~ cells of S. lutea per m~_lliliter. Standard solutions of clindamycin.hydrochloride are plated similarly. The dose levels and responses for the standard solutions are analyzed by least squares regression. The resulting estimates of the parameters of the regression line are used to compute the potency of the unknown samples. ~ Reagents Glycylglycine buffer, 0.1 M, pH 8.0 MgC12, 0.2 M ATP, 0.1 M, pH 8.0 Clindamycin.HCl, 0.27 mM Enzyme, diluted in 20 mM potassium phosphate buffer, pH 7.5 Procedure. The complete reaction mixture in a final volume of 1.0 ml consists of 0.1 ml of glycylglycine buffer, 0.1 ml of MgC12, 0.1 ml of ATP, 0.2 ml of clindamycin.HCl, and 0.5 ml of enzyme. The mixture is incubated for 30 min at 30 °. Reactions are terminated by heating at 85 ° for 5 min. The disappearance of bioactivity is measured by the assay procedure noted above. Under these conditions the rate of reaction is proportional to the concentration of enzyme and linear with time through the 30-rain incubation period. Definition of Unit and Specific Activity. One unit of enzyme is defined as that amount that transforms 1 ~mole of clindamycin.HC1 per minute 1B. J. Magerlein, R. D. Birkenmeyer, and F. Kagan. Antimicrob. Ag. Chemother. p. 727 (1966). J. H. Coats and A. D. Argoudelis.J. Bacteriol. 108, 459 (1971). 8T. :F. Brodasky and W. L. Lummis, Antimicrob. Ag. Chemother. p. 18 (1966).
[58]
CLINDAMYCIN PHOSPHOTRANSFERASE
757
under the assay conditions described. The specific activity is expressed as units per milligram of protein. The method of Lowry et al2 is used to determine protein concentration. Purification Procedure
Step 1. Growth oJ Culture and Preparation of Crude Extract. A seed culture of Streptomyces coelicolor Miiller UC 5240 (NRRL 3532) is prepared in a medium consisting of 25 g of glucose monohydrate per liter and 25 g of cotton seed hydrolysate per liter (Pharmamedia, Traders Oil Mill Co., Fort Worth, Texas). The culture (100 ml of medium in a 500-ml wide-mouth Erlenmeyer flask) is incubated at 28 ° for 72 hr on a rotary shaker (250 rpm, 6-cm stroke). A growth medium is prepared as follows: glucose monohydrate (20 g/liter), yeast extract (2.5 g/liter), NZ-amine B (Sheffield Chemical, Norwich, New York; 5 g/liter), sodium nitrate (1.5 g/liter), ammonium sulfate (1.5 g/liter), calcium carbonate (5.0 g/liter), dipotassium phosphate (1.0 g/liter), potassium chloride (0.5 g/liter), magnesium sulfate (0.5 g/liter), and ferrous sulfate (0.01 g/liter). The medium is adjusted to pH 7.2 before autoclaving; 100 ml aliquots of the medium are dispensed into 500 ml wide-mouth Erlenmeyer flasks. The growth medium is inoculated with 5% (v/v) of the 72-hr seed culture and the cultures incubated for 24 hr at 28 ° on a rotary shaker. Cells are harvested by eentrifugation and washed twice in Cold Spizizen's mineral salts medium2 The washed cells are lysed following the procedure of Hey and Elbein. * The cells are resuspended in 3 volumes of 0.1 M potassium phosphate buffer, pH 7.5, containing egg white lysozyme( Sigma Chemical Co., St. Louis, Missouri) (1 mg/Inl) and EDTA (0.5 mg/ ml) and incubated without stirring at 25 ° for 1 hr. The suspension of lysed cells is centrifuged at 20,000 g for 15 nfin at 0 °. Step 2. Removal o] Nucleoproteins. A 4~ml amount of 1 M MnCI~ is added to each 100 ml of crude lysate. The resulting mixture is kept at 0 ° for 5 rain with occasional stirring. The mixture is then centrifuged at 20,000 g for 15 min, and the precipitate is discarded. Step 3. Ammonium Sul]ate Fractionation. Solid ammonium sulfate is slowly added to the supernatant solution, maintained at 0% to give 30% saturation. The mixture is allowed to stand at 0 ° for 5 rain, after which it is centrifuged at 20,000 g for 20 rain. Additional ammonium sulfate is added to the supernatant fluid to give 60% saturation, and the mixture O. It. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall. J. Biol. Chem. 193, 265 (1951). s j. Spizizen. Proc. Nat. Acad. Sci. U.S. 44, 1072 (1958). 6 A. E. Hey and A. D. Elbein. J. Bacteriol. 96, 105 (1968).
758
ANTIBIOTIC INACTIVATION AND MODIFICATION
[58]
is centrifuged. The precipitate is dissolved in 20 mM potassium phosphate buffer, pH 7.5. Step 4. DEAE-Cellulose Column Chromatography. Diethylaminoethyl cellulose (Cellex, type D; Bio-Rad Laboratories, Richmond, California) is washed sequentially with 0.5 N HC1, distilled water, 0.5 N Na0H, and 20 mM KP04 buffer pH 7.5, after which a column (20 by 127 ram) is prepared. A 200-ml portion of crude lysate is treated with manganese chloride and ammonium sulfate as described above, and the precipitate from the 60% ammonium sulfate treatment is suspended in a final volume of 10 ml. A 7-ml amount of this fraction is applied to the head of the column and washed in with 75 ml of 20 mM KPO, buffer, pH 7.5. Buffers (75 ml each) containing KC1 in 50 mM increments ranging from 0.05 to 0.3 M are added to the column, and 5-ml fractions are collected. The column fractions are assayed for clindamycin phosphotransferase in the reaction mixture described above, but with one-fifth the given amount of clindamycin.HC1 in each 1-ml reaction volume. The reaction tubes are sampled after 5 hr of incubation in a water bath at 30 ° . Samples are tested by disc-plate assay for inhibition of the growth of S. lutea. Tubes containing clindamycin phosphotransferase are pooled (volume, 30 ml), and the protein is precipitated by adding ammonium sulfate to give 60% saturation. The precipitate is suspended in 5 ml of 20 mM potassium phosphate buffer (pH 7.5) for assay. A summary of the purification procedure is shown in the table.
Properties Specificity. Clindamycin phosphotransferase catalyzes the phosphorylation of lincosaminides and related celestosaminidesJ Partially purified enzyme preparations from S. coelicolor Miiller also inactivate streptomycin and several macrolide antibiotics in the presence of ATP and Mg 2÷ ions. These latter activities, however, can either be separated from clindamycin phosphotransferase by DEAE-cellulose chromatography or differentiated by stability to dialysis at pH 6.0. The phosphorylation of clindamycin proceeds with CTP, GTP, UTP, and ADP as well as ATP, however, the rate observed with ATP is much more rapid than that obtained with the other nucleotides. Stability. The enzyme is stable to freeze-drying at the crude lysate stage and can be stored dry for at least 1 year at 0 ° without significant loss of activity. After ammonium sulfate fractionation and DEAE-cellulose chromatography the enzyme is stable to freezing and thawing and can be stored above liquid nitrogen without loss of activity. The enzyme A. D. Argoudelis and T. F. Brodasky, J. Antibiot. 25, 194 (1972).
[591
TOYOCAMYCIN NITRILE HYDROLASE
759
PURIFICATION OF CLINDAMYCIN PHOSPHOTRANSFERASE
Volume Protein (ml) (mg/ml)
Enzyme fraction Crude lysate Manganous chloride -F ammonium sulfate DEAE-cellulose
200 10
6.5 31
5
0.18
Total activity (units X 10-3)
Specific activity Fold (X 10-3) purified
180 114
0.13 0.36
6.2
6.90
1 2.7
Yield (%) 100 63
53
3.4
maintains activity at 25 ° and 30 ° for at least 1 hr. I t displays greater initial activity at 37 ° than at 30 ° but it is inactivated readily at t h a t temperature. Stoichiometry. Although clindamycin A T P stoichiometry would be expected to be mole for mole in the reaction, optimum phosphorylation is obtained with a large excess of A T P over clindamycin. This m a y be explained by the fact t h a t the enzyme preparation still has marked adenosine triphosphatase activity after a m m o n i u m sulfate fractionation.
[59] T o y o c a m y c i n
Nitrile
Hydrolase
By T. UEMATSU and R. J. SUHADOLNIK NHz N/, ~ " ~ -
CN
NH2 N~"'~
I
C-NH, I
Ribo~e
TOYOCAMYCIN
0
Rlbose
SANGIVAMYCIN
Principle. The assay measures the conversion of toyocamycin to sangivamycin in the presence of t o y o c a m y c i n nitrile hydrolase. 1
Assay Reagents. Reaction mixture, 0.1 ml contains: Potassium phosphate buffer, 0.1 M, p H 6.5 [ G - 3 H ] T o y o c a m y c i n , 0.32 mM, 4 Ci/mole T o y o c a m y c i n nitrile hydrolase, 5.7 units 1T. Uematsu and R. J. Suhadolnik, Arch. Biochem. Biophys. 162, 614 (1974).
[591
TOYOCAMYCIN NITRILE HYDROLASE
759
PURIFICATION OF CLINDAMYCIN PHOSPHOTRANSFERASE
Volume Protein (ml) (mg/ml)
Enzyme fraction Crude lysate Manganous chloride -F ammonium sulfate DEAE-cellulose
200 10
6.5 31
5
0.18
Total activity (units X 10-3)
Specific activity Fold (X 10-3) purified
180 114
0.13 0.36
6.2
6.90
1 2.7
Yield (%) 100 63
53
3.4
maintains activity at 25 ° and 30 ° for at least 1 hr. I t displays greater initial activity at 37 ° than at 30 ° but it is inactivated readily at t h a t temperature. Stoichiometry. Although clindamycin A T P stoichiometry would be expected to be mole for mole in the reaction, optimum phosphorylation is obtained with a large excess of A T P over clindamycin. This m a y be explained by the fact t h a t the enzyme preparation still has marked adenosine triphosphatase activity after a m m o n i u m sulfate fractionation.
[59] T o y o c a m y c i n
Nitrile
Hydrolase
By T. UEMATSU and R. J. SUHADOLNIK NHz N/, ~ " ~ -
CN
NH2 N~"'~
I
C-NH, I
Ribo~e
TOYOCAMYCIN
0
Rlbose
SANGIVAMYCIN
Principle. The assay measures the conversion of toyocamycin to sangivamycin in the presence of t o y o c a m y c i n nitrile hydrolase. 1
Assay Reagents. Reaction mixture, 0.1 ml contains: Potassium phosphate buffer, 0.1 M, p H 6.5 [ G - 3 H ] T o y o c a m y c i n , 0.32 mM, 4 Ci/mole T o y o c a m y c i n nitrile hydrolase, 5.7 units 1T. Uematsu and R. J. Suhadolnik, Arch. Biochem. Biophys. 162, 614 (1974).
760
ANTIBIOTIC INACTIVATION AND MODIFICATION
[59]
Procedure. The reaction mixture is incubated at 30 ° for 30 min. Control assays are performed with boiled enzyme. The reactions are terminated by the addition of 0.1 ml ethanol-acetic acid (9:1, v/v) followed by heating at 90 ° in a steam bath for 10 min. The protein is removed by centrifugation. Carrier sangivamycin (30 ~g) is added to the supernatant. The mixture is spotted on a Whatman No. 3 MM paper and developed with water. The area corresponding to sangivamycin (RI 0.80) is cut out and eluted with 0.1 N HC1; an aliquot is counted by liquid scintillation counting. Definition o] Unit and Specific Activity. A unit of enzyme is defined as the formation of 1 nmole of sangivamycin per 30 min at 30 ° in 0.1 ml of incubation mixture. Specific activity is expressed as units per milligram of protein. Protein is determined by the method of Murphy and Kies. 2
Purification Procedure
Step 1. Preparation o] Cell-Free Extract. All purification steps are carried out at 0-4% The cells (Streptomyces rimosus ATCC 14673) are harvested by centrifugation at 4 °, 48 hr after inoculation. The cells then are washed twice with 0.15 M KC1, centrifuged, lyophilized, and stored at --20% The dried cells (5 g), obtained from 600 ml of medium, are suspended in 100 ml of 0.10 M phosphate buffer (pH 7.4) and disrupted with a French press (twice at 16,000 psi). The homogenate is suspended in 50 ml of buffer, stirred for 10 rain, and centrifuged. The sediment is washed with the buffer. The supernatant and washings are combined and dialyzed overnight with 2 liters of 1 mM EDTA (pH 7.4) ; volume 97 ml, 1014 mg protein. Step 2. Ammonium Sul]ate Fractionation. The crude extract (97 ml, 1014 mg protein) is brought to 30% saturation by the :gradual addition of solid ammonium sulfate over a 30-min period with stirring. The precipitate is removed by centrifugation (35,000 g, 10 min). Ammonium sulfate is added to the supernatant to 55% saturation over a 10-rain period, and centrifuged for 10 rain (15,000 g). The precipitate is dissolved in 10 ml of water, and dialyzed overnight with 2 liters of 1 mM EDTA (pH 7.4) ; volume 15 ml, 201 mg protein. Step 3. Protamine Sul]ate Treatment. A 1% protamine sulfate solution (pH 6.8) is added to 15 ml (201 mg protein) until the A2so/A26o of the supernatant becomes 0.90. The solution is centrifuged at 20,000 g, for 10 min. The pellet is suspended in 5 ml of 0.5 M phosphate buffer (pH 6.5). After stirring for 2 hr, the suspension is centrifuged at 20,000 g 2j. B. Murphy and M. W. Kies, Biochim. Biophys. Acta 45, 382 (1960).
[59]
TOYOCAMYCIN
NITRILE
HYDROLASE
©
Z
©
~v
Z
.o
©
761
762
ANTIBIOTIC INACTIVATION AND MODIFICATION
[59]
for 10 min. The pellet is discarded and the supernatant is dialyzed against 3 liters of 1 mM EDTA, 3.5 hr; volume 8.5 ml, 28 mg protein. Step 4. Hydroxyapatite Adsorption. The supernatant (8.5 ml; 28 mg protein) is passed through a hydroxyapatite column (10 ml). The elution of protein is carried out stepwise with 10 ml of 0.05, 0.08, 0.10, 0.12, 0.16, and 0.20 M phosphate buffer (pH 6.8). The hydrolase is eluted with 0.12 M buffer; volume 13.5 ml, 2.2 mg protein. The overall purification is shown in the table.
Properties Specificity. The purified enzyme would only use toyocamycin as the substrate.
Coenzyme, Metal Requirements, and Activators. The enzyme does not require coenzyme, metal ions, nor activators for activity. pH Optimum. The enzyme has an optimum pH at 6.5. Nonenzymic conversion of toyocamycin to sangivamycin occurs only when the pH is more alkaline than 10. Ej~ect o] Metal Ions. Zn 2÷, Mg 2+, Cu ~÷, Fe 2+, and Hg 2+ (all at 2.5 mM) inhibit the enzyme (47, 66, 100, 100 and 100%, respectively). Inhibitors. The following compounds (10-60 mM) inhibit the hydrolase: p-hydroxybenzonitrile, tubercidin, ricinine, tubercidin 5'monophosphate, nicotinonitrile, hydroxytoyocamycin, demethylricinine, O,N-didemethylricinine. The percent inhibition is between 81 and 82%. p-Hydroxybenzonitrile and tubercidin are competitive inhibitors of the hydrolase; K~ = 5.7 and 8.0 mM, respectively. Michaelis Constant. The Km for toyocamycin is 0.5 mM. The curves are linear. At 1.7 mM toyocamycin and 5.3 units of protein, product formation is linear for 60 min at 30°; when the protein is varied between 43 and 172 ~g, there is a linear increase in product formation. Ef]ect of Heat. When the enzyme is heated for 10 rain at 40 ° (0.4 mg protein, 0.6 mM [G-3H]toyocamycin), there is a 30% loss of activity; heating for 10 min at 60 ° results in complete loss of enzyme activity.
[60]
ACTINOMYCINLACTONASE
763
[60] Actinomycin Lactonase By D . PERLMAN
I COOH
0
L-PRO D-VAL
L-PRO
"
D-VAL
I
I
L-THR
L-THR
CO
CO
ells
CHs
I
O
r
CH 3
~-
I
CO
CO
CHs
CH s
,
CH~
~-U~O " /
7
Actinomycinic Acid CO
~ CH 3
CO
N~ N H ~ O" "~ "0 CH~
Actinomycinic Monolactone An induced actinomycin lactonase which opens the lactone linkages of actinomycin has been found in cells of Actinoplanes missouriensis. This enzymic activity is present in growing cultures, washed cells, frozen cells, and cell-free preparations of this organism. Crude enzyme preparations convert actinomycin to actinomycinic acid through actinomycin monolactone; the purified enzyme converts actinomyc'n only to actinomycin monolactone. (There are two isomers possible for the actinomycin monolactone formed during this sequence of reactions, and which one is formed enzymically has not yet been determined. 1) 1K. L. Perlman, J. Walker, and D. Perlman, J. Antibiot. 24, 135 (1971).
764
ANTIBIOTIC INACTIVATION AND MODIFICATION
[60]
Assay Methods
Principle. The enzyme catalyzes the conversion of a neutral molecule to an acidic compound. The assay is based upon the use of radioactive actinomycin. Both product and substratc are extracted from the reaction mixture with an organic solvent at an acid pH. The acidic product is then extracted into aqueous alkaline media, which is counted in a liquid scintillation spectrometer.
Reagents Actinomycin-3H obtained from Schwarz BioResearch. Bray's solution2 Ethyl acetate Tris.HC1 buffer, 0.1 M, pH 7.8
Procedure. The standard reaction mixture contains enzyme preparation, 7.8 nmoles of actinomycin-aH, 6 ~moles of Tris.HC1 buffer, and water to a total volume of 0.20 ml. The reaction mixture is incubated for 60 min at 38 ° using a rotary water bath shaker. The reactions are then terminated by addition of 0.8 ml of 0.5 N HCI. Ethyl acetate (4 ml) is then added and the tubes are shaken vigorously to extract the actinomycin and acidic metabolites into the organic phase. Three milliliters of the ethyl acetate extract arc then transferred to another tube containing 1.5 ml of 0.1 M Tris.HC1, pH 7.8, buffer. After thorough mixing, the ethyl acetate layer is discarded, and the radioactivity in the aqueous layer is determined by liquid scintillation counting of 1-2 ml of sample in 8 ml of Bray's solution. Essentially no degradation of actinomycin is noted if the enzyme is omitted or if boiled enzyme is used. One enzyme unit converts 1 ~g of substrate to acidic product in a 60-min period at 38 ° . Purification Procedures The following procedure is that of Hou and Perlman2 Approximately 100-fold purification is achieved by ammonium sulfate precipitation followed by chromatography on calcium phosphate gel-cellulose, DEAE-cellulose, and Sephadex G-200 columns. Organism and Growth Conditions. The culture of A. missouriensis (IMRU 824) may be obtained from the Institute of Microbiology, Rutgers University, New Brunswick, New Jersey 08903. It is maintained on slants of Berger's agar (1% Contadina tomato paste-l% Heinz Baby 2 G. A. Bray, Anal. Biochem. l , 279 (1960). 3 C. T. Hou and D. Perlman, J. Biol. Chem. 245, 1289 (1970).
[60]
ACTINOMYCIN LACTONASE
765
oatmeal-l.5% ~gar) and grown in liquid culture in a 3% soybean meal-3% glycerol medium. Submerged cultures are started by aseptically transferring cells from slants to 250-ml cotton plugged sterile Erlenmeyer flasks containing 100 ml of the soybean meal-glycerol medium. The inoculated flasks are placed on a rotary shaker (280 rpm, 1-inch displacement) in a 30 ° constant-temperature incubator. Second and subsequent generation fermentations are started by transferring 10 ml of the vegetative growth at 4-6 days to new flasks of the sterile soybean meal-glycerol medium. When actinomycin lactonase activity is to be induced, 1 mg of actinomycin (in methanol or ethanol solution) is added per 100 ml of growing culture 48 hr after inoculation of the fermentation. At the end of the incubation period, the cells arc collected by centrifugation (at room temperature if a refrigerated centrifuge is not available), washed with a volume of 50 mM phosphate buffer (pH 7.0) equivalent to that of the growth media, and then collected for a second time by centrifugation. These washed cells can be frozen at this stage or broken in a French press; the enzyme level of the frozen cells does not decrease appreciably when the cells are stored for up to 2 years at --10 °. If the cells are broken in the French pressure Cell, the debris should be removed by centrifugation and the supernatant solution either subjected immediately to ammonium sulfate fractionation, or frozen. Preparation of Crude Extract. Frozen Actinoplanes cells are thawed and suspended in an equal weight of 50 mM phosphate buffer (pH 7.0) at 4 °. The suspension is passed through a French pressure cell and then centrifuged at 10,000 g for 20 rain at 4 °. Freeze-Thaw Step. The supernatant solution is frozen at --20 ° and then thawed and centrifuged at 10,000 g for 20 rain at 4 °. The precipitate is discarded, as it has very little enzymic activity. Ammonium Sulfate Fractionation. Ammonium sulfate is gradually added to the supcrnatant solution from the preceding step to obtain 25% saturation. After 15 min stirring, the protein precipitate is removed by centrifugation at 10,000 g for 15 rain. Additional ammonium sulfate is added to the supernatant solution to bring the concentration to 50% of saturation. After 15 rain of stirring, the precipitate is collected by centrifugation at 10,000 g, and the supernatant liquid is discarded. The pellet, containing the enzyme, is dissolved in a small amount of 0.5 mM phosphate buffer (pH 7.0) and dialyzed overnight against the same buffer. Calcium Phosphate Gel Chromatography. CaIcium phosphate gel is prepared according to the procedure described by Tsuboi and Hudson, 4 and 4 g of the gel is mixed with 60 g of cellulose powder. After equilibra4K. K. Tsuboi and P. B. Hudson, J. Biol. Chem. 224, 879 (1957).
766
ANTIBIOTIC INACTIVATION AND MODIFICATION
[60]
tion with 0.5 mM phosphate buffer (pH 7.0), the gel-cellulose is packed into a 2 X 80 cm glass column at atmospheric pressure. The enzyme solution is applied to the column, and the actinomycin-degrading activity is eluted stepwise with 1 mM followed by 10 mM phosphate buffer (pH 7.0). The bulk of the enzyme activity is found in the first protein peak (eluted with 1 mM phosphate buffer; these fractions are pooled). DEAE-Cellulose Column Chromatography. The enzyme solution is applied to a DEAE-cellulose 2 X 70 cm column (pH 7.0). The enzyme degrading actinomycin is eluted with a linear gradient formed from 400 ml of 20 mM phosphate buffer (pH 7.0) in the mixing bottle and 400 ml of 90 mM buffer in the reservoir. The elution rate is about 70 ml/hr, and 7-ml fractions are collected. The actinomycin-degrading enzyme activity is found in the first protein peak (between 460 and 700 ml of the gradient). The protein peak eluted with higher concentration of phosphate buffer, e.g., 0.1 to 0.2 M, is the enzyme degrading other peptide antibiotic lactones but is not active on actinomycins. The active material in the first protein fraction is precipitated by adding ammonium sulfate to give 50% saturation and is collected by centrifugation. The pellet is dissolved in a minimum of 10 mM phosphate buffer (pH 7.0) and dialyzed overnight against this buffer. The enzyme solution is concentrated further with Lyphogel (Gelman Instrument Company, Ann Arbor, Michigan). Sephadex G-~O0 Column Chromatography. The concentrated solution is applied to a 2 X 80 cm column of Sephadex G-200 (previously equilibrated with 10 mM phosphate buffer, pH 7.0). The enzyme is eluted with 10 mM phosphate buffer (pH 7.0), and active fractions are pooled. The purification of the actinomycin lactonase activity from 4 kg of Actinoplanes cells is summarized in the table. The overall recovery is 14%, and about 90- to 100-fold purification is achieved. PURIFICATION OF ACTINOMYCINLACTONASE
Step Extrusion through French pressure cell Freeze-thaw (NH4)~SO4 precipitation Calcium phosphate gel DEAE-cellulose column Sephadex G-200 column
Specific activity (units/mg)
Volume (ml)
Protein (mg)
Activity units
Yield (%)
6600
11,134
75,711
6.8
100
6300 125 100 43 22
6,247 2,108 623 89 22
64,344 45,322 28,970 22,072 11,198
10.3 21.5 46.5 248 509
85 59 38 29 14
[61]
PEPTIDE ANTIBIOTIC LACTONASE
767
Properties The known properties of the enzyme include: p H optimum for activity on substrate, 7.8; t e m p e r a t u r e optimum. 38°; Kin, 11.4 ~,M; V ..... 0.267 ~mole per hour per milligram of protein. Significant inhibition with 40 m M silver, cobalt, chromium, copper, iron, mercury, magnesium, sodium, nickel, or zinc was observed. This enzyme can be used in assaying crude materials containing actinomycin, e.g. blood samples, body fluids, to provide specific inactivation. The inactivation product is one of the mixture of actinomycin monolactones and also actinomycinic acid formed by alkaline hydrolysis of actinomycin. ,~ D. Perlman, A. B. Mauger, and H. Weissbach, Antimicrob. Ag. Chemother. 1966, 581 (1967).
[61] P e p t i d e
Antibiotic
Lactonase
B y D. PERLMAN
Peptide lactone ~- H20 --~ linear peptide A constitutive peptide antibiotic lactonase which hydrolyzes lactone linkage of echinomycin, 1 etamycin, ~,3 stendomycin,4, 5 dihydrostaphylomycin S and staphylomycin S, 6,7 thiostrepton, s,9 and vernamycin B 1°,11 has been found in cells of A c t i n o p l a n e s missouriensis ( I M R U 824). Structural formulas of these antibiotics are shown. The lactonase which converts the lactone-containing antibiotics to the corresponding linear 1R. Corbaz. L. Ettlinger, E. G~iumann, W. Keller-Schierlein, F. Kradolfer, L. Neipp, V. Prelog, P. Reusser, and H. Z~ihner, Helv. Chim. Acta 40, 199 (1957). B. Heinemann, A. Gourevitch, J. Lein, D. L. Johnson, M. A. Kaplan, D. Vanas, and I. R. Hooper, Antibiot. Annu. 1954/1955, 728 (1955). 3j. C. Sheehan, H. G. Zachau, and W. B. Lawson, J. Amer. Chem. Soc. 80, 3349 (1958). R. Q. Thompson and M. S. Hughes, J. Antibiot. AI6, 187 (1963). 5 M. Bodanszky, J. Izdebski, and I. Muramatsu, J. Amer. Chem. Soc. 91, 2351 (1969). e p. De Sommer and P. Van Dijck, Antibiot. Chemother. 5, 632 (1955). ' H Vanderhaeghe and G. Parmentier, J. Amer. Chem. Soc. 82, 4414 (1960). 8j. F. Pagano, M. J. Weinstein, H. A. Stout, and R. Donovick, Antibiot. Annu. 1955/1956, 554 (1956). 9B. Anderson, D. C. Hodgkin, and M. A. Viswamitra, Nature (London) 225, 233 (1970). ~oR. Donovick, J. D. Dutcher, L. J. Heuser, and J. F. Pagano, U.S. Patent 2,990,325 (1961). '~ M. Bodanszky and M. A. Ondetti, Antimievob. Ag. Chemother. 1963, 360 (1964).
[61]
PEPTIDE ANTIBIOTIC LACTONASE
767
Properties The known properties of the enzyme include: p H optimum for activity on substrate, 7.8; t e m p e r a t u r e optimum. 38°; Kin, 11.4 ~,M; V ..... 0.267 ~mole per hour per milligram of protein. Significant inhibition with 40 m M silver, cobalt, chromium, copper, iron, mercury, magnesium, sodium, nickel, or zinc was observed. This enzyme can be used in assaying crude materials containing actinomycin, e.g. blood samples, body fluids, to provide specific inactivation. The inactivation product is one of the mixture of actinomycin monolactones and also actinomycinic acid formed by alkaline hydrolysis of actinomycin. ,~ D. Perlman, A. B. Mauger, and H. Weissbach, Antimicrob. Ag. Chemother. 1966, 581 (1967).
[61] P e p t i d e
Antibiotic
Lactonase
B y D. PERLMAN
Peptide lactone ~- H20 --~ linear peptide A constitutive peptide antibiotic lactonase which hydrolyzes lactone linkage of echinomycin, 1 etamycin, ~,3 stendomycin,4, 5 dihydrostaphylomycin S and staphylomycin S, 6,7 thiostrepton, s,9 and vernamycin B 1°,11 has been found in cells of A c t i n o p l a n e s missouriensis ( I M R U 824). Structural formulas of these antibiotics are shown. The lactonase which converts the lactone-containing antibiotics to the corresponding linear 1R. Corbaz. L. Ettlinger, E. G~iumann, W. Keller-Schierlein, F. Kradolfer, L. Neipp, V. Prelog, P. Reusser, and H. Z~ihner, Helv. Chim. Acta 40, 199 (1957). B. Heinemann, A. Gourevitch, J. Lein, D. L. Johnson, M. A. Kaplan, D. Vanas, and I. R. Hooper, Antibiot. Annu. 1954/1955, 728 (1955). 3j. C. Sheehan, H. G. Zachau, and W. B. Lawson, J. Amer. Chem. Soc. 80, 3349 (1958). R. Q. Thompson and M. S. Hughes, J. Antibiot. AI6, 187 (1963). 5 M. Bodanszky, J. Izdebski, and I. Muramatsu, J. Amer. Chem. Soc. 91, 2351 (1969). e p. De Sommer and P. Van Dijck, Antibiot. Chemother. 5, 632 (1955). ' H Vanderhaeghe and G. Parmentier, J. Amer. Chem. Soc. 82, 4414 (1960). 8j. F. Pagano, M. J. Weinstein, H. A. Stout, and R. Donovick, Antibiot. Annu. 1955/1956, 554 (1956). 9B. Anderson, D. C. Hodgkin, and M. A. Viswamitra, Nature (London) 225, 233 (1970). ~oR. Donovick, J. D. Dutcher, L. J. Heuser, and J. F. Pagano, U.S. Patent 2,990,325 (1961). '~ M. Bodanszky and M. A. Ondetti, Antimievob. Ag. Chemother. 1963, 360 (1964).
768
ANTIBIOTIC
INACTIVATION
(L)
CH3
r//-~r/N~ V
N
0 ))
]CO~D-Ser---L-AIa---N--A-~C~.,C ~ L - N -
O
I
n-N-Me-Val
O
(L)
m-Ala~ D-Ser~OC
I
0
~N-.
Me - V a l
I
C--C--N~
II
[61]
AND MODIFICATION
N"
v
CH s
Echinomycin
HyPic
~L-Thr
, D-Leu
•D-aHyPro
I
O i L-PheSar~
Sir L-AIa-- L-~-N-Di-Me- Leu
HyPic = 3-hydroxypicolinic acid PheSar = ~-phenylsarcosine a H y P r o = allo - h y d r o x y p r o l i n e Etamycin
FA
L-Pro
NMe-L-Thr
GIy-o-Val
D-a/le
D-AIa
A-But
D-aThr
D-Val
L-Val
H2
H'c/C'cH' HocHCH~ H H HsC H N--C I ~ I ~ C CH z C O / I~CON--C C O N H C H 2 C O N H - - C - - C O N H - - C - - C O N H - - C HmC/ 2 6 H q l I HsC--H /C~ CHsCH
.,c.
c.,
H H HsC'~ / CH, I { CONH--C--CONH--C--CONH--C--CONH--C--CONH I H l l { H CH s CH CHsCH :C~ I {
,
I
~H,
..c.c.,
.c-cc., CO
CO--C--NHCO--C--NHCO--~--NH I I I H C C H CH.OH s ~NIH~CHz I I CHsN:C~HN:CH z D-alle L-Ser
Stendomycm
L-Ste
~.~OH
H2 CO--NH C~H 5 H2c/C~cH2 I I I (L)CH--CO--NH--CH--CO--N CH (L) I
I
..c
co
OC--CH--NH--CO--CH--N--CO--CH--CH2---< / I
I
CO,_CH 2
8taphylomycin S *Reduced to OH in dihydrostaphy:omycin
')
.-~rh,
[611
P E P T I D E ANTIBIOTIC LACTONASE
O HN
Thiostreptoic acid C/u"-~ [ ~}q~N
769
Pyruvic acid
N
"S 0 I H II
P y r u v i c acid H
~---N/~ ~ ~O ~a:ine~ ~ N N O I H / ~ /~
Alanine NH / [ Isoleucine
•
S
(+)-Cysteme
N
~ 0--~0 Thiostreptine Thiostrepton
N•OH
H2 CO X HC/C'CH2 I I I I CH3--CH--NH--CO--NH--CH--CO--N - - CH I
1
0 I
/ ~
CO
CO CH--NH--CO-- ICH CH2 I CO --
I
N1--CHs N--CO--CH--C~-~ I H2 ~ 7H2 CH~
~N.
R CH s
V e r n a m y c i n Ba ( o s t r e o g r y c i n B) : X = CH~CH3, R = CH s V e r n a m y c i n BB ( o s t r e o g r y c i n B2): X = CH~CHs, R = H V e r n a m y c i n B~/ ( o s t r e o g r y c i n Bt): X = CHs, R = CH s V e r n a m y c i n B 5: X = CH 3, R = H Vernamycin B
compounds is present in growing cultures, washed cells, frozen cells, and cell-free preparations of this organism. Although the enzyme activity is constitutive in this culture, the level can be doubled by addition of suitable substrate antibiotics to the growing culture. ~-14 iz C. T . H o u , D. P e r l m a n , a n d M . R . Schallock, J. A~.tibiot. 23, 35 (1970). ~ D. P e r l m a n , C. T. H o u , a n d A. C a p e k , Antimicrob. Ag. Chemother. 1968, 64 (1969). J~ D. P e r l m a n , A. B. M a u g e r , a n d H. W e i s s b a c h , A~timicl'ob. Ag. Chemothe~'. 1966, 581 (1967).
770
ANTIBIOTIC INACTIVATION AND MODIFICATION
[61]
Assay Methods Principle. The enzyme catalyzes the conversion of a neutral molecule
to an acidic compound. The assay is based on the use of radioactive substrafe or nonlabeled material. If the former is involved, both substrate and product are extracted from the reaction mixture with an organic solvent at an acid pH. The acidic product is then extracted into aqueous alkaline media which is counted in a liquid scintillation spectrometer. If nonlabeled substrate is used, degradation is followed by bioassay of the solvent layer. Reagents and Substrates. [SH]Dihydrostaphylomycin S can be prepared by reduction of staphylomycin S with sodium [3H]borohydride as described by Vanderhaeghe et al. ~5 Echinomycin is available from Hoffmann-LaRoche, Inc. (Nutley, New Jersey), etamycin from Bristol Laboratories (Syracuse, New York), staphylomycin S from Recherche Industrie Therapie (Genval, Belgium), stendomycin from Eli Lilly Company (Indianapolis, Indiana), and thiostrepton and vernamycin B from E. R. Squibb and Sons (Princeton, New Jersey). Procedure. The standard reaction mixture contains enzyme preparation, 22.4 nmoles [3H]dihydrostaphylomycin S (980 cpm/nmole), 6 ~moles of Tris.HCl (pH 7.8), and water to a total volume of 0.2 ml. The reaction mixture is incubated at 38 ° for 60 min using a rotary shaker. At the end of the incubation period, the hydrolysis is terminated by the addition of 0.8 ml of 0.5 N HC1. Four milliliters of ethyl acetate are then added, and the tubes are shaken to extract both the residual dihydrostaphylomycin S and the hydrolysis product into the organic phase. Three milliliters of the ethyl acetate layer are transferred to another tube containing 1.5 ml of 0.1 M Tris.HC1 buffer (pH 7.8); after thorough mixing, the solvent layer is discarded. Under these conditions the linear acidic peptide formed by opening the lactone bond of dihydrostaphylomycin S is extracted into the buffer, while the unchanged dihydrostaphylomycin S remains in the ethyl acetate. A 1.0-ml aliquot of the aqueous buffer phase is assayed for radioactivity by liquid scintillation counting 9 ml of Bray's solution. TM Essentially no degradation of dihydrostaphylomycin S occurs if the enzyme is omitted or if boiled enzyme is substituted for the active enzyme. One unit of enzyme activity converts 1 ~g of substrate to acidic product in 60 min at 38 °. An alternative procedure is the use of nonlabeled antibiotic peptide substrates. In this case an agar diffusion bioassay is used to determine 1~H. Vanderhaeghe, G. Janssen, and F. Compernolle, Verb. Kon. Vlaam. Acad. Geneesk. Belg. 34, 209 (1972). I~G. A. Bray, Anal. Biochem. 1, 279 (1960).
[61]
PEPTIDI~ ANTIBIOTIC LACTONASE
771
residual antibiotic present in the ethyl acetate layer. Sarcina lutea is the test organism for bioassays for echinomycin, etamycin, staphylomycin S, thiostrepton, and vernamycin B. Candida albicans is used for stendomycin assays, in which case the agar medium is adjusted to pH 8.0 (after autoclaving) and 50 mg of tetracycline hydrochloride is added per liter of liquefied agar. Antibiotic potency of samples is calculated from the semilogarithmic plots of the diameter of the inhibition zone vs the log of the concentration of the antibiotic. Boiled enzyme controls and an equal number of standards are included in each bioassay. (For a fuller description of general techniques used in agar diffusion bioassays see Kavanagh 17 and this volume. TM) Purification Procedures
The following procedure is that of Hou et al. ~2 It is similar to the procedure for preparing actinomycin lactonase; in fact, the initial steps are identical. The two enzymes are purified together until the DEAEcellulose column step at which time they are separated. The organism and growth conditions, preparation of crude extract, freeze-thaw step, and ammonium sulfate fractionation of this enzyme have been described for actinomycin lactonase TM and will not be repeated here. Approximately 200-fold purification can be achieved by ammonium sulfate precipitation followed by chromatography on calcium phosphate-cellulose, DEAE-cellulose, and Sephadex G-200 columns. Calcium Phosphate Gel Chromatography. The dialyzed enzyme from the ammonium sulfate fractionation step is applied to a calcium phosphate gel-cellulose column (mixture prepared according to the procedure described by Tsuboi and Hudson 2° where 4 g of the gel is mixed with 60 g of cellulose powder) that has the dimensions 2 X 80 cm. The column is previously equilibrated with 0.5 mM phosphate buffer, pH 7.0. The protein is eluted with phosphate buffer using stepwise increments in concentration from 1 mM to 0.1 M. The antibiotic lactonase activity is eluted with the 1 mM buffer. DEAE-Cellulose Column Chromatography. The active fractions from the calcium phosphate gel column are pooled and applied to a DEAEcellulose column (2 X 70 cm) that had previously been equilibrated with 10 mM phosphate buffer (pH 7.0). The lactonase activity is eluted when the concentration of buffer is raised to between 0.1 and 0.2 M. The active 17F. Kavanagh, Ed., "Analytical Microbiology," Academic Press, New York, 1963. I~This volume [4]. 1, This volume [60]. I(. K. Tsuboi and P. B. Hudson, J. Biol. Chem. 224, 879 (1957).
772
[61]
ANTIBIOTIC INACTIVATION AND MODIFICATION
PURIFICATION
TABLE I OF CONSTITUTIVE PEPTIDE
Step Extrusion through Frenchpressure cell Freeze-thaw (NH4)2S04 precipitation Calcium phosphate gel DEAE-cellulose column Sephadex G-200 column
ANTIBIOTIC
LACTONASE
Specific activity (units/mg)
Volume (ml)
Protein (mg)
Activitya (units)
6600
11,134
126,927
6300 125 109 37 17
6,247 2,108 623
91,205 59,656 42,364 35,856 11,800
27 5
Yield (%)
11.4
100
14.6 28.3 68 1328 2360
78 47 33 28 9
a As determined using [3H]dihydrostaphylomycinS as substrate. fractions are mixed with ammonium sulfate (to give 75% saturation) and the precipitate is collected by centrifugation at 10,000 g. After dialysis against 10 mM phosphate buffer, pH 7.0, the enzyme solution is concentrated further by adding Lyphogel® (Gelman Instrument Company) to a final volume of 2 ml. Sephadex G-200 Column Chromatography. The solution from the DEAE-cellulose column is applied to a Sephadex G-200 column (2 X 80 cm) previously equilibrated with 10 mM phosphate buffer (pH 7.0). The enzyme is eluted with this buffer and the lactonase is found after about 180 ml has passed through the column. The purification of the lactonase activity from 4 kg of Actinoplanes cells is summarized in Table I. The overall recovery is 28% and about 120-fold purification is achieved.
Properties The specific activity of the peptide antibiotic lactonase against various paptide lactone antibiotics is shown in Table II. The lactonase is inactive on actinomycins and is a separate and distinct enzyme from that lactonase. It has a broad specificity as evident from the diverse structures against which it has activity. Other known properties include: pH optimum for activity on substrates, 7.8; temperature optimum, 38°; Kin, 0.373 mM (on dihydrostaphylomycin S); V..... 1.09 t~moles/hr per milligram of protein (on dihydrostaphylomycin S). Significant inhibition occurs with 40 mM cobalt, copper, chromium, iron, mercury, nickel, silver, sodium, and zinc salts. This enzyme can be used in assaying crude materials containing these
[61l
PEPTIDE ANTIBIOTIC LACTONASE
773
TABLE II SUBSTRATE SPECIFICITY OF PEPTIDE ANTIBIOTIC LACTONASE
Substrate
Enzyme activity" (units/mg protein)
Assay method
[3H]Actinomycin Echinomycin
0
l{~dio'tctivity assay
6. 2
Sarcina lutea agar
Etamycin Staphylomycin S Stendomycin
440 185 87
S. hdea agar diffusion S. lutea agar diffusion Candida albicans agar
Thiostrepton Vernamycin B,
0. 3 164
S. lutea agar diffusion S. /ulea agar diffusion
diffusion assay
diffusion assay
a A fraction from the DEAE-eellulose column was used in this study. The enzyme reaction mixture contained 142 tLg of enzyme purified by 1)EAE-cellulose chromatography and 24 t,moles of Tris. HCI buffer (pH 7.0) and antibiotics (64 ~g each for etamycin and thiostrepton, 16 tLg for echinomycin, and 150 ~g for stendomycin, staphylomycin S, and vernamycin B,) in a total volume of 0,8 nil. The reaction was terminated by immersion of the tube in boiling water for 1 rain. a n t i b i o t i c s , e.g., b l o o d s a m p l e s , a n i m a l feeds, etc., to p r o v i d e specific inact i v a t i o n . ( T h e i n a c t i v a t i o n p r o d u c t is t h e linear p e p t i d e also f o r m e d b y w e a k a l k a l i n e h y d r o l y s i s ; the e n z y m e does not affect o t h e r b i o a c t i v e m a t e r i a l s p r e s e n t in such crude s a m p l e s which m i g h t be sensitive to a l k a l i . ) T h e low a c t i v i t y on t h i o s t r e p t o n is p r o b a b l y r e l a t e d to the low w a t e r s o l u b i l i t y of this m a t e r i a l . T h e e n z y m e is also s l i g h t l y a c t i v e on amphomycin.5 t
21M. Bodanszky, G. F. Sigler, and A. Bodanszky, J. Amer. Chem. Soc. 95, 2352 (1973).
AUTHOR INDEX
775
Author Index Numbers in parentheses are references numbers and indicate that an author's work is referred to, although his name is not cited in the text.
A Abbott, B. J., 731 Abdullaev, N. D., 353, 354 Abe, H., 123 Abe, J., 285 Abe, M., 276 Abou-Zeid, A., 204 Abraham, E. P., 70, 72, 73(4), 77(4), 78, 116, 117, 118, 158, 281, 335, 339, 341, 409, 410, 411(16, 17), 414, 418, 423, 471, 472, 473(3, 7), 548, 640, 641, 642(20), 647, 648, 649(20), 650, 651, 652, 728, 729(1), 730(1), 731 Abraham, R. J., 395 Aburaki, S., 354 Acker, R. F., 135 Ackerman, E., 754 Acs, G., 154, 156 Adams, E. P., 648, 651,652 Adams, G. M., 177 Adamson, D. W., 580 Addison, E., 140, 143 Adelberg, E. A., 34 Affonso, O. R., 279 Agarashi, N., 154 Ainsworth, G. C., 17 Aizawa, S., 154, 156 Akamatsu, A. Y., 491 Akasaki, K., 123, 339 Akita, E., 179, 187(36), 188, 200(36), 265, 275, 277(25), 285 Albertini, A., 38, 187, 189 Alberts, A. W., 577, 585, 599 Albuquerque, M. M., 162(244), 163 Alderton, G., 146 Alexander, D. F., 10 Alexander, G. S., 211 Alikhanian, S. I., 36 Allanson, E., 122 Allen, G., 400 Allerhand, A., 392, 414, 416(22)
Allison, D., 115 Altmann, O., 735, 737(4) Alworth, W. L., 515 Amano, Y., 280 Ambler, R. P., 649, 660, 662, 672, 677 Ammann, A., 168 Amper, R., 199 Anderson, B., 767 Anderson, E. S., 54 Anderson, K., 416 Anderson, L., 354,441,448 Anderson, P., 5, 8(4) Andres, W. W., 354 Andrews, P., 486 Andrillon, J., 654 Anet, It., 399 Anet, F. A. L., 399, 405 Anfinsen, C. B., 750 Anker, H. S., 283 Antolik, P., 386 Antosz, F. J., 390(19), 391 Anzai, K., 154, 162 Aoki, H., 147 Aoki, T., 285 Aoyama, T., 254 Arai, M., 137, 139, 162(248), 163, 185 Arai, R., 280 Archer, R. A., 421 Argoudelis, A. D., 119, 125, 144, 253, 339, 448, 756, 758 Arigoni. D., 353 Arima, K., 162 Arkhipova, S. F., 354 Armstrong, R. L., 42 Arnstein, H. R. V., 410, 411(14, 15), 471 Aronson. J. N., 441 Arret, B., 57 Artamonova, O. I., 141, 144, 145 Asai, M., 136, 138, 265, 281, 335 Asano, K., 151 Ascoli, F., 352 Ashii, Y., 154
776
AUTHOR INDEX
Ashton, G. C., 281, 638, 639(3), 640(3) Aszalos, A., 127, 134, 179, 186, 187(33), 190, 193, 195, 207, 341 Atfield, G. N., 281 Atherly, E. G., 642, 648, 649(24) Audubert, R., 279 Auerbach, C., 28 Avramenko, V. G., 561 Awataguchi, S., 285 Axen, R., 717 B
Baas, R., 214, 227 Babad, J., 335, 339 Bacher, A., 515 Backus, E. J., 130, 133 Backus, M. P., 25 Bailey, F,, 307, 308 Bailey, J. V., 122, 126 Balan, J., 109, 164, 168(251) Balasingham, K., 699, 709, 713(16), 714, 717, 721 (27) Balasubramanian, D., 353 Baldwin, J. E., 408, 409, 411(12) Ballio, A., 113, 155, 157 Ballotta, R., 136, 138, 341 Banerjee, A. K., 341 Bannister, B., 342, 448 BarAth, Z., 103, 110(10), 137, 139, 164(167) Barbaro, A. M., 179, 180 Barchas, J., 491 Bardawill, C. J., 495, 500 Barfield, M., 400 Bark, L. S., 184 Barlow, C. B., 354 Barry, G. T., 323, 324, 325, 335, 340, 342 Bartels, C. R., 340 Barth, G., 369 Bartha, K. G., 179 Barton, G. M., 286 Bartz, Q. R., 148, 149, 155, 264, 265, 285, 337, 342 Bassler, G. C., 397 Bastos, M. L., 297 Batchelor, F. R., 113, 699, 701, 714, 716, 720, 721, 726(7) Bati, J., 181 Battersby, A. R., 321, 328, 329(19), 331 (4), 342
Bauer, K., 341, 560, 596, 699, 716 Baumann, F., 724, 726(21), 728 Baxter, R., 577 Bayley, P. M., 353 Bazan, N. G., Jr., 179 Beaven, G. H., 353 Beck, J. R., 390 Becker, E. D., 389, 390, 392(5), 398(8) Bekesi, I., 721 Belec, G., 222 Belloc, A., 145 Benedict, R. G., 5, 8(4), 17 Benner, E. J., 386, 388(15) Bennett, R. E., 115, 118(52), 549, 706, 712(3), 713(3), 714, 715(3), 722, 726(13), 727 Bentley, D. W., 753 Bentley, H. R., 285 Benveniste, R., 611, 619, 623, 625, 626, 627(27), 632, 633, 634 Benz, F., 123 B~rdy, J., 119, 152 Berg, P., 562 Berg, C. M., 45 Berg, P., 477, 569 Berg, T.-L., 573 Berger, J., 336 Bergmann, F. H., 579, 580(3) Bergy, M. E., 158, 339, 342 Berk, B., 127, 134, 341 Berky, J., 340 Berridge, N. J., 339 Betina, V., 102, 103, 105, 106(9), 107, 109, 110, 112, 113, 115(6), 117, 126, 127(103), 130, 131, 137, 139, 156(103), 157, 162(103), 163, 164, 165, 167, 168 (253, 254, 255, 256), 169, 170, 173, 200 Bettinger, G. E., 654 Beyaert, C., 724, 726(19) Beyer, W. F., 253 Beynon, P., 409 Bhacca, N. S., 397 Biagi, G. L., 179, 180 Bickcl, H., 133, 134, 135, 148, 149, 179, 193(34), 281 Biddlecome, S., 624, 625(20) Bier, M., 279 Binningen, H. B., 732 Birch, A. J., 520, 535 Bird, A. E., 180
AUTHOR INDEX
Birkenmeyer, R. D., 756 Birladeaml, L., 402 Bishop, M. N., 337 Blears, D. J., 400 Blinov, N. O., 102, 110(7), 123, 127, 134, 141, 144, 145, 146, 153, 156, 163, 164(7), 169, 170 Bloch, A., 154, 156 Block, I~. M., 259 Bly, 12. K., 135 Bobbitt, J. M., 291 Bodanszky, A., 353, 354, 773 Bodanszky, M., 353, 354, 767, 773 Boeck, L. D., 156, 157 Boegemann, W. H., 130, 133 Boehri, E., 354 Boezi, J. A., 42 Bohonos, N., 130, 133, 140, 143, 342, 6O3, 606 Bomstein, J., 699 Bonacei, A. C., 354, 363, 364 Bonaly, R., 687 Booth, H., 399 Boothroyd, B., 161 Bor~,hers, I., 341 Bord('rs, D. B., 258, 259, 261,263(18), 390 Borisova, V. N., 127 Borowiecka, B., 131, 179, 198 Borowski, E., 127 Borowski, Z. K., 146 Bos, G. J. K., 181, 198(52), 199 Bosshardt, 12, 135 Bossi, R., 151 l~othner-By, A. A., 398 Bouanehaud. D. H., 45, 51, 53, 54, 754 Bouehard-Ewing, J. L., 128 ]~oullon. M. G., 678, 680(8), 682, 683, 684(9), 685 Bovey, F. A., 390, 395(9), 397, 398(9) Bowers, W. F., 202 Bowman, P. B., 215 Boxer, G. E., 81 Boyd, P, S., 158 Bozzi, F., 139, 140(170), 143 Bra(tler, G., 134, 135(146) Bradley, C. H., 409, 423 l¢rammar, W. J., 664 Brandl, E., 714, 723, 726(17) Brannon, D. R., 116, 117, 410 Bray, G. A., 516, 764, 770
777
Brazhnikova, M. G., 127, 144, 335, 567 Bredesen, J. E., 573 Brenner, M, 174, 183, 579, 583(2) Bretze|, G., 723, 724(16), 725(16), 726(16) Brink, N. G., 342 Brittain, P. N., 307, 308 Brockmann, H., 146, 151, 327, 334, 335, 341, 354 Brodasky, T. F., 183, 253, 756, 758 Brodsky, 1~. F., 748, 750(15), 751, 753(15. 17) Bronwen Loder, P,, 471,473 Brooks, S. C., Jr., 335, 337 Brossi, A., 354 Brown, G. M., 515 Brown, 1,. W., 215. 234, 239, 242(23) Briigel, W., 397 Brumann, M., 354 Brunner, tf., 477, 724, 726, 728 Bruno, C. F., 706, 714(6), 717(6), 721(6) Brunsberg, U., 474, 477 Bryan, L. E., 44, 55(14) Brzezinska, M., 611,622, 626 Bueort, R., 352, 353 Bu'Lock, J. D., 520, 533 Bunge, t-l. It., 148, 149,342 Bunnenberg, E., 369 Burachik, M., 331, 344 Bureh, W. J. N., 453 Burckhalter, J. H.. 186, 208(86) Burdhardt, F., 354 Burg, A. W., 515 Burgstahler, A. W., 532, 533(3) Buri, P., 199 Burkhohler, P. R., 6 Burrows. E. P., 135 Burton, H. S., 158, 335, 341 Burton, R. B., 130 Bush, C. A., 348 Bush, I. E., 184 Busse, M. J.. 304 Butte, J. C., 491, 494(12) Butterfield, A. G., 318 Butterworth, D., 716 Button, A. C., 390 Buysk(', 1). A., 139, 143, 173(173) Buzzetti, F., 128, 129 Byeroft, B. W., 258 Byrde, ]2. J. W., 729 Bystrov, B. F., 353
778
AUTHOR INDEX
C Caffau, S., 139, 143 Cajori, F. J., 335 Caldwell, D. J., 348(12), 349 Calendar, R., 562, 569 Callow, R. K., 339, 548 Cameron-Wood, J., 701, 716 Campbell, J. N., 688 Capek, A., 151, 769 Caplis, M. E., 384 Caro, L., 45 Caron, E. L., 155, 335, 505 Carpenter, F. H., 328, 340 Carter, H. E., 152, 258, 260 (6), 338 Carver, D. D., 411 Cassani, A., 189 Cassani, G., 38, 187 Caswell, A. H., 354, 364 Cecere, F., 207 Ceder, O. J., 135 Cee, A., 184 Celmer, W. D., 133, 339, 341 Cerdh-Olmedo, E., 34, 35 Cerny, G., 622 Cetrulo, S. D., 27 Chabbert, Y. A., 51, 53, 54, 619 Chaiet, L., 337 Chain, E. B., 70, 113, 699, 714, 716, 720, 721, 726(7) Chakrabarty, A. M., 52 Chamberlain, N. F., 397 Chandler, C. D., 301 Chaney, M. O., 353 Chang, A. C. Y., 48, 49, 50 Chang, C. J., 409 Chang, J., 331 Chanter, K. V., 82, 85(29), 678, 679, 6S0(7) Chapman, H. R., 207 Chapman, O. L., 393 Charm, S. E., 708 Charpentier, G., 145 Chase, M. W., 99 Chassey, B. M., 154 Chauvette, R. R., 699 Cheatle, E., 618 Chedekel, M., 297 Cheeseman, G. C., 339
Chen, G. C. C., 34 Chesters, C. G. C., 23 Chevereau, M., 624 Chiang, C., 706, 712(3), 713(3), 714, 715(3) Chidester, C. G., 214 Childers, R. F., 392 Chit Maung, 482, 485(1), 486 Chmara, H., 127 Chou, J., 673 Christian, W., 500 Churchill, B. W., 16 Ciak, J., 51 Ciferri, O., 38, 187, 189, 580 Ciganek, E., 135 Citri, N., 95, 640, 641, 642(8), 647, 648, 649(18), 650, 652, 662, 664, 670(1), 671 Claesen, M., 722, 723(14), 726(14), 727 Claridge, C. A., 720, 721, 726(2), 727 Clark, R. G., 140, 143 Cline, J. C., 156, 157 Clowes, R. C., 48, 50(5), 51(5) Clubb, M. E., 410, 411(14) Coats, J. H., 756 Coatzee, J. N., 53, 54(30) Cochran, D. W., 414, 415, 416(22) Cohen, A. I., 397 Cohen, S. N., 48, 49, 50 Cole, M., 75, 110, 111, 114, 698, 699, 700, 701, 702(3), 704, 705, 706(1, 2), 707(2), 708(2), 714, 715(2), 716, 717, 720, 721, 722, 726(11) Collie, J. N., 520 Collins, G. R., 533 Collins, J. F., 83, 111, 112(42), 641, 653, 662(3) Compernolle, F., 770 Conchie, J., 639 Conconi, F., 451, 458(5) Cone, J., 515 Conlon, R. D., 301 Cookson, R. C., 398 Cooper, D., 126 Cooper, D. J., 264 Cooper, K. E., 61 Cooper, R. D. G., 416, 421 Cope, A. C., 135 Copius-Peereboom, J. W., 102, 173, 175 Corbaz, R., 131, 134, 337, 767 Corcke, C. T., 135
AUTHOR INDEX
Corcoran, J. W., 487, 488, 491, 492(13), 494(12) Cordes, E. H., 418 Corio, P. L., 390 Cornell, N. W., 354 Cornish, D. W., 301 Coronelli, C., 136, 138 Cosloy, S. D., 48 Courtois, Y., 354 Crabb, T. A., 398 Crabb(~, P., 348, 349(1), 351(1) Craig, D., 320, 343(2) Craig, J. A., 577 Craig, L. C., 146, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329(19), 330, 331, 335, 337, 340, 341, 342, 343(2, 5), 344, 345, 353, 548, 559, 560, 561 Crane, L. J., 654, 662, 663(18) Craske, J. D., t77 Crast, L. B., 66 Crawford, K., 158, 640 Cremer, E., 179 Crew, M. C., 211 Crompton, B., 640 Cron, M. J., 449 Crook, K. E., Jr., 340 Cross, T., 5 Crothers, D. M., 352 Crum, G. F., 169, 336 CsSnyi, V., 83, 641, 649 Csob{m, G., 107 Cuatrecasas, P., 750 Cullen, W. P., 342 Cunningham, K. G., 285 Cur|e, S. A., 118 Currie, S. A., 110
D Daehne, W. V., 353 Dailey, B. P., 397 Dalgleish, D. G, 647, 649, 652(38) Dallas, M. S. J., 183 Danie|s, E. E., 258 DanieIs, M. J., 147 Daniels, P., 126 Daniels, P. J. L., 264, 624 Darken, M. A., 127
779
Da Salete Barros Cavalcanti, M., 162 (244), 163 Date, M., 37 Datta, N., 53, 54, 70, 674, 676(8), 677, 738, 748(5), 755(5) David, M. M., 495, 500 Davidson, N., 42 Davies, J., 42, 611, 619, 620, 622, 623, 624, 626, 627(27), 632, 633, 634 Davies, M. C., 261,263(18) Davies, R., 75, 647, 648, 649(20), 650, 651, 652(20, 44) Davies, R. B., 641 Davis, B. D., 38 Davis, F. A., 264, 271 Davis, R. E., 402 Davis, R. W., 42 Davis, S, 179, 187(33), 190, 193, 195 Dawson, R. M. C., 141, 160 Day, L. E., 114 Day, R. A., 641, 648, 649(16) Deak, S. B., 340 de Albuquerque, M. M. F., 162(246), 163 de Barros Coelho, J. S., 162(244, 246), 163 de Beauveser, J. C., 182 De Boer, C., 136, 138, 155, 169, 335 de Chezelles, N., 145 D~cio de Andrade Lyra, F., 162, 163 de Crombrugghe, B., 739, 743(9), 745(9) Degani, H., 354 Deguchi, T., 491, 720 Dekhuizen, H. M., 107 DeLaHiguera, N., 408, 411(12) De|aroff, V., 352 Delie, V., 34, 35 Della Penna, G., 207 DcLong, D. C.. 156, 157 Demain, A, L., 14, 15, 28, 39, 116, 117(62), 431,638, 639, 640(7), 732 Demarco. P. V., 353, 416, 421, 495 de Mende, S., 279 Dement|eva, S. P., 204 De Muria, J., 130, 133 Denne, D. W., 411 de Oliveira, L. L., 162(246), 163 DePalma, R. E., 491 DeRose, A. F., 335 d~, Ro~i, P., 287, 288 DeSantis, P., 352
780
AUTHO~ INDEX
Desaty, D., 406 DeSerres, F. J., 32 Deshmukh, P. V., 258 Deshpande, G. R., 135 De Sommer, P., 158, 159, 767 Despois, R., 145 Determann, H., 180 De Thomas, A. V., 178 De Thomas, C. R., 178 Detre, G., 406 De Valesi, A. G., 171 De Vault, R. L., 337 Devenyi, T., 181 De Vries, W. H., 169, 336 De Zeeuw, R. A., 182, 183 Dhaese, A., 720, 722 Dhont, J. H., 182 Di Accadia, F. D., 113 Diass, P. A., 108 Di Carlo, F. J., 211 Dieckman, J. F., 304, 305 DieM, P., 395 Dietz, A., 119, 121, 169 DiGirolamo, A. B., 580 DiGirolamo, M., 580 Dill, W. A., 159, 336 Dimroth, P., 521, 524, 528, 529, 530(6), 533 Dion, H. W., 120, 122(84), 264, 265, 285 Djerassi, C., 348, 353, 369 Doan, L., 383, 384 Dobias, J., 109, 137, 139, 164(30, 167) Dobrecky, J., 186, 199 Doctor, V. M., 338 Doddrell, D., 414, 415, 418(22) Doerner, K., 185 Doersehuk, A. P., 19, 40 Doery, H. M., 281,339, 341 Doi, O., 622, 626, 627 Dole~ilov£, L., 158, 487 Dolliver, M. A., 333, 340, 342 Domaradzki, M., 179 Donaldson, B. R., 393, 396(30) Donin, M. N., 341 Donovan, F. W., 520 Donoviek, R., 767 Dornberg, K., 134, 135(146) Dornbush, A. C., 130, 133, 140, 143 Dosko~ilov'~, D., 169, 385 Dougall, D., 281
Dovovick, R., 146 Doyle, F. P., 113 Drake, N. A., 107 Drost, G., 134 Drozen, V., 170 Drummond, G. I., 441 Dubnau, D. A., 663 Dubost, M., 145 DudiIlskaya, A. A., 561 Duez, C., 688 Dulaney, D. D., 37, 38(20) Dulaney, E. L., 37, 38 Dulbecco, R., 109 Dumont, E., 179 Duncan, D. T., 127, 152(111), 153(111), 154(111), 158(111), 203 Duncombe, R. E., 73 Dunnigan, D. A., 353 Dunnill, P., 699, 706, 708, 709(15), 714, 721(18, 27) Duquette, L. G., 353 Durkovsky, J., 109 Dusart, J., 688 Dusinsky, G., 386 Dutcher, J. D., 286, 341, 767 du Vigneaud, V, 328, 340 Dworschack, R. G., 337 Dyer, J. R., 258, 260(6) Dykhovichnaya, D. E., 168
E
Eastwood, F. W., 341 Ebata, M., 261 Eberhardt, H., 735, 737(4) Eble, T. E., 158, 337, 339, 342 Ebringer, L., 109, 164(30) Eckle, M., 393, 396(31) Eda, J., 627 Edwards, R. A., 177 Efremov, E. F., 353 Egan, R. S., 353, 354, 359 Egawa, Y., 353 Eggers, F., 353 Egorov, C. A., 260 Ehrimger, D. J., 491 Eilers, N. J., 137, 158 Eisenberg, F, 128, 129 Elander, R. P., 39, 158, 159
AUTHOR INDEX
Elbein, A. D., 621, 757 Eliel, E. L., 532, 533(3) Elliott, D. C., 141, 160(183) Elliott, W. H., 141, 160(183) Ellis, J. R., 540, 542(3) Ellis, P. D., 416 Elhnan, C., 483 Elson, S., 75 Ekstner, E. F., 409, 515 Emsl(,y, J. W., 390, 395(6) Endo, S., 111, 113 Endo, T., 253 Epsiein, W. W., 390 Erickson, R. C., 115, 118(52), 722, 726(13), 727 Ericsson, H. M., 59, 66(10) Ermolova, O. B., 214 Ernbiick, S., 717 Ettling(,r, L., 131, 134, 767 Eustace, G. C., 716 Evans, tl. S., 286 Evans, W. G., 699 Evere(I, P. M., 81 Ewing, G. W., 301 Eyring, H. E., 348(12), 349 Ezaki, N., 265, 285
F
Fiihner, 133 Falkow, S., 738 Falliek, G. J., 301 Fantes, K. H., 23, 638, 639(3), 640(3) Farr, A. L., 456, 507, 679, 757 Farran, J. D., 116 Farrar, T. C., 389, 392(5) Farrer, W. E., 84 Fawcett, P., 409, 423 Feeney, J., 390, 395(6) Felber, E., 311,312, 732 Feldman. L. I., 140, 143 Fennell, D. I., 8, 9(8) Feren~.z, I., 641, 649 Ferguson, R. C., 390 Ferrari, A., 75 Fielding, A. H., 729 Fieser, L. F., 369 Fies~r, M., 369 Figenschou, K. J., 573
7S1
Finland, M., 642, 652(22) Fisehbach, H., 141, 198 Fischer, L. J., 212 Fischer, R. D., 389, 401(4) Fisher, M. W., 281 Flato, J., 375 Fleming, J., 640, 647, 651 Fleming, P. C., 678 Florey, H. W., 335 Floss, H. G., 409, 499 Flynn, E. H., 728,730(2) Foley, C., 5, 8(4) Folkers, K., 337, 339, 342, 448, 449 Ford, J. H., 85, 337, 504 Forhardt, R. P., 155 Forrester, P. 1., 541, 542, 543(8), 546(8), 548(4, 8) Forsyth, W. G. C., 336 Fossdal, K., 382 Foster, M. C., 281 Foster, T. J., 754, 755 Fox, J. A., 158, 339 Francov/~, V., 159 Fraser. R. R., 392, 402(25) Freeman, H. P., 177 Frei, R. W., 181 Freifelder, D., 42, 43 French, J. C., 148, 149, 264, 342 Fr~re, J. M., 688, 690, 692(14), 696, 698 Freyburger, W. A., 214 Fried, J., 333, 342, 638 Friedman, H. L., 354 Friedman, P. A., 354 Friedman, S. M., 491, 492 Friend. E. J., 34, 35(11) Fries, I,., 134, 497 Fries, N., 38 Frodyma, M. M., 181 FrChlm, L. O., 208, 573, 585 Fr0yshov, O., 570, 576, 577(12), 586 Frolov, I. I., 301 Fromageot, P., 354 Frost, D., 179, 187(33), 190, 193, 195 Fugner, R., 134, 135(146) Fujii, A., 286, 287(29) Fujikawa, K., 340, 579 Fujimoto, Y., 162(245), 163 Fujita, H., 151 Fukasawa, T., 45, 51 Fukalsu, S., 264
782
AUTHOR INDEX
Fukugawa, Y., 154, 156 Fullbrook, P. D., 75 Funayama, S., 628, 631(5), 632(5) Funk, T., 353 Furukama, M., 151 Furumai, 145 Fusari, S. A., 120, 122(84), 155, 265, 281 Fust, B., 354
G Gaeumann, E., 123, 148, 149 G~iumann, E., 131, 133, 134, 179, 193(34), 281, 767 Gale, P., 339 Gale, R., 131, 495 Gale, R. M., 158, 159, 338 Gallo, G. G., 409 Gamba, M. F., 180 Gang, D. M., 699 Ganguli, B. N., 338 Ganskirt, H., 182 Garber, N., 641,649(18), 753 Garbisch, E. W., 395 Gardner, J. A. F., 286 Gardner, J. F., 341 Garofalo, M., 248 Garrett, E. R., 64, 66 Gasparic, J., 184 Gatenbeck, S., 474, 477 Gaucher, G. M., 541, 543(8), 546(8), 548(4, 8) Gause, G. F., 567 Gavan, T., 618 Gazso, J., 179 Geiersbach, H. J., 108 Geiss, F., 182, 183 Gerband, G. R., 54 German, V. F., 391 Gerzon, K., 131, 156, 157, 495 Gevers, W., 549, 558, 577, 585, 586, 594 Ghosh, B. K., 663 Ghuysen, J. M., 687, 688, 690, 691, 692(14), 696, 698 Gibbons, W. A., 331 Giddings, G. J., 102 Gilhuus-Moe, C., 576, 577(12), 578, 586 Gilliland, J. M., 570 Gillin, F. D., 642, 648, 649(24)
Gillis, B. T., 135 Glass, D. G., 678 Glazko, A. J., 159, 336, 735 Godzeski, C. W., 116 Godzesky, C, 340 Goegelman, R. T., 267, 275(5), 276(5) Goko, H., 285 Goldberg, I. H., 354 Goldman, P., 599 Goldner, M., 678 Goldstein, A. W., 353, 498 Golovkina, L. M., 170 Golumbic, C., 327 Gonqalves de Lima, O., 162(244, 246), 163 Goodall, R. R., 75, 101, 110(1) Goodlett, V. W., 403 Goodman, J. J., 19 Goodman, R. A., 392 Gordee, E. L., 114 Gordee, R. S., 156, 157, 158, 159 Gore, B. H., 298 Gorini, L., 38 Gorman, M., 39, 66, 156, 157, 495 Gornall, A. G., 495, 500 Goss, W. A., 119, 125 Gotfredsen, W. O., 353 Goto, T., 337 Gottlieb, D., 18, 119, 125, 152, 168, 264, 338 Gourevitch, A., 720, 767 Graber, R. P., 448, 449 Grainger, A., 107 Grant, H. N., 128, 129 Grant, P. T., 410, 411(15) Gray, E., 18, 579, 580, 581(6), 583(2) Gray, P. P., 708, 709(15) Grazi, E., 451, 458(5) Greco, A. M., 136, 138, 341 Green, A. A., 748 Greenough, R. C., 211 Gregory, F. J., 151 Gregory, J. D., 325, 335, 337, 342, 345 Grell, E., 353 Greull, G., 528 Grindley, J. N., 54 Grindley, N. D. F., 54 Grinnan, E., 122 Gritter, R. J., 291 GrSne, H., 151, 335 Grosjean, M., 348
&UTHOR INDEX
783
Hara, N., 280 Hara, T., 275, 277(25), 289, 290, 309 Harada, H., 116 Harada, N., 354 Harada, S., 136, 138 Harads, Y., 153, 335 Hardy, C. J., 175 Hardy, T. L., 699 Harfenist, E. J., 335, 548 Hargie, M. P., 281 Harpootlian, H., 128 Harris, A. M., 70, 82, 85(29), 678, 679, 680(7), 683, 685(1), 686(1) Harris, R. K., 395 Hart, M. V., 716 Harvey, R. A., 515 Harwood, J. H., 625, 739 Hasegawa, T., 136, 138 H Hash, J. H., 606 Hashimoto, T., 273 Haapala, D. K., 738 Haskell, T. H., 148, 149, 155, 337, 342 Haas, M. J., 611,622, 623(16) Hassall, C. H., 339 Habeeb, A. F. S. A., 742 Hata, T., 134, 153, 264, 353 Haber, A., 409 Haupt, I., 722, 726(8) Habgood, H. W., 175 Hausmann, W. K., 258, 259, 261, 263(18), Haehimori, A., 622 326, 330, 335, 337, 340, 341, 345. Hacker, A. K., 354, 364 548 Hackman, C., 151 Hayamizu, K., 397 Haensler, C. M., 135 Haynes, W. C., 4 Hagaman, E. W., 409 Heatley, N. G., 158, 339 Hager, L. P., 528 Hedges, R. W., 53, 54 Hahn, F. E., 51 Heftmann, E., 102, 279, Hais, I. M., 102, 159 Heinemann, B., 108, 134, 152(22), 767 Hale, C. W., 117, 158, 281, 335, 414 Heinrich, G., 549 Hall, D. A., 388 Hellberg, H., 111, 114 Hall, J. R., 649 Hendess, R. W., 154 Halpern, O., 353 Hendlin, D., 15, 17, 116, 118 Hamada, H., 120, 122(83) Hamada, M., 154, 156, 264, 269, 273, 285, Hennessey, T. D., 678, 679, 680, 684(4), 685(4, 10) 289, 625 Hennis, H. E., 353 Hamano, K., 185 Henry, R. A., 304, 305 Hamasaki, T., 409, 414, 416(25) Henry, R. J., 78, 79 Hamdan, A., 265 Heppel, L. A., 620, 673 Hamill, R. L., 39, 495 Herbert, M., 23 Hamilton, D., 520, 533 Hernandez, S., 110, 118 Hamilton, M. A., 335 Herold, M., 140, 143 Hamilton-Miller, J. M., 81,698, 700 Herr, R. R., 158, 339, 342 Hanawalt, P. C., 34 Herrmann, E. C., 109 Hanka, L. J., llO Hervey, A., 337 Hannig, K., 289 Herzog, H. L., 265 Hansson, E,, 114
Gross, E., 330, 331(23), 339, 342 Grostic, M. F., 390 Grove, D. C., 83, 106, 607 Grundy, W. E., 13 Gruner, J., 311, 312, 732 Grunert, R. R., 478 Grutzner, J. B., 405, 408, 409 Guerola, N., 35 Guerra, M. C., 179, 180 Guiney, D. G., Jr., 354 Guiochon, G., 301 Gunsalus, I. C., 52 Guroff, G., 515 Guschlbauer, W., 354 Gyanchandani, N. D., 179
784
AUTHOR INDEX
Heslot, H., 28 Hesseltine, C. W., 4, 5, 8(4) Hetman, J. S., 385 Hettinger, T. P., 146, 335, 337, 559, 560 Heuser, L. J., 767 Hewitt, V. A., 114, 700 Hey, A. E., 621, 757 Hichens, M., 391 Hickey, R. J., 335, 513, 548 Hidaka, T., 154, 156 Higgins, C. E., 146 Higuchi, T., 353, 362 Hinman, J. W., 155, 335, 505 Hinners, T. A., 353 Hiranaka, H., 150, 274 Hirata, Y., 337 Hirs, C. H. W., 570 Hirsch, U., 40 Hishta, C., 248 Hjert~n, S., 537 Hnilica, V. S., 435, 457, 458(18) Hochstein, F. A., 339 Hockenhull, D. J. D., 16, 23, 638, 639(3), 640 Hockey, J. A., 183 Hodgkin, D. C., 767 Hoehn, M. M., 116, 145, 146 Hoeksema, H., 169, 336, 505 Hoener, B.-A., 354, 364 Hoette, I., 134 Hoff, D. A., 549 Hoffhine, C. E., Jr., 339, 448, 449 Hogeboom, G. H., 323, 328, 340 Hollis, D. P., 397 Hollister, Z. J., 108, 152(108) Holly, F. W., 449 Holmes, R. E., 156, 157 Homer, R. B., 354 Hong, D. D., 186, 208(86) Hoogerheide, J. C., 134 Hook, D. J., 409 Hooper, I. R., 134, 337, 340, 449, 767 Hopwood, D. A., 34, 35, 36 Horak, E., 177 Hori, M., 154, 156, 299 Horii, S., 254, 265, 273, 275(16) Hornemann, U., 499 Horowitz, M. I., 123 Horsk~, O., 140, 143 Horton, D. R., 116
Horv~th, I., 130, 131(129) Hosoya, S., 280, 337, 339, 341 Hou, C. T., 764, 769, 771 Hou, J. P., 66, 77, 78 Housewright, R. D., 78, 79 Houtman, R. L., 237 Howard, A. J., 108, 152(22) Howison, P. W., 352, 365, 366 Hsu, L., 48, 49, 50 Huang, H. T., 720 Huber, F. M., 116, 117(63), 699 Huber, G., 134, 497 Huckstep, L. L., 408, 411(12) Hudec, J. J., 398 Hudson, P. B., 765, 771 Hiitter, R., 131, 133, 134, 151, 179, 193(34) Hughes, D. W., 179, 222, 318 Hughes, M. S., 341, 767 Hughes, W. L., 748 Hulme, M. S., 520, 533 Hurley, L. H., 499 Hursky, V. S., 109 Hutchison, D., 339 Hutchison, J. D., 354, 364 Huxtable, R. J., 409 Hwang, S., 9
Ichikawa, T., 37, 155 Ichiyanagi, T., 275, 285 Igarashi, N., 156 IglSy, M., 130, 131 Iguchi, S., 254 Iida, Y., 111, 113 Iinuma, K., 264, 272, 289 Ijichi, K., 169, 170(261) Ikeda, D., 122 Ikeda, G. J., 128 Ikeda, Y., 266, 270(4), 272, 285 Ikekawa, T., 148, 150, 179, 187, 188, 200, 274 Ikushima, H., 147 Imam, G. M., 424 Imanaka, H., 147 Imsande, J., 640, 642, 648, 649(24) Inamine, E., 431, 638, 639, 640(7) Ingraham, J. L., 35 Inoue, A., 151
AUTHOR INDEX Inouye, H., 340 Inouye, S., 220, 265, 276, 611 Inuzuka, M., 51 Isbell, B. E., 353 Ishibashi, K., 342 Ishid'b H., 283 Ishida, N., 119, 121, 125, 145, 169, 170 Ishii, Y., 335 Ishikura, T., 37, 155 Ishizuka, M., 285 Isono, K., 153, 154, 155, 156, 162, 338 Ito, E., 154 Ito, G., 628, 631(5), 632(5) Ito, K., 154, 156 Ito, T., 264 Ito, Y., 145, 200, 285 ltoh, H., 569, 578, 585 Iwmov, V. T., 353, 354 Iwami, F., 150, 179, 187(36), 188, 200(36), 274 lwasa, T., 265 Izdebski. J., 767
J Jack, G. W., 91, 92, 96(8), 673, 676, 677, 679 Jaekman, L. M., 390, 398(7), 399(7) Jackson, B. G., 699 Jackson, L. J., 78 Jackson, M., 110, 118 Jackson, W. G., 342 Jacobaeei, S., 139, 143 Jacob< A., 754 Ja('o})sen, E., 382 Jago, M., 70, 73(4), 77(4), 78, 640 Jahnlee, H., 342 Jakubowski, Z. L,, 155 James, K. C., 185 Jang, I{., 729, 730(4), 731(4) Jansen, E. F., 729, 730(4), 731(4) ,lanssen, G., 232, 770 ,lan~on, J. A. T., 85 J{~rai, M., 154, 156 ,lsret, R. S., 264 Jarvis, F. G., 722, 724 J'mt('l'~t, M., 414 •layar.maen, K., 579, 581(1), 582(1), 583(1)
785
Jeffery, J. D'A., 116, 118, 728, 729(1), 730(1), 731 Jelinek, M., 341 Jensen, E., 40 Jereezek-Morawska, E., 127 Jimeno de Ossd, F., 177 Jirgensons, B., 348 Joel, C. D., 179 Johnson, A. W., 123, 258 Johnson, B. A., 283 Johnson, D. L., 767 Johnson, D. P., 57 Johnson, F., 353 Johnson, H. E., 135 Johnson, J. L., 504 Johnson, K., 688, 696 Johnson, L. E., 214 Johnson, L. F., 397, 408, 409, 414, 416(25) Johnson, L. R. F., 421 Johnson, M. J., 110, 115, 475, 476(2), 483. 720, 722, 724 Johnson, R. D., 409 Johnson, S., 19 Johri, B. N., 201 Jones, H., 62 Jones, I. F., 388 Jones, K. M., 141, 160(183) Jones, N. D., 353 Jones, P. H, 353 Jones, R. G., 395 Jork, H. Z., 181 Joyner, C., 265 Jhzs'L G., 154, 156 Jukofsky, D., 297 Juvarkar, J. V., 354
K
Kaczmarek, D. K., 515, 516(3) Kaeding, W. W., 533 Kagan, F., 756 Kaiser, D. G., 125, 214, 237 Kakinuma, K., 409 Kalinina, M. V., 186 Kaliszan, R., 385 Kalkstein, A., 640, 641(8), 642(8) Kamada, H., 162(245), 163 Kamala, T., 162 Kamata, Z., 164
786
AUTHOR INDEX
Kameda, Y., 254, 265, 273, 275(16) 134, 141, 144, 145, 146, 153, 156, 163, Kanamori, H., 134 164(7), 169(7), 170, 258, 259(10), 260, Kandler, O., 687 274 Kaneda, T., 491, 492(13), 494 Khokhlova, Y. M., 141, 144(184), 145, Kaneko, T., 285 153, 169, 170 Kano, T., 520 Khryaseheva, K. M., 170 Kantor, N., 122 Kies, M. W., 516, 760 Kaplan, M. A., 767 Kikuchi, M., 145 Karasawa, K., 162(248), 163, 339 Kiltz, H. H., 330, 342 Karg, H., 181 Kimura, Y., 203 Kariya, M., 511, 513(6), 515 King, N. K., 281 Kariyone, K., 116 King, R. W., 393 Karnovsky, M. L., 110, 115 King, T. J., 258 Kasamatsu, H., 738 King, T. P., 320, 330, 331, 342, 353 Kashelikar, D. V., 548 Kinnear, J. E., 44, 55(14) Kasukabe, Y., 123 Kinney, R. W., 140, 141(180), 143 Katagiri, K., 152, 203 Kinoshita, M., 354 Katagiri, M., 264 Kinstle, T. H., 390 Kato, E., 162(245), 163 Kirby, J. P., 261,263(18) Kato, F., 720 Kirby, S. M., 72, 73, 82, 85(7, 29), 89, Katz, E., 202, 209 670, 679, 680, 684(17) Kaufman, H., 38 Kirchner, J. G., 173 Kaufmann, W., 699, 716 Kirsch, E. J., 500 Kautz, F., 352 Kirshbaum, A., 57 Kavanagh, F., 56, 57, 58, 59(1, 2, 8, 9), Kishi, T., 136, 138, 285 60, 61, 62, 63(1, 2), 64, 66(8, 19, 20), Kiss, P., 181 67(19), 68(19, 22), 69(19, 22), 106, Kitagawa, T., 123, 258, 259 Kitazawa, E., 122 337, 771 Kiyohara, H., 465, 469 Kavanagh, K., 122 Kawabe, H., 634 Klein, F. K., 177 Kawaguchi, H., 119, 121, 125, 130, 133, Kleinkauf, H., 549, 558, 577, 585, 586, 594 Kluender, H., 409, 423 265, 336 Kawaguchi, K., 520 Kluepfel, D., 136, 139 Kawahara, K., 254, 265, 273, 275(16) Klyuyeva, L. M., 127 Knoll, W. M. J., 409 Kawaji, S., 265, 272(1), 273(1), 276 Kawamata, J., 151 Knox, N. G., 39 Kawasaki, M., 162 Knox, R., 81 Kay, G., 706, 714 Kobaru, S., 265 Kellerhals, H., 395 Kobayashi, F., 122, 627, 634 Keller-Schierlein, W., 128, 129, 131, 133, Kobinata, K., 155 134, 151, 767 Koch, K. F., 264, 271, 285 Kelley, J. M., 75 Koe, B. K., 339 Kelly, B. W., 158 Koga, F., 134 Kelly, L., 664 Kogan, G. A., 354 Kelly, R. G., 139, 143, 173(173) Kogut, M., 641, 642 Kenig, M. D., 114, 700 Kohsaka, M., 147 Kennedy, B. P. C., 540 Koike, M., 275, 277(25) Kern, D. L., 120, 122(84), 265 Kojuma, K., 611 Keutmann, E. H., 130 Kollar, G., 638, 639, 640(8) Khokhlov, A. S., 102, 110(7), 123, 127, Koll£r, J., 154, 156
AUTHOR INDEX
Komatsu, N., 280, 337, 339, 341 Kominek, L. A., 160, 502 Kondo, E., 738 Kondo, H., 275, 277(25), 285 Kondo, S., 264, 265, 266, 268, 270(4), 272, 273(1), 274, 275, 278(27), 279, 281(8), 282(8), 283, 285, 289, 290, 619, 622, 625, 626, 634 Konecny, J., 311,312, 732 Konigsberg, W., 326, 335 Konishi, M., 265 Kono, Y., 123 Konstantinova, N. V., 144 Korehagin, V. B., 160, 161, 204 Korshalla, J. D., 500 Koshi, T., 627 Koshiyama, H., 130, 133, 336 Kosower, E. M., 354 Kov55, ~., 109, 164, 168(251) KovaSiSov'£, L., 164, 169 Kovacs, J., 181 Kova;ik, L., 140, 143 Kowsharowa, I. N., 152 Kowszyk-Gindifer, Z., 490 Koyama, G., 285 Kozatani, J., 116, 118 Kozuki, S., 261 Kradolfer, F., 131, 767 Krakower, G. W., 108 Kramer, M., 98, 99 Krasnobrizhii, N. Ya., 354 Krause, J. M., 84 Krauss, D., 390 Kristensen. T., 578 Kroschwitz, J. I., 414, 416(26) Krueger, W. C., 354, 367 Krugliak, E. V., 127, 335 Kubo, N., 51 Kudo, S., 162(245), 163 Kuehl, F. A., 254 Kuehl, F. A., Jr., 337 Kugelman, M., 335, 337 K~bnle. E., 549 Kuijpers, G. G., 182 Kukolja, S.. 353 Kukuda. D. S., 116, 117(63) Kumagai, K., 145 Kumon, S., 136, 139 Kunstman, M. P., 127, 354
787
Kunugita, K., 147 Kurahashi, K., 569,571,575, 577, 578, 579, 585 Kurath, P., 353 Kurita, M., 116 Kurylo-Borowska, Z., 559, 560, 561, 564 Kurz, W., 524, 536 Kusaka, T., 285 Kushner, D. J., 654, 663(8) Kutkov.~, M., 164, 168(251) Kutzbach, C., 709, 713(17), 714, 716(17) Kuwabara, S., 640, 641, 642, 648, 651,652 Kuzel, N.. 56 Kyburz, E., 131 Kyotani, Y., 220, 264, 265 L Labia, R., 654 Lago, B., 638, 639(4) Laiken, S. L., 331,353 Laine, I. A., 354 Laland, S. G., 208, 568, 570, 572, 573, 575, 576, 577(2, 12), 578, 585, 586 Lamotte, H., 178 Lampen, J. O., 654, 662, 663, 685 Lancaster, J. E., 258, 261(4) Lancini, G., 409 Langlykke, A. F., 638, 639(1) Langman, R. E., 570 Lanz, P., 354 Lapidus, J. B., 352, 365 Lardy, H. A., 448 Large. C. M., 337, 342 Larsen, S. H., 156, 157 Larson, L. M., 123 Larson, M., H., 137, 139 Laszlo, P., 396 Lavrova, M. F., 127 Law, J. L., 491 Lawson, W. B., 767 Layne, E., 510, 523 Lazar, R., 178 Leach, B. E., 337, 339 I, each, R. H., 185 Lechev'dier, H., 135 Lederberg, E. M., 38 I, ederberg, J., 38 Lederer, M., 279 Lee, B. K., 706. 714(6), 717(6), 721(6)
788
AUTHOR INDEX
Lee, S. G., 560, 586, 596 Lees, K. A., 304 Lees, T. M., 130, 133 Legator, M., 18 LeGoffic, F., 624, 654 Legrand, M., 348, 352, 353, 354 Lein, J., 134, 720, 721, 726(2), 727, 767 Leitner, F., 66 Lemanski, P., 134 Lemke, P. A., 408, 409, 410, 411, 414 Lener, M., 352 Lenkinski, R. E., 402 Leonard, R., 154, 156 Lergier, W., 354 Lesnkova, A. V., 170 Leung, D. C., 577 Levenberg, B., 515, 516(3) Levi, A. A., 101, 110(1) Levin, 0., 537 Levine, J., 141, 198 Levvy, G. A., 639 Levy, G. B., 383 Levy, G. C., 391, 405 Lewis, C. N., 169 Lewis, J. C., 169, 170(261) Leyh-Bouille, M., 687, 688, 690, 692(14), 696, 698 Li, T.-K., 354 Libinson, G. S., 304 Lichtenberg, E., 159 Lietman, P., 739 Lieu, V. T., 181 Light, R. J., 521, 528, 530, 531(1), 533, 534(8, 9), 535, 536(1) Lightbown, J. W., 287, 288 Lilligren, B. L., 735, 736(2), 737(2) Lilly, M. D., 699, 706, 708, 709, 713(16), 714, 717, 721(18, 27) Lin, W., 335 Lincks, M., 606 Lindberg, J. G., 394, 401(32), 403(32) Lindberg, M., 49 Lindenfelser, L. A., 5, 8(4), 337 Lindstrom, E. B., 75 Ling, N.-S., 259 Lingens, F., 515, 735, 737(4) Lins de Oliveira, L., 162(244), 163 Linzer, R., 584 Lipkind, G. M., 354
Lipmann, F., 549, 558, 560, 577, 584, 585, 586, 594, 596 Lippmaa, E., 416 Lisboa, B. P., 184 Lisowski, J., 353 Lively, D. H., 156, 157 Ljones, T., 585 Lloyd, P. H., 649, 652 Loder, B., 116, 117(53), 118, 281 Lodi, L., 186 Loeber, G., 354 LSliger, J., 408, 411(12) Loettler, K., 318 Lomakina, N. N., 127 Lombardi, N. N., 186 Longe, H., 335 Loseva, G. I., 214 Lowe, L. A., 147 Lowry, D. L, 78 Lowry, O. H., 456, 507, 679, 757 Lu, S. H., 515 Luben, G., 180 Lucente, G., 716 Luck, G., 354 Ludescher, U., 354 Luedemann, G. M., 21, 119, 121, 125, 128, 129, 130 Lummis, W. L., 108, 756 Lunel, J., 145 Lustig, E., 395 Luttinger, J. R., 721, 726(2), 727 Lynen, F., 521, 522, 524(6), 528, 529(6), 530(5, 6), 533, 560, 585
M
Mabe, J. A., 39, 116, 117 McAlpine, T. S., 488 McCalla, T. M., 540, 542(3) McCormack, R. B., 333, 342 McCormick, J. R. D., 19, 40 McCoy, E., 37 MacDonnell, L. R., 729, 730(4), 731(4) Macek, K., 102, 104, 159 McFadden, H., Jr., 618 McFarlane, W., 390, 396(13) McGahren, W. J., 354 McGilveray, I. J., 179 McGuire, J. M., 146
AUTHOR INDEX
789
Marsh, W. S., 341 Maciel, G. E., 416 Marshall, A. C., 180 Maciel, G. M., 162(244, 246), 163 Marshall, M. J., 70, 82, 85(29), 678, 679, Maciel, M. C. N., 162(246), 163 680(7), 683, 685(1), 686(1) MeInnes, A. G., 406 Marston, R. Q., 341 McInnis, D. J., 214, 227(1) Martin, E., 340 McKee, C. M., 62, 341 Martin, J. H., 140, 143, 342, 603 MacKellar, F. A., 390, 391, 399 Martin, J. R., 353, 359, 498 McMahan, J. R., 63 Martin, J. S., 397 McMahon, 1R. E., 116, 117(59) Martin, L. L., 409 Maemorine, H. G., 111 Martin, M., 301 McNall, E. G., 161 Martinelli, E., 409 MeNary, J. E., 258 Maruino, H., 162(245), 163 Madaiah, I., 648 Maeda, K., 148, 150, 264, 265, 268, 269, Mason, D. J., 119, 121 272, 273(1), 279, 281, 282(8), 283, 285, Massey-Westropp, R. A., 535 Masuda, T., 281 286, 289, 634 Mata, J. M., 110, 118 Maehr, H., 274, 275, 276(24), 277 Mathis, G., 190, 193, 196, 197 Magerlein, B. J., 756 Mathur, H. H., 390(19), 391 Magni, L., 114 Matrishin, M., 19 Magnus, K. E., 339 Matsubara, T., 116, 117, 118, 208 Maheshwari, M. L., 390(19), 391 Matsumae, A., 153 Mahler, H. R., 418 Matsuo, A., 465 Majer, J., 487 Matsuzaki, M., 130, 133, 336 Majerus, P. W., 577, 585, 599(6) Matteo, C. C., 708 Majors, R. E., 301, 318 Majumdar, M. K., 119, 120(76, 77), Matter, U. E., 397 Matthew, M., 70, 678, 679(1), 683(1), 684, 122(77), 126, 611 685(1), 686(1) Majumdar, S. K., 119, 120(76, 77), Matthews, T. R., 156, 157 122(77), 126, 611 Mauger, A. B., 202, 313, 352, 767, 769 Makino, S., 123 Maurer, H. R., 554 Maksimova, T. S., 152 Mayfield, J.. 342 Malczewska-Koneeka, W., 490 Mays, D. L., 248 Malera, A., 353 Meadway, tl. J., 649, 654, 660, 661,662(10), Malik, J. M, 409 672 Malik, V. S., 735, 737(1) Meinardi, G., 186 Malling, H. V., 32 Meleney, F. L., 283 Malysz, D., 131 Melling, J., 641, 642(20), 648(20), Man~.her, R. A., 151 649(20), 652(20) Mancy, D., 145 Merkenschlager, M., 524, 536 Mandel, M., 34 Meseck, E., 265 Mandelstam, J., 641, 653, 662(3) Mesentsev, A. S., 335 Mann, R. L., 338 Meshcheryakova, E. N., 354 Mansford, K. l:l., 699 Messina, N., 339 Manzke, H., 185 Meuhnan, P. A., 136, 138 Maral, R., 145 Meyer, C, E., 341, 342 Marconi, W., 207 Meyers, E., 128 Margosis, M., 222, 228, 232 Meynell, G. G., 42, 50(10), 51(10) Marquet, A., 353, 688 Michael, W. R., 337 Marquez, J., 265 Marquez, J. A., 120, 126, 130, 171, 172 Michaelis, A. F., 301
790
AUTHOR INDEX
Miekel, S., 738 Mighton, It,, 327 Migliore, D., 265 Miida, T., 220 Mik6s, O., 291 Milavetz, B., 409 Mile, I., 641, 649 Miller, A. K., 337 Miller, A. L., 450, 466, 628, 630, 631 Miller, G. A., 414 Miller, G. H., 64 Miller, I. M., 116, 117(62), 732 Miller, M. M., 122 Miller, P., 340 Miller, P. A., 140, 143, 158, 342, 603, 606 Miller, R. F., 354, 364 Miller, R. P., 116, 117 Mine, Y., 117, 208 Miroshnikov, A. I., 353 Misawa, M., 720 Misiek, M., 66, 134 Mistretta, A. G., 147 Mitidieri, E., 279 Mitscher, L. A., 127, 140, 143, 258, 261, 342, 352, 353, 354, 359, 363, 364, 365, 366, 603 Mitsuhashi, S., 41, 45, 50(2), 51(2), 122, 627, 634 Miura, K., 264 Miyairi, N., 147 Miyaki, T., 119, 121, 125, 130, 133, 336 Miyamoto, M., 274, 627 Miyazaki, J., 119, 121, 125, 162 Miyazaki, T., 465 Miyoshi, T., 147 Mizoguchi, T., 469 Mizsei, A., 130, 131(129) Mizuno, K., 127, 136, 138, 265, 285 Mochales, S., 110, 118 Moeller, H., 186 Molho, D., 735, 737(3) Molho-Lacroix, L., 735, 737(3) Monnikendam, P., 261 Monreal, J., 579, 581(1), 582(1), 583(1) Morawiec, J., 353 Morell, J. L., 330, 331(23), 339 Moreno, R., 688 Mori, T., 220, 264, 265, 275, 285 Morin, R. B., 495 Morisi, F., 207
Moriyama, K., 283 Morozowieh, W., 306 Morris, A., 72, 73, 85(7), 89, 670, 680, 684(17) Morris, C. J. O. R., 281, 320 Morris, D., 471 Morris, P., 320 Morton, J., 264 Mosher, H. S., 369 Moye, C. J., 535 Mozingo, R., 449 Mueller, W., 352 Muggleton, P. W., 80, 85(26), 117, 678, 679(5), 657(5), 732 Mukherjee, P. P., 514 Mul~, S. J., 297 Muller, N., 390 Munakata, K., 275, 285 Muntz, R. L., 392 Mural, K., 133, 339, 341 Muramatsu, I., 767 Muraoka, Y., 286, 287(29) Murase, M., 264, 265, 272(1), 273(1), 276 Murata, T., 45 Murawski, A., 265 Murphy, C. F., 416 Murphy, J. B., 516, 760 Muroi, M., 136, 138, 285 MurrelL J. N., 395 i Nagahama, N., 200 Naganawa, It., 264, 269, 279, 281(8), 282(8), 283, 285, 625, 626, 634 Nagarajan, R., 117, 353 Nagasawa, H. T., 211 Nagata, A., 285 Nagatsu, C., 123 Nagatsu, J., 155 Naghizadeh-Nouniaz, H., 178 Nakagawa, A., 353 Nakagawa, Y., 265 Nakajima, M., 285 Nakamura, A., 45 Nakamura, G., 154, 162 Nakamura, S., 51, 145, 162(248), 163 Nakanishi, K., 354 Nakano, K., 185 Nakatsu, K., 285
AUTHOR INDEX Nakaya, R., 45 Nakyama, M., 264 Nakel, M., 696 Namba, M., 200 Namikawa, K., 416 Nanda, G., 144 Nandi, P., 144, 341 Napier, E. J., 161 Nara, T., 720 Narasimhachari, N., 135 Narasimha Murthy, S. V. K., 511, 513(6), 515(6) Narayanan, C. R., 403 Nash, C. H., 408, 409, 411, 414 Nash, H. A., 147 Nathorst-Westfelt, L., 698, 716 Navolotskaya, T. I., 214 Navon, G., 3M Nayler, J. H. C., 113 Neelakanthan, L., 353 Neher, R., 293, 294 Neidlemann, S. L., 140, 141(180), 143 Neipp, L., 131, 134, 151,767 Nelson, G. L., 391 Nelson, J. D., 156, 157 Nemec, P., 103, 109, 110(10), 130, 137, 139, 164, 165, 168(256), 169 Nescot, R., 160, 161 Neu, H. C., 620, 673 Neuhaus, F. C., 584 Neuss, N., 408, 409, 411(12) Newkirk, J. F., 14, 15, 116, 117 (62), 732 Newsam, S. W. B., 683 Newton, G. G. F., 72, 116, 117, 118, 281, 335, 339, 410, 411(16, 17), 414, 423(16, 17), 471, 472, 473(7), 548, 640, 728, 729(1), 730(1), 731 Nichol, C., 154, 156 Nicolaus, B. J. R., 136, 138 Niederwieser, A., 174, 183 Niemeyer, J., 146 Nieto, M., 688, 690, 692(14), 696, 698 Niida, T., 265, 285, 611 Nimi, O., 465, 469, 628, 631(5), 632(5) Ninet, L., 145 Nishida, N., 116, 117, 118, 208 Nishikawa, M., 285 Nishikawa, Y., 391 Nishimoto, Y., 199
791
Nishimura, J. S., 202 Nitta, K., 268 Niwa, T., 285 Noda, T., 285 Noggle, J. H., 389 Nomi, R., 465, 469, 628, 631(5), 632(5) Nomura, S., 153 Nordstrom, K., 75, 84(15) Nose, K., 185 Noseworthy, M. M., 140, 143 Novick, R. P., 45, 53(21), 55(21), 75, 91, 665, 666(4), 689 Nukada, K., 397 Numazaki, Y., 283 Nussberger, G., 281
O O'Brien, E., 131 O'Callaghan, C. H., 72, 73, 80, 82, 85(7, 26, 29), 89, 117, 670, 678, 679, 680, 684(17), 687(5), 732 0chab, S., 131,179, 198 Oda, A., 109 Oda, T., 220, 264, 265, 275, 285 Oden, E. M., 21, 119, 122, 125, 128, 129 Oden, G. H., 121 O'Dom, G. W., 375 Oesterhelt, D., 585 Oesterling, T. O., 242 0ffe, H. A., 699, 716 Ogata, K., 520 Ogawa, H., 276 Ogston, A. G., 424, 649 Ogura, M., 627 Ohashi, Y., 285 Ohata, Y., 469, 628, 631(5), 632(5) 0hkuma, K., 154, 335 Ohmori, T., 130, 133, 336 Ohnishi, M., 389, 391(2), 397 Ohno, M., 285 Ohtake, T., 275, 278(26) Oishi, M., 48 Oka, Y., 283 Okachi, R., 720 Okada, K., 339 Okami, Y., 148, 150, 265, 268, 272(1), 273, 283
792
AUTHOR INDEX
Okamoto, S., 119, 121, 125, 169, 170(263), 208, 737 Okanishi, M., 119, 121, 130, 133, 336 Okanishi, M. S., 626 Okazaki, Y., 272, 285 O'Keefe, A. E., 333, 342 Okimoto, Y., 162 Okuda, T., 145, 200, 285, 353 Okui, M., 116, 117, 118, 208 Oldfield, E., 392 Olpinska, E. Z., 204 Olson, B. H., 127, 152(111), 153(111), 154(111), 158(111), 203, 281, 342 Olson, E. C., 337, 390 Omachi, K., 119, 121, 162, 164, 169 Omoto, S., 220, 264, 285 Omura, S., 264, 309, 353 Onaga, J., 120, 122(84), 265 Ondetti, M. A., 767 Oostendorp, J. G., 181, 198(52), 199, 700 Oparysheva, E. F., 134, 170 Orchinnikov, Yu. A., 353 Ormand, R., 254 Ormond, R. E., 148, 149 Ortengren, B., 698, 716 Osaki, A., 37 Osato, T., 130 Osato, Y., 268 Osborn, M., 602, 685 Osborne, D. N., 298 Osono, T., 283 Osteryoung, R. A., 375 Otake, N., 154, 155, 156, 264 Otani, T. T., 335 Otsuka, H., 261 Ott, J. L., 116 Ottake, T., 307 Ottinger, R., 396 Ozaki, K., 155 Ozanne, B., 626, 627(27), 634 P Pagano, J. F., 146, 341, 767 Page, J. E., 388 Pal, A., 144 PaleSkov£, F., 158 Pan, S. C., 286 Pandey, R. C., 391
Pansy, F. E., 127 Paris, R., 159 Parmentier, G., 722, 723(14), 726(14), 727, 767 Parr, C. W., 259 Parry, E. P., 375 Paschal, J. W., 353 Pascua], C., 397 Pass, L., 575 Pastan, I., 739, 743(9), 745(9) Patil, G. V., 641, 648(16), 649(16) Patt, P., 341 Pattabiraman, T. N., 514 Patterson, T. L., 130, 133, 258, 259, 261 Paul, I. C., 392 Paulus, H., 18, 579, 580, 581, 582(1), 583, 584(7) Paunez, J. K., 181 Pavey, D., 116 Pazdera, H. J., 131 Peaeoeke, A. R., 649, 652(38) Peck, R. L., 339, 448, 449 Peel, E. W., 448, 449 Pehk, T., 416 Peleak, E., 140, 143 Pereira, J. N., 119, 122 Perkins, H. R., 688, 690, 692(14), 696, 698 Perlman, D., 108, 131, 151, 354, 638, 639(1), 763, 764, 767, 769, 771(12) Perlman, K. L., 354, 763 Perret, C. J., 74, 91, 642, 679 Perry, J. J., 504 Perun, T. J., 353, 359, 497 Pestka, S., 508 Peterson, D. H., 136, 138 Peterson, W. H., 123, 137, 139, 342 Pettinga, C. W., 130, 131(125), 133, 495 Pfaender, P., 549 Pfeifer, S., 203 Pfenning, N., 327, 334, 335 Philip, J. E., 281 Philipson, L., 49 Phillips, P. H., 478 Phillips, W. D., 390 Phillips, W. F., 335, 344 Pidacks, C., 305, 308(16) Piekarska, Z., 490 Pieper, S. W., 414 Pier, E. A., 397 Pike, J. E., 399
AUTHOR INDEX Piltitov£, L., 107, 167 Pinnert, S., 145 Pinsky, A., 335, 339 Pirkle, W. H., 392 Pitcher, R. 'G., 409 Pitt'a, C., 354 Pittenger, R. C., 37 Pla, L. C., 452 Plaut, G. W. E., 333, 342, 515 Pletnev, V. Z., 354 Plimmer, R. H. A., 453 Plund, N. J., 318 Poddukanaya, N. A., 354 Podojil, M., 169 PSlter, H., 202 Pogell, B. M., 508, 509, 510(3), 511,513(2, 5, 6), 514, 515 Pohl, L., 393, 396(31) Pollard, F. H., 175 Pollock, M. R., 74, 87, 91, 93(9), 94, 95, 98, 99, 640, 641, 642, 647, 648, 650(3), 652, 653, 654, 658, 659, 660, 662, 663, 664, 670(1), 671 Polsinelli, M., 38 Pomazkova, V. A., 144 Poole, J. W., 66, 77, 78 Poore, G. A., 156, 157 Pope, C. G., 17 Popov, E. M., 354 Porath, J., 717 Porter, J. N., 127 Porter, 12. F., 135 Post, O., 344 Posternak, T., 440, 443(5) Powell, A. J., 520, 533 Prath, J., 146 Prelog, V., 131, 134, 337, 767 Prescott, G. C., 504 Presti, A. E., 125 Prets~h, E., 397 Preud'homme, J., 145 Price, K. E., 66 Pridham, T. G., 5, 8(4), 337 Printz, M. P., 331,353 Probst, G. W., 145 Prokop, J. F., 13 Proshlyakova, U. V., 152 Pross, A., 397 Pruess, D. L., 409, 475, 476(2), 483, 720 Pschigoda, L. M, 354, 367
793
Puchnina, A. V., 170 Pugh, C. T., 116 Purdy, S. J., 185 Pursiano, T. A., 66 Putter, I., 125 Puza, M., 487
O Quadrifoglio, F., 353
R
Radecka, C., 185 Rafelson, M. E., 39 Rahalker, P. W., 258 Rake, G., 62 Ramachandran, L. K., 326, 337 Randall, R. J., 456, 507, 679, 757 Randall, W. A., 83, 106, 607 Randerath, E., 180 Randerath, K., 173, 179(4), 180, 181 Rangone, R., 136, 138, 159, 160, 161 Rao, K. V., 335, 337, 341, 342, 500 Rao, M. M., 508, 509(2), 511(2), 513(2) }-taper, K. B., 10 Rapoport, H., 177 Rappe, C., 416 Rappuoli, B., 207 Rasmussen, C. E., 353, 362 Rastetter, W., 408, 411(12) l~atner, D. I., 352 Rauenbusch, E., 709, 713(17), 714, 716(17) Raymond, S., 343 Reaut, J., 145 Rebello, P. F., 508, 509(2), 511(2), 513(2), 514 Rebenstorf, M. A, 399 Reber, V. H., 159 Rebstock, M. C., 159, 336 Regna, P. P., 139, 339 Regosz, A., 385 Reehfi~ek, Z., 169 Reich, E., 154, 156 Reich, H. J., 414, 416(26) Reichl, E. R., 184 Reimann, H., 264 Reio, L., 169
794
AUTHOR INDEX
Reissi, J., 396 Renn, D. W., 335 Renneberg, K.-H., 341 Reshetov, P. D., 123, 258, 259(10), 260, 274 Ressler, C., 548 Reusser, F., 354, 367 Reusser, P., 134, 281, 767 Reuwer, J. F. 539 Reynolds, P. E., 688 Rheinwald, J. G., 52 Rhoades, J. A., 264, 271, 285 Rhodes, A., 15 Rhodes, C. T., 388 Ribeiro, L. P., 279 Rice-Evans, C., 353 Richards, M., 714, 716, 720, 721, 726(7) Richmond, M. H., 70, 87, 88, 91, 92, 93, 94, 95, 96, 641, 653, 662(3), 664, 667, 670(6), 672, 673, 674, 676, 677, 679, 680, 685(10) Ridgway, J. D., 207 Riedel, B. E., 383, 384 Riegelman, S., 212 Riehen, J. N., 732 Rinehart, K. L., Jr., 119, 125, 264, 390, 391, 409 Rinert, K. E., 354 Rivard, D. E., 532, 533(3) Robbins, W. J., 337 Roberts, J. D., 414, 416(26) Robertson, J. H., 214, 217, 218, 220(2), 227, 228(10), 243, 245(25), 246, 247(25), 251, 253, 301, 309, 312(21), 314, 315 Robison, R. S., 127, 134 Rock, S., 397 Rodinov, V. M., 561 Roegner, F. R., 33 RShr, M., 111, 113(39), 114, 477, 724, 726, 728 Rolinson, G. N., 23, 111, 113, 114(38), 699, 714, 716, 720, 721, 726(7) Romano, A. A., 337 Romanova, N. B., 36 Romeo, A., 716 Ronayne, J., 393 Roncari, G., 561 Ron-Zenziper, E., 647 Rosa De Carnevale Bonino, C. D., 186
Rosdahl, V. T., 94, 95(13), 664 Rosebrough, N. J., 456, 507, 679, 757 Rosenbrook, W., Jr., 354 Roskoski, R., Jr., 549, 558(6), 560, 586, 594, 596 Rosner, J. L., 739, 743(9), 745(9) Ross, G. W., 70, 80, 82, 85(26, 29), 678, 679, 680(7, 8), 682, 683, 684(9), 685(1, 8), 686(1), 687(5) Ross, J., 335 Rosselet, J. P., 109, 265 Rossi, D., 716 Rossi, E., 186 Rothrock, J. W., 125, 267, 275, 276 Rousos, G. G., 151 Rousselet, M. R., 159 Rownd, R., 42, 45, 738 Rozanova, T. M., 134 Rubin, F. A., 77, 78 Rudin, L., 49 Rudinger, J., 467 Rudzik, M. B., 640, 642(9) Ruiter, A., 353 Russo-Alesi, F. M., 75 Ruttenberg, M. A., 331, 353 Rutten-Pinckaers, A., 687, 688(3) Ryan, A. J., 520 Ryan, C. W., 66 Ryan, F. J., 27 Ryder, A., 155 Rydon, H. N., 286 Ryu, D. Y., 706, 714(6), 717(6), 721(6) Rzeszotarski, W. J., 313
Sabath, L., 70, 73(4), 77(4), 78, 642, 652 Sabol, S. L., 352 Saburi, Y., 123, 337, 339 Sackmann, W., 133, 134, 179, 193(34) Saffer, E., 297 Safferman, R. S., 135 Saito, A., 119 Saito, K., 265 Sakagami, Y., 136, 137, 139, 622 Sakai, H., 147, 162, 338 Sakakibara, H., 285 Sakamoto, Y., 579 Sakurai, Y., 285
AUTHOR INDEX Sakurazawa, M., 285 Sallee, T. L., 177 San Clemente, C. L., 342 Sands, L. C., 738, 747, 748(5), 751, 752(18), 753, 755(5, 13, 18) Sankaran, L., 509, 510(3), 511, 513(5, 6), 515(6) Sano, Y., 338 Sargent, M. G., 74, 663, 685 Sarma, M. R., 403 Sarngadharan, M. G., 515 Sasaki, K., 155, 390, 391 Sato, E., 578 Sato, Y., 323, 324, 340 Satoh, K., 275, 277(25) Saturnelli, A., 603 Saunders, D. L., 178 Savage, G. M., 136, 138, 158, 169 Savino, M, 352 Savitsky, G. B., 416 Sawa, T., 154, 156, 285 Sawada, Y., 123, 258, 259, 354 Sawai, T., 662 663(18) S~wazaki, T., 162 Sax, K. J., 258, 261 Saxholm, H., 578 Saz, A. K., 78 Scanes, F. S., 336 Scavizzi, M. R., 51, 53, 54 Schaad, L. J., 539 Schacht, R. J., 390(19), 391 Schaefer, T., 400 Schaffner, C. P., 119, 123, 125, 135, 152, 258, 274, 275, 276(24), 277, 338 Schallock, M. R., 769, 771(12) Schenek, J. R., 281 Schenk, J., 354 Schenk, R., 335 Scherr, G. H., 39 Schimauchi, Y., 154 Schirmer. R. E., 389 Schlegel, R., 135 Schleifer, K. H., 687 Schlenk, F., 491 Schlitt, H., 182, 183 Schlossmann, K., 524, 536 Schmidt, J., 148, 149, 151 Schmidt, R., 265 Schmidt-K~.stner, G., 148, 149, 151 Schmit, J. A., 304, 305
795
Schmitt, J. P., 190, 193, 196, 197 Schmitz, H., 134, 337, 340 Schneider, H. G., 335 Schoeher, A. J., 336 Schuurmans, D. M., 127, 152(111), 153(111), 154(111), 158(111), 203, 281, 342 Schwaier, R., 35 Schwarting, A. E., 291 Schwarz, E., 549 Schwarz, J. C. P., 393, 396(30) Schwiezer, E., 585 Schwyzer, R., 337, 354 Scott, A. I., 529, 542, 543(5), 544(5), 545 (5), 548(5) Scott, J. R., 739 Scott, P. M., 540 Sebek, O. K., 22, 214 Sedkowska, J., 560 Seghal, S. N., 136, 139, 724, 726(18) Seidl, E., 179 Seiler, N., 186 Seino, A., 123 Sela, M., 641, 649(18) Self, D. A., 706, 714 Seizer, G. B., 78, 122, 140, 143 Semaka, S. D., 44, 55(14) Semar, J. B., 108 Sen, G. P., 341 Sensi, P., 136, 138, 149, 341 Senyavina, L. B., 353, 354 Sergeyeva, L. N,, 141, 144 Serizawa, Y., 162 Sermonti, G., 155, 157 Serova, L. I., 204 Seto, H., 406, 408 Seto, T. A., 720 ~ev5ik, V., 169 Seyffert, R., 529 Sezaki, M., 275, 277(25) Sferruzza, G., 159, 160, 161 Sferruzza, S., 136, 138 Shafer, H., 254 Shaikh, K., 699 Shankel, D. M., 36 Shapiro, S. K., 491 Sharon, N., 335, 339 Shaw, W. V., 737, 738, 739, 743(1, 9), 745(9), 747, 748, 750(15), 751, 752(18, 19), 753, 754, 755
796
AUTHOR INDEX
Shay, A. J., 127 Sheehan, J. C., 767 Shell, J. W., 337 Shemin, D., 522 Shepel, E. N., 353 Shepherd, D., 520, 533 Sherma, J., 179, 267 Sherratt, D., 653, 661 Sherris, J. C., 59, 66(10) Shiba, T., 285 Shibahara, S., 264, 285 Shier, W. T., 119, 125, 264 Shimada, K., 36 Shimauchi, Y., 155, 156 Shimi, I. R., 424 Shimizu, G., 162(245), 163 Shimura, M., 275, 277(25) Shingler, A. H., 72, 85(7), 89, 670, 680, 684(17) Shirato, S., 123 Shiratori, T., 169, 170(263) Shirota, F. N., 211 Shockman, G. D., 106 Shoii, J., 152 Shoji, J., 180, 261,265 Shomura, T., 265, 285 Shoolery, J. N., 397 Shotwell, O. L., 337 Shu, P., 140, 143, 261,342 Shukailo, N. I., 214 Shull, G. M., 720 Shutova, K. I., 123, 260 Sieber, P., 337 Siegerman, H. D., 375 Siemion, I. Z., 353 Sigler, G. F., 773 Sih, C. J., 409, 423 Silverstein, I~. M., 397 Silvestri, S., 385 Siminoff, P., 109, 136, 137, 138 Simlot, M. M., 549 Simmons, R. J., 65 Simon, E. J., 522 Simon, M., 42 Simon, W., 397 Simpson, R. M., 297 Sinclair, A. C., 119, 121 Singh, K., 724, 726(18) Sinsheimer, J. E., 186, 208(86) Sisenwine, S. F., 179
Sjoberg, B., 698, 716 SjSstrSm, J. E., 49 Sjolander, N. O., 19, 40 Skorvaga, M., 458, 466, 468(5), 469(5, 6), 470(5), 629, 633, 634, 636(2), 637(2) Slater, B. J., 353, 354, 364 Slater-Eng, B., 354, 363, 364(16) Slomp, G., 390, 391, 394, 395, 399 Slusarchyk, D. S., 128 Slusarchyk, W. A., 128 Smalley, H. M., 533 Smashey, A. R., 147 Smith, A. L., 622 Smith, B., 418, 472, 473 Smith, C. G., 110 Smith, D. H., 77, 78, 622, 625, 739 Smith, E. L., 115 Smith, G. N., 520, 533, 735, 736(2), 737 Smith, J. T., 81 Smith, K., 45 Smith, N., 158, 335 Smith, P. W. G., 286 Smith, R. L., 20, 21(25) Smith, R. M., 119, 121, 136, 138, 504 Smith, S. L., 390 Snatzke, F., 353 Sneath, P. H. A., 83, 111, 112(42), 641, 653, 662(3) Snell, B. K., 341 Snell, E. E., 577 Snell, N., 146, 169, 170 Snyder, L. R., 178, 291, 301 Sobic~ewski, W., 179 Sobin, B. A., 133, 339 Soeda, M., 280, 339, 341 Sohler, A., 119, 125 Sokoloski, T. D., 352, 354, 363, 364, 365, 366 Sokolski, W. T., 136, 137, 138, 158, 214 Solomons, I. A., 139, 339 Somerfield, G. A., 161 Sonoda, Y., 280 Souto, J., 171 Spaeren, U., 208 Spangle, L. A., 416 Specht, D., 549 Speedie, M. K., 499 Speert, A., 396 Spencer, B., 477, 482, 485(1) Spicer, L. D., 390
AUTHOR INDEX Spiegelberg, H., 336 Spies, J. R., 554 Spi~ek, J., 158 Spizizen, J., 757 Spring, F. S., 285 Spry, D. O., 353 Stadtman, E. R.. 482, 743 St,dfl, E., 179 Stahmann, M. A., 33 Standiford, J., 337 Stanek, J., 168 Stanley, A. R., 549 Stapley, E. 0., 110, 118, 148, 149, 153 Starcher, B. C., 353 Stark, W. M, 20, 21(25), 39, 146, 156, 157 Starke, W. M, 130, 131(125), 133 Stauffer, J. F., 16, 25, 33 Steigler, A., 134, 497 Steinman, I. D., 135 Stephen, M. J., 400 Stephens, J., 107 Stepushkin~, V. V., 160, 161 Stern, A., 331 Sternberg, H., 123 Sternhell, S., 390, 397, 398(7), 399(7), 400 Stewart, S., 354 Stiller, E. T, 333, 342 Stivers, E. C., 539 Stodola. F. H., 17, 337 Stone, R. W., 340 Stothers, J. B., 391,392, 402(25), 405, 408 Stout, H. A., 146, 767 Straeher, A., 330 StrauchovS, O., 137, 139(169) Strominger, J. L., 690, 691(15) Strong, F. M., 335 Stroshane, t/. M., 409 Struck, H., 181 Struder, R. O., 354 Struyk, A. P., 134 Sugamara, K., 341 Suhadolnik, R, J., 154, 409, 515, 759 Suhara, Y., 148, 150, 273, 274 Sukapure, I~. S., 258 Suketa, Y., 340 Sullivan. H. R., 116, 117(59) Surikova, E. I., 186 Surkova, K. I., 304 Sutcliffe, L. H., 390, 395(6) Sutherland, R., 700, 701(16)
797
Suvorov, N. N., 561 Suzaki, K., 283 Suzuki, M., 353 Suzuki, S., 153, 154, 155, 156, 162, 338 Suzuki, T., 340, 397 Suzuki, Y., 182, 208, 309, 353, 737 Swain, C. G., 539 Swart, E. A., 339, 342 Sweeley, C. C., 258 Sweeney, M. J., 156, 157 Sykes, R. B., 70, 75, 84(15), 88, 95(6), 672, 673, 674(1), 676(1). 677, 679, 685(12) Sylnons, R. H., 570 Synge, R, L. M., 285 Szabo, E.. 641, 649(14, 19) Szab5, G., 107 Szab6, I., 120 Szasz, G., 174 Szetirmai, A., 706, 725 SzilSgyi, I., 120 Szybalski, W., 42 Szymanski. H. A., 397
T Taber, W. A., 135, 337 Tada, M., 521, 530(5) Tadanier, J., 353 Takagi, Y., 36 Takahashi, B., 280, 337 Takahashi, H., 578 Takahashi, S., 285 Takaishi, N., 622 Takano, T., 116 Takasawa, S., 119, 121 Take, T., 285 Takemura, S., 258 Takeshita, R., 178 Takeuchi, M., 397 Takeuchi, S., 123 Takeuchi, T., 148, 150, 154, 156, 182, 264, 265, 268, 272(1), "273, 275, 278(27), 285, 619, 625, 626 Takita, T.. 150, 154, 264, 269, 274, 286, 287(29) Talati, P. G., 699 Tan, C. T., 392, 402(25) Tanabe, M., 406, 408, 409, 414, 416(25)
798
AUTHOR INDEX
Tanaka, N., 622, 626, 627 Taniguchi, M., 409 Tanis, R. J., 642, 648, 649(24) Taniyama, H., 123, 258, 259, 354 Tanner, F. W., 133, 341 Taraszka, A. J., 237 Tardrew, P. L., 483 Tate, M. E., 414 Tatsuoka, S., 354 Tatsuta, K., 122 Tatum, E. L., 564 Taubman, S. B., 491, 494(12) Tausig, F., 337 Taylor, E. C., 154 Teeters, C. M., 339 Tendler, M. D., 6 Tessier, J., 352 Testa, R. T., 171,172 Teuber, M., 622 Thatcher, D. R., 647, 648, 649(32), 651, 662 Thirumalachar, M. J., 258 Thomas, D., 409, 414, 416(25) Thomas, R., 113, 114(43), 128 Thompson, R. Q., 125, 341, 767 Thorne, C. B., 342 Thrum, H., 134, 135, 148, 149, 354, 722, 726(8) Timbal, M. T., 148, 149 Tinelli, R., 687 Tio, C. O., 179 Tipper, D. J., 626, 627(27), 634, 687, 690, 691(15) Tiselius, A., 537 Tishchenko, G. N., 354 Tishler, M., 264 Titus, E., 327, 333, 342, 638 Titus, E. 0., 210 Tkach, R., 126 Tobkes, M., 140, 143 Tochikura, T., 520 Todd, A., 341 TSnjes, H., 202 Toennies, G., 106 Tokunaga, K., 275, 285 Tolstykh, N. V., 127 Tome, J., 340 Tomino, S., 569, 578, 585 Tomioka, K., 162(245), 163 Tomiyama, Y., 162, 338
Tomoeda, M., 51 Toner, G. M., 335 Torriani, A. M., 74, 641, 642(17) Totsugawa, K., 265 Townsend, M. E., 36 Toyoshima, S., 199 Tozer, B. T., 336 Trappe, W., 293 Tresner, H. D., 513 Tridgell, E. J., 641, 642(13, 17) Trown, P. W., 414, 418 Trudnikova, I. N., 134 Truitt, S. T., 409 Truter, E. V., 185 Tsiganov, V. A., 339 Tsuboi, K. K., 765, 771 Tsuchida, E., 199 Tsuchiya, T., 120, 122, 272, 285 Tsuji, K., 214, 217, 218, 220(2), 222, 224, 227, 228(10), 243, 245(25), 246, 247(25), 251, 253, 278, 301, 309, 312(21), 314, 315 Tsukiura, H., 119, 121, 125, 130, 133, 265 Tsurumaki, Y., 136, 139 Tsuruoka, T., 265, 285 Tunin, D., 122 Turner, W. B., 540, 541(1) Turner-Graft, R., 335, 339 Twigg, G. H., 78 Tyran, B., 353 U Uchida, K., 155 Ueda, A., 136, 137, 139 Ueda, K., 162 Ueda, M., 265, 268, 272(1), 273(1) Uematsu, T., 759 Umezawa, H., 119, 120, 121, 122(83), 130, 145, 148, 150, 153, 154, 156, 162(248), 163, 179, 187, !88, 200(36), 264, 265, 266, 268, 269, 270(4), 271, 2.72(1), 273, 274, 275, 278(27), 279, 281(8), 282(8), 283, 285, 286, 287(29), 289, 290, 339, 619, 622, 625, 626, 627, 634 Umezawa, S., 120, 122, 264, 272, 285, 354 Umino, K., 264, 285 Uramoto, M., 264 Uri, J., 162, 164, 721
AUTHOR INDEX
Urry, D. W., 353, 389, 391(2) Urx, M., 140, 143 Usdin, E., 106 Usher, J. J., 471 Usui, N., 264, 269 Utahara, R., 119, 121, 268, 626, 634 Uzu, K., 153, 335
V Vaage, O., 585 Vagelos, P. R., 577, 585, 599 Vagina, I. I., 204 Valdebouze, P., 139, 140(170), 143 Valu, G., 721 Van Abeele, F. R., 130, 131(125), 133, 338 Vanas, D., 767 Vandamme, E. J., 720, 722, 724, 725(20), 726(19) Van den Elzen, H. M., 44, 55(14) Vanderhaeghe, H., 158, 159, 232, 722, 723(14), 726(14), 727, 767, 770 Vanderheuvel, W. J. A., 254 Van Dijck, P., 158, 159, 767 van Eck, T., 134 Vanek, Z., 158, 168, 487 Vangedal, S., 353 van Giessen, B,, 224 van Leeuwenhoek, A., 700 Van Tamelen, E. E., 258, 260(6) Vazquez, E. A., 199 Velluz, L., 348 Venkatasubramanian, K., 706, 714(6), 717(6), 721 (6) Vergine, V. J., 304 Verrastro, D., 178 Vetlugina, L. A., 153, 169 Vezina, C., 136, 139, 724, 726(18) Vignau, M., 352 Vining, L. C., 135, 151, 337, 406, 735, 737(1) Vink, H., 175 Vischer, E., 133, 134, 148, 149, 179, 193(34), 281 Viswamitra, M. A., 767 Vivilecchia, R., 301 Vlietinck, A., 722, 723(14), 726(14), 727 Voelter, W., 369 Voets, J. P., 720, 722, 724, 725(20), 726(19)
799
Vogel, G., 531, 533(2), 534(2), 538(2) Vogler, K., 354 Voinova, Z. E., 160, 161 yon Ammon R., 389, 401(4) Vondr~Sek, M., 158, 169 Vondr£Skov'£, J., 137, 139(169), 140, 143 yon E. Doering, W,, 402 yon Laer, V., 35 Voser, W., 128, 129, 133, 134, 179, 193(34), 281 Vrit~kov~, A., 169 Vtorova, Z. I., 204
W Waehnert, U., 354 Wagman, G. H., 21, 102, 106, 1J0(8), 115(8), 119, 120, 121, 122, 125, 126, 128, 129, 130, 138, 171, 172, 201, 291 Wagner, R. L., 339 Wahlqvist, S., 114 Waisviz, J. M., 134 Wakae, M., 119, 121, 125 Wakaki, S., 153, 162(245), 163 Wakamiya, T., 285 Wakazawa, T., 276 Waksman, S. A., 8, 135, 151, 339 Waldi, D., 212 Waldschmidt-Leitz, E., 723, 724(16), 725(16), 726(16) Walker, J., 763 Walker, J. B., 431, 433, 434, 435, 436(2), 437(5), 438(5), 440, 441, 442, 443(10), 444, 445(2), 448, 449(14), 450, 451, 452, 453(3, 9), 454(2, 6, 7), 455(2, 4, 7, 8), 457, 458, 459, 460(2), 461(2, 4), 462, 463(2), 464, 465(2, 8), 466, 468(4, 5), 469(4, 5, 6), 470, 621, 628, 629, 630, 631, 632(4), 633, 634, 636(2), 637(2) Walker, M. S., 440, 441, 444, 445(2), 448, 449(14), 450, 451,452(3), 453(3), 458, 459, 460(2), 461(2), 462, 463(2), 464(2), 465(2), 466, 468(4), 469(4), 470(4), 621, 628, 629, 630(7), 631(2), 632(4), 633, 634 Waller, G. R., 154 , Waller, R. E., 73 Wallh~usser, K. H., 134, 497
800
AUTHOR INDEX
Walsh, J. M., 177 Walter, H., 521, 524(6), 528(6), 529(6), 530(6), 533 Walti, A., 448 Walton, R. B., 116, 117(62), 720, 732 Warburg, O., 500 Warburton, D., 699, 709, 713(16), 714, 717, 721(18, 27) Warren, B., 179 Warren, H. B., Jr., 13 Warren, S. C., 410, 411(16, 17), 423(16, 17), 472, 473(7) Wasson, J. R., 390 Watanabe, H., 44, 265 Watanabe, I., 120, 122(83), 220, 265 Watanabe, M., 339 Watanabe, S., 283, 339 Watanabe, T., 45, 51, 111, 113, 134 Waters, J. L., 301 Webb, K. H., 400 Webber, B. B., 32 Weber, K., 802, 685 Weidenmiiller, H. L., 134, 497 Weigert, F. J., 414, 416(26) Weinstein, M. J., 21, 102, 106, 110(8), 115(8), 119, 120, 121, 122, 125, 126, 128, 129, 130, 138, 139, 146, 171, 172, 201, 291, 767 Weisenborn, F. L., 128, 140, 141(180), 143 Weisiger, J. R., 326, 330(12), 335, 337, 548 Weiss, F. A., 4 Weissbach, H., 202, 209, 767, 769 Welsch, M., 687, 688 Weltzien, H. C., 107 Wenkert, E., 409 West, C. A., 258 Westhead, E. W., 540 Westley, J. W., 123, 258, 409 Wettstein, A., 131, 133, 134, 179, 193(34), 281 Wetzel, E. R., 258, 259, 261, 263(18) Whaley, H. A., 130, 133, 258, 260(6) Whitaker, J. R., 279 White, R. J., 409 Whitehead, B. Y., 23, 638, 639(3), 640(3) Whitehouse, R. L. S., 44, 55(14) Whitfield, G. B., 258, 260(6) Whitney, J. G., 116, 117 Wicker, K. J., 116, 117(64) Wickerham, L. J., 4
Wieland, T., 180 Wiele, H., 177 Wiley, P. F., 131, 495 Wilgus, R. M., 39, 158, 159 Wilkins, J. R., 169 Wilkinson, R. G., 140, 143 Wilkinson, S., 147 Willcott, M. R., III, 402 Willecke, K., 585 Williams, C. A., 99 Williams, D. E., 153 Williams, D. H., 393 Williams, J. H., 140, 143 Williams, R. C., 304, 305, 331 Williams, R. G., 306 Williams, R. H., 156, 157 Williams, S. T., 5 Willis, M., 57 Willmer, N. E., 120, 122(84), 265 Wilson, A. N., 153 Wilson, W. L., 185, 222, 318 Winfield, A. F., 119, 121 Winlder, M. F., 515 Winshell, E., 751, 752(19), 753(19) Wise, W. S., 78 Witchit~, J. L., 54 Wnuk, R. J., 136, 138 Wolf, E. J., 160, 161 Wolf, F. J., 153, 254, 267, 275(5), 276(5), 337 Wolfe, R. N., 146 Woo, P. W. K., 120, 122(84), 265, 285 Wood, G. C., 354 Woodruff, H. B., 110, 118 Woods, B. L., 145 Work, T. S., 339, 548 Worrel, C. S., 735, 736(2), 737 Wren, J. J., 259 Wright, H. M., 36 Wright, W. W., 78, 140, 141, 143 Wunderly, C., 279 Y Yacobson, L. M., 214 Yagashita, K., 130 Yagi, A., 279, 281(8), 282(8), 283, 285 Yagisawa, M., 274, 275, 278(27), 619, 625, 626
AUTHOR I N D E X
Yagisawa, N., 353 Yagishita, K., 265, 268, 272(1), 273(1) Yaguchi, M., 275, 278(26) Yakovleva, E. P., 339 Yakubov, G. Z., 141, 144, 145, 153, 169 Ya,nabayashi, S., 136, 137, 139 Yamada, H., 136, 138, 577 Yamada, M., 569, 571, 575, 577(5), 578, 585 Yamagishi, M., 281,627, 634 Yamaguchi, T., 145, 200 Yamamoto, H., 153, 264, 266, 270(4), 272, 275, 278(27), 619, 626 Yamamoto, M., 720 Yamamoto, O., 397 Yamamoto, T., 109 Yamaoka, K., 352, 354 Yamashita, S., 162, 338 Yamatodani, S., 113 Yamauchi, Y., 123 Yamazaki, Y., 182 Yanagisaws, M., 397 Yaniaguchi, M., 122 Yashin, Y. I., 301 Ycager, R. L., 227 Yehaskel, A. S., 264 Yclin, R. E., 397 Yokoo, M., 281 Yokose, K., 274 Yokota, Y., 117, 208 Yokoyama, T., 264 Yonehara, H., 123, 154, 155, 156, 162(248), 163, 253, 264, 335, 339 Yoshida, H., 285 Yoshida, T., 152, 203 Yoshimura, Y., 627 Yoshioka, H., 285
801
Young, R. W., 19 Ysern, X., 392 Yunsten, H., 154, 335 Yurina, M. S., 127
Z Zaborsky, O., 717 Zachau, H. G., 767 Ziihner, H., 128, 129, 131, 134, 151, 179, 193(34), 281, 767 Zaffaroni, A., 130 Zahner, H., 337 Zak, A. F., 214 Zanotti, G., 716 Zappia, V., 491 Zarnach, J., 202, 203 Zbinovsky, V., 259 Zderie, J. A., 353 Zepf, K. H., 151 Zhdanov, V. G., 36 Ziffer, H., 352, 354 Ziffer, J., 16 Zimmer, C., 354 Zimmer, T.-L., 568, 570, 572, 575, 576. 577(2, 12), 578, 585, 586 Zimmerman, F. K., 35 Zimmerman, S. B., 110, 118 Zinner, M., 477, 726 Zipser, J., 753 Zuidweg, M. H. J., 181, 198(52), 199 Zurcher, R. F., 397 Zweig, G., 179, 267 Zydek-Cwick, C. R, 491 Zyk, N., 75, 83 Zykalov'~, K. A., 354
802
SUBJECT INDEX
Subject Index A Aabomycin, 137 7-ACA, see 7-Aminocephalosporanic acid 3'-Acetamido-3'-deoxyadenosine, 154 ['4C] Acetoxy-chloramphenicol derivatives, 743 Acetyl-n-alanyl-D-glutamic acid, 697 ['~C]Acetyl chloramphenicol, 743-746, see also Chloramphenicol acetyltransferase, radioactive assay Acetyl-coenzyme A, 521, 540 Acetylesterase, cephalosporin, see Cephalosporin acetylesterase N~-fl-l,4-N-Acetylglucosaminyl-N-acetyl-
muramyl-L-alanyl-D-isoglutaminyl)N ~-(pentaglycyl)-L-lysyl-D-alanyl-Dalanine, 697 N%Acetyl-L-lysyl-D-alanyl-D-alanine, 697 N-Acetylneomycins, 120 Acetyltransferases, see specific acetyltransferase A c h r o m o b a c t e r sp. fungal penicillin acylase from, 722 penicillin acylase from, 719 Acridine orange as curing agent, 51 Acridine as mutagen, 35 Actidione, see Cycloheximide Actinamine, 216-217 Actinoidin, 127 Actinomycetes antibiotic fermentation by, 18-21, s e e also specific antibiotic media for maintaining, 5-7, see also Media for maintaining Actinomycetes soil culture for, 8 Actinomycin chiroptical studies of, 352 circular thin-layer chromatography, 180 countercurrent distribution, 327 high-pressure liquid chromatography, 313-319
paper chromatography, 150-151 solvent systems for countercurrent distribution, 334 synthesis, 763-767, see also Actinomycin lactonase thin-layer chromatography, 187, 202 Actinomycin lactonase, 763-767 Actinoplanes, 765-766 ammonium sulfate fractionation, 765 assay, 764 calcium phosphate gel chromatography, 765-766 crude extract preparation, 765 culture preparation, 764-765 DEAE-cellulose column chromatography, 766 inactivation product, 767 properties, 767 purification, 764-766 reaction scheme, 763 Sephadex G-200 column chromatography, 766 A c t i n o p l a n e s missouriensis actinomycin from, 763, see also Actino-
mycin lactonase peptide antibiotic lactonase from, 767, see also Peptide antibiotic lactonase Actinospectacin paper chromatographic data of, 121 solvent system for, 119-120 Actinospectinoic acid, 216-217 Actithiazic acid, 334 Acumycin, 134 Acyl-CoA:6-aminopenicillanic acid acyltransferase, 474-476 activators, 476 ammonium sulfate precipitation, 475 assay, 474-475 hydroxypatite gel adsorption and elution, 475 P e n i c i l l i u m c h r y s o g e n u m preparation, 475
SUBJECT INDEX pH effect, 476 purification, 475-476 Sephadex G-200 column chromatography, 475 specific activity, 474 specificity, 476 unit definition, 474 Aeylhydroxamate, 477 Acyltransferase, see Acyl-CoA:6-aminopenieillanic acid acyltransterase ['y2~P]Adenosine triphosphate, inosamine kinase assay, 451 S-Adenosylmethionine (SAM) puromycin assay, 509 SAM : dedimethylamino-4-aminoanhydrotetracycline N-methyltransferase 603-606 SAM:erythromycin C O-methyltransferase, 487-498 SAM :indolepyruvate 3-methyltransferase, 498-502 S-Adenosylmethione: dedimethylamino4-aminoanhydrotetracycline N-methyltransferase, 603-606 assay, 603-605 properties, 605-606 reaction scheme, 603 Streptomyces aureo]aciens preparation, 605 Streptornyces rimosus preparation, 604 S-Adenosylmethionine: erythromycin C O-methyltransferase (SAM :EaDM transmethylase), 487--498 S-adenosylmetrionine, 490-491 ammonium sulfate precipitation, 492, 494 assay, 488-491 cell-free extract, 492 cellular location, 495-496 L-cladinose moiety, radioactivity of, 489 erythromycin A derivatization of, 494 measurement of formation from erythromycin C, 489 recrystallization, 490 erythromycin C (EaDM) and its spiroketal (6 --~ 9:12 --> 9), 490 incubation, 488-489 inhibitors, 496
803
kinetic properties, 496-497 partition chromatography, 492-493 pH effect, 496 purification, 491-492 radioactivity measurement, 495 scintillation system, 495 specificity, 497 Streptomyces erythreus preparation, 491 temperature dependence, 496 thin-layer chromatography, 493-494 transmethylase reaction product, 495 transmethylase reaction reversibility, 495 S-Adenosylmetrionine : indolepyruvate 3-methyltransferase, 498-502 ammonium sulfate treatment, 500-501 assay, 499 Bio-Gel A-Sm chromatography, 501 crude extract, 500 DEAE-Sephadex chromatography, 501 inhibitors, 502 kinetic properties, 502 molecular weight, 502 pH effect, 501-502 purification, 500-501 reaction scheme, 498 Sephadex chromatography, 501 specificity, 502 Streptomyces griseus preparation, 500 unit definition, 500 Adenylstreptomycin, 121 Adsorption chromatography, 173-174 calculation, 174 Aerobacter aerogenes, fl-lactamase in, 673 Aerobic euactinomycetes, 687-688 Agar Bennett's, 6 Czapek's solution, 4 dilution assay, 62 malt extract, 5 N-Z amine-starch-glucose, 7 nutrient, 5 oatmeal, 6 potato dextrose, 4 TGY, See Tryptone glucose yeast extract tomato paste oatmeal, 6
804
SUBJECT INDEX
trypticase-yeast extract, 6 yeast extract, 5 Agar gel electrophoresis, 287-289 relative mobilities of antibiotics on, 288 Aglycones of olivomycin, 334 Aglycosidic nonpolyene macrolides, 136-137 Agrobacterium tume]aciens, chloramphenicol acetyltransferase in, 738 L-Alanine: 1D-l-guanidino-l-deoxy-3keto-scyllo-inositol aminotransferase, 462-465 assay, 462-464 biological distribution, 464 inosamine transaminase separation, 463-464 specificity, 464 stability, 465 Albomycetin, 131 Alcaligenes ]aecalis, penicillin acylase in, 718 Alicyclic antibiotics, 156-159 Alkylating agents, mutation induction by, 32-35, see also specific agent Alternariol, 163 Amaromycin, 131 Amicetin paper chromatography, 153 solvent system for countercurrent distribution, 334 Amidase, 208 Amidinomycin, 145-146 Amidinotransferase, 451-458, see also T.-Arginine :inosamine-P amidinotransferase Amino acid antibiotics, paper chromatography, 144-152, see also specific antibiotic 5,-(a-Aminoadipyl) cysteinylvaline, 471 -~-(a-Aminoadipyl)cysteinylvaline synthetase, 471-473 assay, 471 Cephalosporium acremonium preparation, 473 specificity, 473 DL-a-Aminoadipic acid ethyl amide, 411--412 7-Aminocephalosporanic acid (7-ACA), 699
bioautography, 117 CMR spectroscopy, 411-412 fl-lactamase hydrolysis, 690 paper chromatography, 116-118 solvent systems, 116 Aminocyclitol antibiotics, 215-217, see also Aminoglycoside-modifying antibiotics 7-Aminodeacetylcephalosporanic acid, 730 3-Amino-3-deoxy-D-glucose, 272 2-Amino-2-deoxy-neo-inositol-5-P, 457 9- (3'-Amino-3'-deoxy-fl-D-ribofuranosyl) 6-dimethylamino-9H-purine, 211 Aminodeoxy-scyllo-inositol, 432 ATP :inosamine phosphotransferase assay, 445-448 L-glutamine : keto-scyllo-inositol aminotransferase assay, 440 rayo-inositol : N A D + 2-oxidoreductase assay, 434 1-Amino-l-deoxy-scyllo-inositol-4-P, 432 1D-I-Amino-l-deoxy-scy llo-inositol-6-P ,
432 5-Amino-4-deoxy-a,a-trehalose, 289-270, 272 3-Amino-4,7-dihydroxy-8-methyl coumarin, 506 Aminoglycoside antibiotics, see also Aminoglycoside-modifying antibiotics gas-liquid chromatography 217-218 high-pressure liquid chromatography, 278 ion-exchange chromatography, 263-278, see also specific antibiotic Amberlite CG-50 column, 266 ammonia gradient, 270 carboxylic acid resins, 268 commercial cation exchangers, 266 Dowex l-X2, 287 phosphonic resins, 272-273 resin columns preparation, 267-268 sulfonic acid resins, 272-273 nonionic adsorption chromatography, 275-280 paper chromatography, 119-122, see also specific antibiotic Aminoglycoside-modifying enzymes, 611-632, see also specific enzyme
SUBJECT INDEX assay, 612-619 cofactors, 617 crude extract preparation, 620 DEAE-cellulose chromatograph)', 622-623 enzymic lysis by lysozyme E s c h e r i c h i a coli, 621 enzymic lysis by lysostaphin, 621-622 French pressure cell, 621 nlolecular weight, 623 osmotic shock, 620 t)olymyxin B, 622 properties, 623--628 purification, 622-623 sonication, 620 Streptomycetes, growth medium preparation, 619 1D-1 -Amino-3-guanidino-l,3-deoxys c y U o - i n o s i t o l - 6 - P , 432 6-Aminopenicillanie acid (6-APA) chromatographic assay, 700 differential pulse polarography, 387 hydroxylamine assay, 699-705, see also Penicillin aeylase a-lactamase hydrolysis, 690 paper chromatography, 110-116, see olso Penicillin, paper chromatography penicillins separated from, 115-116 quantitative estimation, 115-116 6-Aminopenicillanic acid aeyltransferase, 474-476, see also Aeyl-CoA:aminopenieillanic acid acyltransferase Aminotransferase, see L-Alanine :ID-Iguanidino-l-deoxy-3-keto-scylloinositol aminotransferase, L-Glutamine:keto-scyllo-inositol aminotransferase Amphotericin A, 135 Amphotericin B, 136, 200 Ampieillin differential pulse polarography, 382 high-pressure liquid chromatography, 309-311, 319 penicillin a:.ylase assay, 701 Angolamycin, 134 Angustinycin, 154 Anhydrochlorotet racycline, 199 Anhydrocycloheximide, 158 Anhydroerythromycin, 309
805
Anhydrooxytetracycline, 199 Anhydrotetracycline, 199, 317 Anion-exchange resins in nonionic adsorption chromatography, 275-280 Anthelvencin, 145-146 Anthracycline, 141-144 Anthracyclinone, 141-144 Antibiotic interaction with biopolymers, 367-369 Antibiotic-producing microorganism, 3-21, see also Antibiotic production in liquid culture, Culture, Media Antibiotic production in liquid culture, 11-14 fermentation, 12 inoculum, 11-12 medium A-4, 13 medium A-9, 14 medium A-12, 14 Antibiotics relative inhibitory coefficient of, 66--69 screening for antitumor activity, 203 Antiflmgal polypeptide, 334 Antimycin A, 135, 253 Antimycins chiroptical studies, 352 paper chromatography, 137 solvent systems for countercurrent distribmion, 334 Antist,q)hylococcal penicillinase, 87 Antitumor activity of antibiotics, screening, 263 6-APA, see 6-Aminopenicillanic acid Apoxytetracyclines, 199 L-Arginine :inosamine-P amidinotransferase, 451-458 assay, L-[ guanidino-i~C] arginine, 452-454 canavanine, 454-455 hydroxylamine, 455-456 ATP :inosamine phosphotransferase assay, 447 biological distribution, 457 canavanine assay, 454-455 canavanine :amlnonium hydroxide transamination, 458 chemically phosphorylated inosamine derivative, 453-454 L-[ guanidino-~C ] arginine assay, 452-454
806
SUBJECT I N D E X
hydroxylamine assay, 455-456 inhibitors, 458 natural acceptors preparation, 453 properties, 457-458 purification, 453, 456-457 specific activity unit, 456 specificity, 457 unit definition, 456 Arginine: ornithine exchange reaction, 452 Aromatic antibiotics, see also specific antibiotic gas-liquid chromatography, 228-234 high-pressure liquid chromatography, 301-305 paper chromatography, 159-163 solvent systems, 163 Ascosin, 136 AspergiIlus ochraceus, 721 AspergiUus ustus, 323 Asperlin, 409 Assays, antibiotic, general discussion, 55-69 diffusion, 60-61 dilution, 62-65, see also Dilution assay of diverse biological samples, 65-66 microbiological, 55-69 organisms used in, 57-58 photometric method, 63-65 quantitative aspects, 58-59 relative inhibitory coefficient, 66-69 screening methods, 58 theoretical equations, 66-67 ATP:Dihydrostreytomycin-6-P 3'a-phosphotransferase, 634-637, see also Dihydrostreptomycin-6-P 3'a-kinase ATP :Inosamine phosphotransferase, 444-451, see also Inosamine kinase ATP :Streptomycin 3"-phosphotransferase, 632-634, see also Streptomycin 3" kinase ATP :Streptomycin 3'-prosphotransferase, 628-632, see also Streptomycin 6-kinase [~22P]ATP, insomaine kinase assay, see [722P]Adenosine triphosphate, inosamine kinase assay Aureofungin, 136 Aureolic acid, 127 Aureomycin, 334
AUTOTURB system, 56, 69 Auxotrophs, 38 Auxotrophy as genetic marker, 44 Avilamicin, 128 Azacolutin, 136 Azalomycin, 137 Azoxyketone, 352
B BA-9O, 912, solvent system, 334 BA-181,314, solvent system, 334 BA-6903, solvent system, 334 Bacillus brevis
edeines, 560, 564 gramicidin S, 567 tyrocidine synthetase, 587-588 Bacillus cereus, fl-lactamase, 98, 640-652, see also fl-Lactamase from Bacillus cereus Bacillus circulans
aminoglycoside antibiotics, 265 circulin, 579 Bacillus licheni]ormis
bacitracin, 548 fl-lactamase, 93, 94, 653-664, see also fl-Lactamase from Bacillus licheni]ormis Bacillus megat~rium, penicillin acylase, 711-721, see also Penicillin acylase from Bacillus megaterium Bacillus mycoides, hydrolase in, 735 Bacillus polymyxa, polymyxin, 17-18,
579, 580-581 Bacillus subtilis
aminoglycosidic antibiotic, 120 cephalosporin acetylesterase, 731-734, see also Cephalosporin acetylesterase, from Bacillus subtilis cephalosporin, 117 enzyme A, 735 everninomicin group, 128 fl-lactamase, 247 lincomycin group, 128 media for, 16-17 penicillin, 111 tetracycline, 140 Bacitracin biosynthesis, 548--559, see also Bacitracin synthetase
SUBJECT
countercurrent distribution, 326, 330 solvent system, 334 for structural studies of, 330 high-pressure liquid chromatography, 314, 319 paper chromatography, 146 structure, 548 Baeitracin synthetase, 548-559 ammonium sulfate precipitation, 558 assay, 551-556 ATP-3~PP1 exchange measurement, 551, 553, 555, 558-559 Bcwillus licheni]ormis preparation, 198 cell lysis, 556 cells, harvesting and storage of, 556 DEAE-cellulose chromatography, 557 diafiltration and concentration, 557 disc gel electrophoresis, 554, 558 fermentation, 554-556 Micrococcus flavus preparation, 551 MiUipore filter test, 552, 553, 558 molecular weight, 555 protein determination, 554 purification, 556-558 radio thin-layer chromatography, 552, 553-554 reaction scheme, 549 SDS disc gel electrophoresis, 554 Sephadex G-50 chromatography, 556 Sephadex G-200 chromatography, 557 sucrose density gradient centrifugation, 554 unit definition, 549 Bacterial penicillin acylase, 705-725, see also Penicillin acylase of Bacillus megatarium, Penicillin acylase of Escherichia coli
Bacteriophage P1 in Escherichia coli, 45-47 Bacteriophage P22 in Salmonella typhimurium, 47 Base analogs as mutagen, 35 BB-K8 (Amikacin), structure, 612 Benzylpenicillin, 476 6-aminopenicillanic acid converted to, 111 K,,, and Vm~xvalues, 689 /8-1actamase assay, 71 penicillin acylase assay, 699 BenzylpenieiUin-enzyme complex, 688
807
INDEX
Bioautography, 105-110, see also specific antibiotic antibiotics antibacterial, 106-107 antifungal, 107-108 antileptospiral, 109 antiprotozoal, 109 antiviral, 109 cytotoxic, 108-109 correlative assays, 110 paper electrophoresis, 282 penicillin, 111 phage-inducing antibiotics, 108 thin-layer chromatography, 179-186 Biosynthetic 6-methylsalicylic acid, 533-535 Biosynthetic penicillins, 115 2,2'-Bipyridine, 502 Blasticidin S, 47 Bleomycin group, 148, 150 Bluensidine, 429 moiety of, equation, 430, 444 Bluensomycin biosynthesis inosamine kinase, 444-451, see also Inosamine kinase of the guanidinated inositol moieties of, 429-433 bluensidine moiety, 430, 444 chiroptical studies of, 352 solvent system, 119-120 structure, 429 Bostrycoidin, 334 Brevicid, 202 Butirosin, solvent system, 119-120 structure, 613 6'-N-t -Butyloxycarbonylkanamycin, 289-290 C Caffeine, as mutagen, 36 Calonectria, 721
Canavanine, 454-455 Canavanine :ammonium hydroxide transamination, 458 Candicidin, 136 Candida albicans, stendomycin assays, 771
808
SUBJECT INDEX
Capreomycin, 146 Carbohydrate antibiotics, 119-129, see also specific antibiotic Carbomycin, 131, 198 Carbon molecular resonance spectroscopy, see CMR spectroscopy Carrier-free continuous electrophoresis, 289-290 CAT, see Chloramphenicol acetyltransferase Cefazolin, 41, 73 Celesticetin, 336 Celestosaminide, 253 Cephalexin, 73, 411-412 Cephaloglycin, 117, 387 Cephaloridine, 73 hydrolysis by Enterobacter cloacae P99 cephalosporinase, 687 interaction with staphylococcal penicillinase, 671 ~8-1actamase assay, 71 thin-layer chromatography in pharmacokineties studies, 211 Cephalosporoic acid, 71 Cephalosporin C, see also Cephalosporins bioautography for, 117 CMR spectroscopy, 408, 410, 411-417, 418-425, see also CMR spectroscopy high-pressure liquid chromatography, 311-312, 319 macroreticular resin chromatography, 299 paper chromatography, 117-118 Cephalosporin P series, see Alicyclic antibiotics Cephalosporin acetylesterase, from Bacillus subtilis, 731-734 assay, 732-733 culture preparation, 733 inhibitors, 734 kinetic properties, 734 molecular weight, 734 pH optimum, 734 purification, 733-734 specific activity, 733 stability, 734 substrates, 734 unit definition, 733
Cephalosporin acetylesterase, citrus, 728-731 activators, 730 assay, 728-730 manometric, 728-729 potentiometric, 729 crude extract preparation, 729 kinetic properties, 731 pH optimum, 731 properties, 730-731 reaction scheme, 728 specific activity, 729 specificity, 730 stability, 730-731 unit definition, 729 Cephalosporinase assays, see fl-Lactamase Enterobacter species, 678, see also fl-Lactamase from Enterobacter species R factor-mediated, 678 specific anti-fl-lactamase sera for quantitative study of, 86-100, see also Immunological techniques Cephalosporins bioautography, 117 fl-lactamase assays, 71 paper chromatography, 116-118, 159 solvent systems for countercurrent distribution, 334 thin-layer chromatography, 207-208 Cephalosporium acremonium
~-(L-a-aminoadipyl)-L-cysteinyl-Dvaline, 471 carbon molecular resonance spectroscopy, 411,414 fungal penicillin acylase from, 721 media for, 14 Cephalothin, 72-73 bioautography, 117 interaction with staphylococcal penicillinase, 671 Cephamandole, 73 Cephamycins, 71, 110 bioautography, 117 paper chromatography, 116-118 Cephem antibiotics, 410 Champamycin, 136 Chelocardin, 367
SUBJECT INDEX
Chiroptical methods, see Speetropolarimetry Chloramphenieol o-acetoxy derivatives of, 738 acetyltransferase, see Chloramphenieol acetyltransferase biotransformation of, 208 chiroptical studies, 352 diger(mtial pulse polarography, 375-376, 377, 380, 382--383 enantiolneric p-phenyl analog of, 366 gas liquid chromatography, 228-232 calculation, 232 hydrolysis at amide bond, 734-737 paper chromatography, 159-160, 162 preparative thin-layer chromatography, 185 resistance, 737 solution spectrophotometry with thinlayer chromatography, 186 solvent system for eountercurrent distribution, 336 spectropolarimety, 364-366 Streptomgces venezuelae, media for, 18 strncture of compounds related to, 737 Chloramphenicol acetyltransferase, 739-754 acetylation techniques, 739 I'~C]aeetyl chloramphenicol direct measurement, 743-746 activators, 755 acyl acceptor, specificity for, 753 acyl donor, specificity for, 753 affinity chromatography, 750 alumina gel procedure, 750 ammonium sulfate precipitation, 748 assay, 740-746, see also specific assay reaction sequence, 740 bacterial growth, 747-748 eatabolite repression of synthesis of, 738-739 chromatographic detection of acetylation, 740-741 eoenzyme A sulfhydryl group, 742 crude extract preparation, 748 DEAE-cellulose chromatography, 749 gel filtration, 749 genetic mode of synthesis, 754 heat stability, 754
809
hybrid formation, 755 inhibitors, 755 Km values, 754 molecular weight, 752 native tetrameric ehloramphenicol acetyltransferase, 755 phages carrying gene for, 739 pH optimum, 752 properties, 752-754 purification, 746-752 from Escherichia coli, 747-751 from other bacterial species, 751-752 radioactive assay, 739, 743-746 [uC]acetyl-coenzyme A, 744 culture preparation, 745-746 pyl'uwtte-kinase system, 744 specificity, 753 spectrophotometrie assay, 739, 742-743 stability, 755 from Staphylococcus spp., 751-752 streptomycin sulfate precipitation, 748 structural gene, 738 thin-layer chromatography, 739, 740-741 Chloramphenicol hydrolase, Streptomyces, 734-737 acetone powder, 736 assay, 735 culture preparation, 736 properties, 736 purification, 736 reaction scheme, 734 Chloramphenicol palmitate, 59 Chloramphenicol-resistant bacteria, chloramphenicol aeetyltransferase from, 737-754, see also Chloramphenicol acetyltransferase Chloramphenicol stearate, 159 Chloramphenicol suecinate, 159 7-Chloro-6-demethyl-5a, 6-anydrotetraeycline, 140 7-Chloro-6-deinethyl-4-didemethylaminotetraeycline, 140 Chloroform, as solvent for thin-layer chromatography, 205 Chloronitrophenols, 168 Chlorothriein, 409 Chlortetracycline electroanalytical techniques, 385 high-pressure liquid chromatography, 317
810
SUBJECT I N D E X
paper chromatography, 140 media, 19 thin-layer chromatography, 199 Chromatograms paper, see Paper chromatography pH, 164-168 salting-out, 162-164 Chromatography adsorption, 173-174 circular thin-layer, 180 gas-liquid, 213-256, see also Gas-liquid chromatography gradient, 179-180 high-pressure liquid, 278, 300-32, see also High-pressure liquid chromatography ion-exchange, 256-278, see also Ionexchange chromatography macroreticular resin, 296-299, see also Macroreticular resin chromatography nonionic adsorption chromatography, 275-280, see also Nonionic adsorption chromatography paper, see also Paper chromatography partition, 174-175 programmed multiple development (PMD), 180 reversed-phase partition, 180 silica gel, 291-296, see also Silica gel chromatography thin-layer, 172-213, see also Thin-layer chromatography Chromin, 135 Chromomycin As, 127 Chromomycin, 352 Cinerubin, 145 Circular dichroism (CD), see also Spectropolarimetry equations, 355 Circular thin-layer chromatography, 180 Circulin, 579 Cirramycin, 336 Citrus cephalosporin acetylesterase, 728-731, see also Cephalosporin acetylesterase, citrus "~C-labeling in study of antibiotic biosynthesis, 404425, see also CMR spectroscopy S t r e p t o m y c e s aureolacien~,
L-Cladinose moiety of erythromycin A, 489 Clindamycin biosynthesis, 755-759, see also Clindamycin phosphotransferase gas-liquid chromatography, 237-239 phosphorylation, 755-759, see also Clindamycin phosphotransferase Clindamycin palmitate, 243-245 Clindamycin phosphate gas-liquid chromatography, 242-243 high-pressure liquid chromatography, 306-307, 319 Clindamycin 3-phosphate, 756 Clindamycin phosphotransferase, 755-759 ammonium sulfate fractionation, 757-758 assay, 756-757 crude extract preparation, 757 DEAE-cellulose column chromatography, 758 nucleoproteins removal, 757 properties, 758-759 purification, 757-759 reaction scheme, 755 specific activity, 757 specificity, 758 stability, 758 stoichiometry, 759 unit definition, 756 Cloxacillin, 671 Clupeine, 336 CMR spectroscopy ~3C-enriched precursor, 407, 411, 417 cephalosporin C biosynthesis, 408-409, 411-417, 418-425 chemical shifts in chephalosporin models, 415 in spectra, 415-417 experimental conditions, 406-408 fermentation, 411,414 high field spectrometer, 405 instrumental requirements, 405-406 isolation, 411, 414 labeling conditions, 407-409, 411, 414 fl-lactam antibiotics biosynthesis, 410-425 model compounds synthesis, 411 n~tural abundance CMR spectrum, 4O7
SUBJECT I N D E X
Nuclear Overhauser effect, 407 penicillin V biosynthesis, 414, 418-425 pH dependence of resonance in cephalosporin C, 415-416 preliminary ~*C experiment, 407 proton noise decoupling, 406 pulsed Fourier transform, 406 satellite method, 406 spectrum assignments of chemical shifts in, 415-416 recording, 414-415 nC Nuclear magnetic resonance spectroscopy Conjugation of antibiotics, 210 Conjugative resistance plasmids, 42, s e e also Resistance plasmids Cordycepin, 154 Correlative assays, 110 Cotton effects, 349 Coumeromycins, 336 Countercurrent distribution, 320-346 apparatus, 341-346 bacitracin, 326 distribution assembly, 344 isolation from growth medium, 320-324 molecular weight determination, partial substitution, 328-330 nonideality, 331 phenol as solvent for highly polar solutes, 333-334 polypeptide antibiotics, 324-327 Post trains, 343-344 purification, 324-327 purity, testing for, 327-328 Raymond trains, 343 rotatory evaporation apparatus, 345-346 structur,d studies, 330-331 solvent systems, 331-341 tube design of counter-double-current distribution train, 345 Culture collections American Type Culture Collection, 3 Centraalbureau voor Schimmelcultures, 3 Commonwealth Mycological Institute, 3 Institute for Fermentation, 3
811
National Collection of Industrial Bacteria, 3 Northern Utilization Research and Development Division, 3 liquid, 11-14, s e e also Antibiotic production in liquid culture maintenance, 3-8, see also Media media, see Media preservation, 8-11 freezing, 9 liquid nitrogen, 9 lyophilization, 9-11 mineral oil, 9 periodic transfer, 8 soil culture, 8 Curamicin, 128 Curing agents, 49-52, see also specific agent Cyanein (brefeldin A), 137 Cyathin, 201 Cycloheximide (actidione) gas-liquid chromatography, 234-237 paper chromatography, 156-158 solvent systems for countercurrent distribution, 336 Cyclopaldic acid, 162 Cystamine effect on 1 - g u a n i d i n o - l - d e o x y - s c y l l o inositol-4-P phosphohydrolase, 461 as inosamine-P amidinostransferase inhibitor, 458 Cystine as inosamine-P amidinotransferase inhibitor, 458
D Daunomycin, 144, 318, 319 dc Polarography, see Direct current polarography DD-Carboxypeptidase alanine, chemic.al estimation of free, 690-691 D-alanine, enzymic estimation of, 691 assay, 690-692 crude extraet preparation, 692 fluorodinitrobenzene assay, 690 incubation conditions, 690 radioactive [l~C]Ac=,-L-lys-D-aladipeptide estimation, 691-692
812
SUBJECT INDEX
S t r e p t o m y c e s preparation, 692 substrates, 690 unit definition, 690 Deacetylcephaloglycin, 117 Deacetylcephalosporanic acid, 730 Deacetylcephalosporin C, 117, 730 Deacetyleephalothin (7fl-thienylacetamidocephalosporadesic acid), 730 Deacetylrifampin, 305 Dechlorogriseofulvin, 234 Dedimethylamino-4-aminoanhydrotetracycline, see S-Adenosylmethionine: dedimethylamino-4-aminoanhydrotetracycline N-methyltransferase Deformino LL-AC541, 258-260 Dehydrocycloheximide, 158 Dehydrogriseofulvin, 234 Demethyldecarbamylnovobiocin, 304 O-Demethylpuromycin, 509-513, see also Puromycin S-adenosylmethionine: O-demethylpuromycin O-methyltransferase Demetric acid, 336 3'-Deoxydihydrostreptomycin-6-P, 634 2-Deoxystreptamine, 444--445, 449-450 2-Deoxystreptamine derivatives, 124 Destomycin, 277 Deuterochloroform, 393 N ", N~-Diacetyl-L-lysyloD-alanyl-D-ala nine, 697 1,4-Diaminobutylphosphonic acid, 458 L-2,4-Diaminobutyrate activating enzyme, 579, see also Polymyxin synthetase :L-2,4-diaminobutyrate activating enzyme 1D-1,3-Diamino-l,3-dideoxy-scyUoinositol-2-P, 432 1D-1,3-Diamino-l,2,3-trideoxy-scyllo-
inositol-6-P, 432 Diazomycin, 336 3',4'-Dideoxykanamycin B, 271-272, 278 Diethylpyrocarbonate, 540 Differential pulse polarography, 373-388, see also specific antibiotic advantages, 375-377 analysis procedure, 380-381 dropping mercury electrode, 374-375 equations, 380-381 equipment, 377-379 limitations, 375-377
Diffusion assays, 60-61 1,3-Diguanidino-l,3-dideoxy-scyllo-inosi.
tol, 432 1D-1,3-Diguanidino-l,3-dideoxy-scylloinositol-6-P, 432 paper chromatographic data of, 121 streptomycin 6-phosphotransferase, 629, 631 structure, 429, 614 Dihydrostreptomycin-3" -P, 637 Dihydrostreptomycin-6-P, 634 [3'a2H] Dihydrostrept omycin-6P, 466 Dihydrostreptomycin-6-P 3'a-kinase, 634-637 assay, 635-636 biological distribution, 636-637 properties, 636-637 reaction scheme, 634 specificity, 637 stability, 636 S t r e p t o m y c e s bikiniensis, preparation, 636 Diketopiperazine derivatives, 144 Dilution assay, 62-65 agar method, 62 photometric method, 63-65, see also Photometric methods for dilution assays serial dilution in tubes, 62-63 d~-Dimethyl formamide, 393 d6-Dimethyl sulfoxide, 393 2:4-Dinitrostyrene, 72 Diplococcus p n e u m o n i a e , chloramphenicol acetyltransferase from, 738 Direct current (de) polarogaphy, 374 Distamycin A, 145-146 N",N~-Disuccinyl-L-lysyl-D-alanyl-p-gluetamic acid, 697 5,5'-Dithiobis-2-nitrobenzoid acid (DTNB), 742-743 Diumycin, 128 DME, see Dropping mercury electrode DMF, see d~-Dimethyl formamide DMSO, see d~-Dimethyl sulfoxide DNA, requirements for transformation, 49 DNP-bacitracin, 336 DNP-streptothricin, 336 DPP, see Differential pulse polarography Doxycycline, 385
SUBJECT INDEX
Dropping mercury electrode (DME), 374-375 Drosopholin, 336 Duramycin, 336 E
Echinomycin paper chromatography, 150, 152 peptide antibiotic lactonase, 767-773 Sarcina lutea, 771 structure, 768 Edeine biosynthesis, 562-564 countercurrent distribution, 335, 339 paper chromatography, 146 structure, 337 Edeine A biosynthesis, 562-564 structure, 559-560 Edeine B biosynthesis, 562-564 structure, 559-560 Edeine synthetase, 559-567 activation reaction, 560 amino acids activation, 562 assay, 560-564 Bacillus bevis preparation, 564 binding reaction, 561 biosynthesis of edeines A and B, 562-564 crude extract preparation, 564 DEAE-cellulose chromatography, 564-565 inhibitors, 566 molecular weight, 567 pH optimum, 566 polymerization reaction, 561 properties, 566-567 purification, 563-566 Sephadex G-20O filtration, 565-567 stability, 566 EDTA, see Ethylenediaminetetraacetate Electrophoresis, 279-291 agar gel, 287-289 carrier-free continuous, 289-290 paper, 280-286, see also Paper electrophoresis thin-layer, 286-287 Eluotropic series, 292-293
813
Emericellopsis minima, fungal penicillin
acylase from, 723 EMS, see ethylmethanesulfonate Enantiomeric p-phenyl analog of chloramphenicol, 366 Endomycin, 135, 336 Enterobacter cephalosporinase, 678-679, see also ~-Lactamase from Entero~ bacter species Enterobacter ~-lactamase, see ~-Lactamase from Enterobacter species 4-Epianhydrotetracycline, 199, 317 Epidermophyton floccosum, fungal penicillin acylase from, 721 6-Epihetacillin, 362-363 4-Epitetracycline, 199, 317 Erythromycin biosynthesis, 487-498, see also S-Adenosylmethionine:erythromycin C O-methyltransferase gas-liquid chromatography, 245-248 calculation, 247 euteric coated tablet, 246 high-pressure liquid chromatography, 308-309, 319 proton magnetic resonance spectroscopy, 391 Streptomyces erythreus, media for, 20 thin-layer chromatography, 198, 204 Erythromycin A, 490 derivatization, 494 partition chromatography, 492-493 structure, 488 thin-layer chromatography, 493-494 Erythromycin B paper chromatography, 131 structure, 488 Erythromycin C partition chromatography, 492--493 spiroketal (6 --> 9 : 12 --->9), 490 structure, 488 thin-layer chromatography, 493-494 Erythromycin methyltransferase, see S-Adenosylmethionine :erythromycin C O-methyltransferase Erwinia aroideae
fungal penicillin acylase from, 722 penicillin acylase from, 718 Escherichia coli
aminoglycosidic antibiotics, 120
814
SUBJSCT INDEX
bacteriophage P1 kc, 45-47 chloramphenicol acetyltransferase, 741, see also Chloramphenicol acetyltransferase chloramphenicol hydrolase, 735 gentamicin adenylytransferase, 625 kanamycin acetyltransferase, 623-624 fl-lactamase, 672-677, see also IIIa fl-Lactamase neomycin phosphotransferase, 626 penicillin acylase, 705--721,see also Penicillin acylase from Escheriehia coli
streptomycin phosphotransferase, 627 streptomycin-spectinomycin adenylyltransferase, 625 for transduction, 46 transformation of resistance plasmids, 48 Esterase, cephalosporin, see Cephalosporin acetylesterase Etamycin countercurrent distribution, 336 peptide antibiotic lactonase action on, 773, see also Peptide antibiotic lactonase structure, 768 Ethidium bromide as curing agent, 51 Ethylenediaminetetraacetate, as inhibitor of streptomycin-6-P phosphohydrolase, 470 Ethyleneimine, 36 N-Ethylmaleimide, 502 Ethylmethanesulfonate, as mutagen, 666 Eubacteria aminoglycoside-modifying mechanisms, 611 antibiotic fermentations by, 16-18 media for maintaining, 5, see also Media, for eubacteria paper chromatography, 135 Everninomicin, 128-129 Exogenous penicillin V, 114 Exfoliatin, 128
F
Fermentation in mutation program, 25-26
thin-layer chromatography for study of, 201-206, see also Thinlayer chromatography, fermentation Fervenulin, 336 Flaveolin, 336 Flavacid, 135 Flavipin, 163 Flavobacterium, 737 Flavofungin, 136 Folic acid, 520 Folimycin, 137 Fomecin A, 163 Formamidine, 451, 461 Formamidine disulfide.2HC1 as inosamine-P amidinotransferase inhibitor, 458 Formycins, 154, 156 3-Formyl rifampin, 305 Foromacidin, 131 Framicetin, 211 Fungal penicillin acylase, see also Penicillium chrysogenum acylase, Penicillium ]usarium acylase Fungi antibiotic fermentation by, 14-16 media for maintaining, 4, see also Media, for fungi Fungichromin, 136 Fusarium avenceum, penicillin acylase from, 723 Fusarium monili]orme, penicillin acylase from, 723 Fusarium semitectum, penicillin aeylase from, 723-725, see also Penicillium ]usarium acylase Fusidine, 204 Fusidic acid, 159, 352
G
Gabromycin, 200 Gas-liquid chromatography, 213-256, see also specific antibiotic Aminocyclitol antibiotics, 217-218 antimycin A, 253 aromatic antibiotics, 228-234 celestosaminide, 253 chloramphenicol, 228-232 clindamycin, 239-242
SUBJECT INDEX clindamycin palmitate, 243-245 cycloheximide, 234-237 erythromycin, 245--248 gentamicin, 217-218 griseofulvin, 232-234 glutarimide antibiotic, 234-237 kanamycin, 218-220 lincomycin-clindamycin family, 237-245 lividomycin, 220 neomycin, 220-228 paromomycin, 228 penicillin, 248-251 phosphonomycin, 254 spectinomycin, 215-217 tetracycline, 251-253 thiamphenicol, 254 validamycin, 254-265 GAT:, see Gentamicin acetyltransferase I
GAT:,, see Gentamicin acetyltransferase II GAT:::, see Gentamicin acetyltransferase III Geldanamycin biosynthesis, 409 paper chromatography, 137 proton magnetic resonance spectroscopy, 390-391 structure, 397-398 Geldanamycin acetate, 401, 403 Geminal couplings, 398 Genetic transfer of R factor, 43--49, see also Resistance plasmids Gentamicin biosynthesis, 615-618, see also Aminoglycoside modifying enzymes gas liquid chromatogaphy, 217-218 Micromonospora purpurea, media, 21 relative mobilities of, 126 solvent system, 119-120 structure, 613 thin-layer chromatography, 200 Gentamicin C complex, 274 Gentamicin acetyltransferase assay, 615-618 from Escherichia coli, 624 from Pseudomonas aeruginosa, 624 from Providencia, 624 Gentamicin acetyltransferase I, 616
815
Gentamicin acetyltransferase II, 616 Gentamicin acetyltransferase III, 616, 625 Gentamicin adenylyltransferase, 616, 625 Gentisylalcohol dehydrogenase assay, 544 Geodin, 163 Gladiolic acid, 163 Glebomycin bioautography, 42 paper chromatographic data, 121 structure, 429 L-[14C]Glutamine, 443 L-Glutamine :keto-scyllo-inositol aminotransferase, 439-443, 462 assay, 440-443 L-[1'C]glutamine, 443 myo-inositol 2-dehydrogenase,443 biological distribution, 442 ion-exchange chromatography, 441 properties, 442-443 reaction scheme, 439 specificity, 442-443 stability, 442 Glutarimide antibiotics, 234-237 Glycylglycine, 161 Gradient thin-layer chromatography, 179-180 Gramicidin countercurrent distribution, 324-327 polymyxins compared with, 584 solvent systems, 336 Gramicidin S biosynthesis, 567, see also Gramicidin S synthetase molecular weight determination, 328-330 solvent system for countercurrent distribution, 336 structure, 567 thin-layer chromatography, 208-209 Gramicidin S synthetase, 567-579 affinity chromatography, 575 amino acid-dependent ATP-[14C]AMP exchange, 569-570 amino acid-dependent ATP22PP, exchange, 569, 576 aminosulfate precipitation, 573 assay, 568-571 Bacillus brevis preparation, 571-572 crude extract preparation, 573
816
SUBJECT INDEX
DEAE Sephadex A-50 chromatography, 573, 574 heavy enzymes physical properties, 577 specificity, 577 inhibitors, 578 incubation mixture, 568-569 kinetic constants, 578 light enzyme properties, 577-578 molecular weight, 577 4'-phosphopantetheine, 577 properties, 576-579 purification, 571-576 purity, 576 racemization of phenylalanine, 571 reaction scheme, 568 Sephadex G-200 chromatography, 573, 574 separation of light and heavy enzymes, 575 specificity, 577 stability, 577 streptomycin sulfate precipitation, 573 thio ester-bound amino acids, 570 thiotemplate mechanism, 568 Gresein, 336 Griseofulvin chiroptical studies of, 352 chromatographic date, 163 gas liquid chromatography, 232-234 high-pressure liquid chromatography, 307-308, 319 paper chromatography, 161 Penicillium species media, 15 thin-layer chromatography, 200 in pharmacokinetic studies of, 212-213 Griseoluteins, 336 GTP, see Guanosine triphosphate Guanidinated inositol moieties of streptomycin, 429-433 1D-1-Guanidino-3-amino-l,3-dideoxyscyllo-inositol, 432 1D-1-Guanidino-3-amino-l,3-dideoxy-
1L-l- [14C] Guanidino-3-amino-l,3-dideoxy-scyUo-inositol, 465 L- [ Glanidino-~C ] arginine, 447 ATP :inosamine phosphotransferase assay, 447 inosamine-P amidinotransferase assay, 452-454
scyllo-inositol-6-P, 432 1-[~C]Guanidino-l-deoxy-scyllo-inositol -
H-277 (streptothricin-like), 336 Halomicins, 128 Hamycin, 136, 338 Helvolic acid, 159 Heptaene, 136
4-P, 460 1D.1- [14C] Guanidino-3-amino-l,3-dideoxy-scyllo-inositol, 463
1D-1-Guanidino-l-deoxy-3-keto-scylloinositol, 432, 463
1-Guanidino-l-deoxy-scyllo-inositol-4-P phosphoydrolase, 459-461 assay, 459-461 biological distribution, 461 inhibitors, 461
2-Guanidino-l-deoxy-neo-inositol, 432 2-Guanidino-2-deoxy-neo-inositol-5-P, 432
1-Guanidino-l-deoxy-scyllo-inositol-4-P, 432
Guanidinodeoxy-scyllo-inositol, 432 Guanosine triphosphate, 515 Guanosine triphosphat e-8-formylhydrolase, 515-520 activators, 517 ammonium sulfate fractionation, 517 assay, 516 cell-free extracts, 516 DE-52 cellulose column chromatography, 517 dissociation of, 519 inhibitors, 517 Michaelis constant, 519 molecular weight, 519 purification, 516-518 Sephadex G-200 column chromatography, 517 specificity, 517 specific activity, 516 Streptomyces rimosu~ preparation, 516 unit definition, 516 N-guan-Streptolidylgulosamine, 123
H
SUBJECT INDEX Hetacillin, 362-363 Heteromer peptide, 146 Heterocyclic antibiotics, 157 High-pressure liquid chromatography, 278, 300-320 actinomycin, 313, 319 ampieillin, 309-311, 319 aromatic antibiotics with nitrogen, 301-305 bacitracin, 314, 319 cephalosporin C, 311-312, 319 clindamycin phosphate, 306-307, 319 daunomycin, 318, 319 erythromycin, 308-309, 319 griseofulvin, 307-308, 319 kanamycin, 307-308, 319 leucomycin, 309, 319 novobiocin, 301-304, 319 oxytetracycline, 318--319 penicillin G, 312-313, 319 penicillin V, 313, 319 rifampin, 304-305, 319 tetracycline, 315-318, 319 virginiamycin, 305, 319 High-voltage electrophoresis, 281-282 Hikizimyein, 153 Homomycin, 338 Homopeptide, 145-146 Hondamyein, 137 Hybridimycin, 119-120 Hydrolysis fragment, 260-263 Hydroxylamine as mutagen, 35 m-Hydroxybenzyl-alcohol dehydrogenase, 540-548 assay, 544 calculation, 544 cell-free extract, 543 gentisyl alcohol dehydrogenase assay, 544 [ 1-"C ] m-hydroxybenzaldehyde assay, 544 inhibitors, 546 kinetic properties, 546 Michaelis-Menten plots, 546 molecular weight, 546 P e n i c i l l i u m urticae preparation, 542 pH optimum, 546 Polyclar AT treatment, 543, 545 polyketides, 541 properties, 545-548
817
purification, 544-545 reaction scheme, 541 specificity, 547 unit definition, 544 3-Hydroxybenzyl-alcohol: NADP oxidoreductase, see m-Hydroxybenzylalcohol dehydrogenase [ 1-'C ] m-Hydroxybenzaldehyde, 544 4-Hydroxy-3 (3-methyl-2-butenyl) benzoic acid, 506 Hydroxystreptomycin, 121 Hygromycin, 338
Ikutamycin, 137 Immunological techniques, t~-lactamase, 86-100 antisera inoculation program with crude enzyme, 89-90 preparation, 86-90 antistaphylococcal penicillinase, 87 crude enzyme preparation, 87-90 isolation of fl-lactamase-less mutants,89 N-methyl-N-nitro-N-nitrosoguanidine, 89 neutralization analysis, 90-97 mixtures of ~-lactamases, analysis of composition of, 96 molecular variants, 94-95 mutations, laboratory-induced, 96 natural variants, detection of, 94-95 neutralization curve, 92 time com~e of reaction, 92 precipitation analysis, 98-100 Bacill~s cereus, 98 gel precipitation, 99 immune electrophoresis, 99 purified enzyme preparation, 86-87 staphylococcal penicillinase, 87 Indolepyruvate, see S-Adenosylmethethionine :indolepyruvate 3-methyltransferase Indolmycin, biosynthesis, 498-502, see also S-Adenosylmethionine :indolepyruvate 3-methyltransferase Inhibitory coefficient of antibiotics, 66-69
818
SUBJECT INDEX
Inosamine derivatives, chemically phosphorylated, 453-454 Inosamine kinase, 444--451 aminodeoxy-scyllo-inositol, 448 L-arginine :inosamine-P amidinotransferase assay, 447 assay method I, 445--447 extract preparation, 446 mycelia growth, 446 L-ornithine in incubation mixture, 445 paper chromatography, 446-447 method II, 447-450 L-arginine :insoamine-P amidinotransferase, 447 radioehemical method, 451 [7-~P] ATP assay, 451 biological distribution, 450 2-deoxystreptamine from kanamycin, 449-450 equations, 444 inhibitors, 451 monoamidinated streptamines, 449 paper chromatography, 446-447 properties, 450--451 purification, 450 specificity, 450 streptamine and streptidine from dihydristreptomycin, 448-449 Inositol, chromatography, 432 Iodine, as inactivator of ~-lactamase, 662 Iodoacetamide, inhibitor of 6-methysalicyclic acid synthetase, 528 Ion-exchange chromatography, 256-278, see also specific antibiotic Amberlite CG-50 resin, 268, 270, 272, 274 Amberlite, IR-120 resin, 266, 272, 273, 277
Amberlite, IRC-50 resin, 266, 269 Aminex A-27, A-28, 266, 275 4-amino-4-deoxy-a,~-trehalose, 269-270 aminoglycoside antibiotics, 263--278, see also Aminoglycoside antibiotics, specific antibiotic ammonia gradient, 270 carboxymethyl cellulose, 273-274 cation interference, 206 cellulose exchanger, 273-275
3',4'-dideoxykanamycin B, 271-272 Dowex l-X2, 267, 275, 276, 277 Dowex 50, 272, 273 Duolite c-62, 272 gentamicin C complex, 274 high-pressure liquid chromatography, 278 kanamycin, 268-269 kasugamycin, 273 Lewatit SP-120, 272 lividomycin, 275 nebramycin, 271 nonionic adsorption chromatography, 275-280, see also Nonionic adsorption chromatography phosphonic acid resin, 272 CM-Sephadex C-25, 266, 273-274, 275 SE-Sephadex C-25, 266, 273-274 streptothricin-like antibiotics, 256-263, see also Streptothricin-like antibiotics hydrolysis fragments, 260-262 intact antibiotics, 258-260 sulfonic acid resins, 272 validamycins, 273 Ion-exchange thin-layer chromatography, 180-181 Ionizing radiation, mutation induction by, 31-32 Isochlorotetracycline, 199 Isogriseofulvin, 234 Isonovobiocin, 304
K Kanamycin biosynthesis, 615-618, see also Aminoglycoside modifying enzymes biotransformation of, 208 carrier-free continuous electrophoresis, 289-290 2-deoxystreptamine from, 449-450 gas liquid chromatography, 218--220 high-pressure liquid chromatography, 278, 307-308, 319 ion-exchange chromatography, 268-269, 272 Amberlite-IR-120 resin, 272 Duolite C-62, 272-273
SUBJECT INDEX
nonionic adsorption chromatography, 276-277 solvent system, 119-120 thin-layer chromatography, 211 Kanamycin A kinetic data, 623 structure, 612 Kanamycin acetyltransferase, 616 assay, 615-618 from Escherichia cell activity, 623 buffer, 623 kinetic data, 623 pH optimum, 623 stability, 623 from P s e u d o m o n a s aeruginosa, 624 Kasugamycin, 272-273 KAT, see Kanamycin acetyltransferase 1 D - 4 - K e t o - m y o - i n o s i t o l , 442-443 Keto-scyllo-inositol, 432, 440-441, see also L - G l u t a m i n e :keto-scyIlo-
inositol aminotransferase L-guanidino-3-keto-scyllo-inositol
aminotransferase assay, 465 m y o - i n o s i t o l : N A D ÷ 2-oxidoreductase
assay, 434, 437-438 Kikumycin A, 145-146 KlebsielIa aerogenes, tMactamase in, 673, 678 Kojic acid, 154 L LL-AC 541, 258-260 LL-AB 664, 258 /~-Lactam antibiotics chiroptical studies of, 352 CMR spectroscopy in biosynthesis of, 410-425, see also specific antibiotic paper chromatography of, 110-119, see also specific antibiotic screening, 52 ~-Lact am'~se antis(~ra preparation, 86-90 crude enzyme preparation, 87-88 inoculation program for crude enzyme, 89-90 isolation of fl-lactamase-less mutants, 89 purified enzyme preparation, 86-87
819
antisera speeifc, 90-100 assay acidimetric methods, 77-80, 83-84 alkimetric methods, 77-80 biological, 81-82 hydroxylamine, 81, 85 indicator method, 78-79 iodometric method, 74-77, 83-84 macroiodometric determination, 74-76 macroiodometrie method of Perret, 83 manometric measurement of CO2, 7940 microbiological, 81-82, 83 microiodometric determination, 76-77 pH star titration method, 79 fluorescence for testing antibiotics resistance to, 208 immunological techniques, 86-100, see also Immunological techniques molecular variants, detection of, 94-95 neutralization analysis, 90-97, see also hnmunological techniques in penicillin acylase preparations, 700-701 precipitation analysis, 98-100 reaction scheme, 70 unit definition, 74 /3-Lactamase from Actinomycetes species, 687-698, see also O-Laetamase fl'om S t r e p t o m y c e s fl-Lactamase from Bacillus cerm~, 640-652, see also fl-Lactamase I, ¢~-Lactamase II concentration in culture, 643-644 culture preparation, 642-643 /~-la(.tamase II isolated from fl-lactamaso I. 640-641,644 1)urification, 641-644, 646 specific activity, 642 substrate profile of extracellular, 647 fl-La('tamase I from Bacilbts cereus amino acid cmnposition of, 648 chemi(:al properties, 647-648 conformational changes, 649-650 kinetic properties, 647 modification reaction, 649 molecul:lr weight, 649
820
SUBJECT INDEX
properties, 644-650 physical, 644-650 purification, 641-644, 646 stability, 644, 647 fl-Lactamase I I from Bacillus cereus amino acid composition of, 651 chemical properties, 650-652 inhibitors, 652 kinetic properties, 650 modification, 652 molecular weight, 652 physical properties, 652 purification, 641-644, 646 stability, 650 substrate profile of extracellular, 651 thiol group modification of, 652 fi-Lactamase from Bacillus licheni]ormis, 653-664 affinity chromatography, 654 amino acid sequence, 661 Bacillus licheni]ormis preparation, 654 cell-bound, purification of, 655--658 chemical properties, 660 culture preparation, 654-655 extracellular, purification of, 655 heterogeneity, 661 hydrolysis of penicillin and cephalosporin derivatives, 659 immunology, 662 iodine reaction, 662 kinetic properties, 658-659 Michaelis constants, 659 modification, 662-664 molecular weight, 662 physical properties, 662 physiological efficiency, 660 plasma membrane bound, purification of, 663 purification, 653-658 secretion, 663 specific activity, 659 stability, 658 tetranitromethane modification reaction, 662 trypsin release of, 663 fi-Lactamase from Enterobacter species, 678-687
antiserum, 684 assay, 679-680 cross-reaction, 684
dialysis and concentration, 681 Enterobacter cloacae, preparation, 680 homogeneity, 685 hydrolysis of cephaloridine, 687 induction, 684 inhibition, 686-687 isoelectric focusing patterns, 685 kinetic properties, 686 molecular weight, 685 polyacrylamide disc electrophoresis, 683 properties, 683-687 purification, 680-683 QAE-Sephadex chromatography, 681-682 Sephadex G-50 chromatography, 681 specific activity, 679 substrate specificity, 686 temperature effect, 686 ultrasonic disruption in purification of, 681 unit definition, 679 fl-Lactamase from Escherichia coli, 672-677, see also IIa fl-Lactamase /3-Lactamase from Staphylococcus aureus, 664-672 activity, 670-671 6-aminopenicillanic acid induction, 666 cell-bound, 670 constitutive mutants, 666-667 cloxacillin induction, 666 culture preparation, 665-666 ethylmethanesulfonate as mutagen, 666 N-methyl-N-nitro-N-nitrosoguanidine as mutagen, 666 molecular properties, 671-672 purification, 667-670 spot tests, 670 specificity, 671 stability, 670 variants, 664-665 t~-Lactamase from Streptomyces albus, 687-698 assay, 689 concentration of enzyme preparations, 693 crude extract preparation, 692 culture media, 688 hydrolysis of fl-lactam antibiotics, 689-690, 697
SUBJECT INDEX
inhibitors, 697 iodine, sensitivity to, 696 isolation from strain Albus G, 695-696 isolation from strain R 39, 693, 695 K~ and Vma. values, 689-690 kinetic properties, 689-690 metal ion requirements, 696 pH optimum, 696 phosphate buffer, 693 physical properties, 696--697 polyacrylamide gel electrophoresis, 693 properties, 696-698 purification, 692-696 - - S H group reagents, sensitivity to, 696 specific activity, 693 stability, 696 specificity, 697 unit definition, 689 Ia fl-Lactamase, 673, see also fi-Lactamase from E n t e r o b a c t e r species IIIa ~-Lactamase, 672-677 centrifugation steps, 675 DEAE-cellulose column chromatography, 676 EDTA in sucrose purification, 673 Escherichia coli K12, growth medium, 673 location in cells, 672-673 molecular weight, 676-677 properties, 676-677 P s e u d o m o n a s aeruginosa, growth medium, 674 purification, 673-676 R factor-mediated, 673 G-75 Sephadex column, 676 specificity, 676-677 stability, 676 ultrasonic treatment, 675 fi-Laetamase-less mutants, 89 Lactobacillus helveticus, gramicidin S from, 577 Lactonase actinomycin, 763-767, see also Actinomycin lactonase peptide antibiotic, 767-773, see also Peptide antibiotic lactonase -y-Lactone, 72 Lemacidin B1, 123 Leucomycin, 134, 309, 319
821
Levomycin, 338 Levorin, 136, 338 Licheniformins, 128, 548, see also Bacitracin synthetase Lincomycin group bioautography, 128 clindamycin from, 756 gas liquid chromatography, 237-239 paper chromatography, 128 proton magnetic resonance spectroscopy, 390 solvent systems for countercurrent distribution, 338 thin-layer chromatography in pharmacokinetie studies, 211 Lincomycin tetrakis-TMS derivatives, 239 Lincosaminide, 755, see also Clindamycin phosphotransferase Liquid culture, 11-14, see also Antibiotic production in liquid culture Litmocidin, 168 Lividomycin gas-liquid chromatography, 220 ion-exchange chromatography, Amberlite CG-50 column, 269 CM-Sephadex, 275 solvent system, 119-120 structure, 614 Lividomycin A, 278 Lividomycin B, 269 Lividomycin phosphotransferase, 627 Luteomycin, 338 Lyophilization, preservation of cultures, 3 L-f~-Lysine, 123 Lysostaphin, 621,752 Lysozyme Escherichia coli, 621 L-Lysyl-D-glutamyl-D-alanine, 697
M
Macrocyclic antibiotics, 139 Macrocyclic lactone antibiotics, 129-137, ~ee also specific antibiotic Macrolide antibiotics, see also specific antibiotic chiroptical studies of, 352 gas-liquid chromatography, 245-248
822
SUBJECT INDEX
high-pressure liquid chromatography, 308-309, 319 thin-layer chromatography, 193, 198 Macroreticular resin chromatography, 296--299 adsorption, 298 cephalosporin C isolation from fermentation broth, 299 clean-up of new resin, 298 desorption of compounds, 298 regeneration of resin, 298 rehydration of resin, 298 properties of resin, 297 XAD-2, 297 Magnamycin, 131, 338 Magnetic circular dichroism, 347, see also Spectropolarimetry Malonyl coenzyme A 6-methylsalicylic acid biosynthesis, 521 patulin biosynthesis, 521, 540 a-D-Mannosidase enzyme, see Mannosidostreptomycin hydrolase Mannosidostreptomycin biosynthesis, 637-640, s e e also Mannosidostreptomycin hydrolase paper chromatographic data, 121 structure, 614 Mannosidostreptomycin hydrolase, 637-640 assay, 638-639 inhibitors, 639 location, 639 metal requirement, 639 pH optimum, 639 reaction scheme, 637 specificity, 639 stimulators, 639-640 substrate source, 639 unit definition, 639 Mannosyl glucosaminide, 272 MCD, see Magnetic circular dichroism Media, see also specific antibiotic for antinomycetes, 5-7 N-Z amine-starch-glucose medium, 7 Bennett's agar, 6 oatmeat agar, 6 tomato paste oatmeal agar, 6 trypticase-yeast extract agar, 6 yeast extract agar, 5
aeration effect on, 7 for eubacteria, 5 nutrient agar, 5 tryptone glucose yeast extract agar,, 5 for fungi, 4 Czapek's solution agar, 4 malt extract agar, 5 potato dextrose agar, 4 light effect on, 7--8 liquid culture, 11-14 fermentation, 12-13 inoculum, 11 pH effect on, 7 temperature effect on, 7 Melinacidin, 144 Methacycline hydrochloride, 385 Methicillin, 243 L-Methionine, 487 8-Methoxypsoralen, 36 Methyl bis(3-chloroethyl)amine (HN2), 33-34 3-Methylindolepyruvate, 500 N-Methyl-N-nitro-N-nitrosoguanidine,
as mutagen, 34-35, 89, 666 3-Methyl-7-(2-phenoxyacetamido)-3cephem, 411--412 6-Methylsalicylic acid chemical synthesis, 532--533 decarboxylase activity, 530 patulin biosynthesis, 520-521,540 reaction sequence, 520-521 preparation of, 535 [14C]6-Methylsalicylic acid, 531, 533, 535 6-Methylsalicylic acid (2,6-cresotic acid) decarboxylase, 530-540 ammonium sulfate fractionation, 536-537 assay, 531-532 biosynthetic 6-methylsalicylic acid isolation of, 533 preparation of, 535 chemical synthesis of 6-MSA, 532-533 DEAE-Sephadex A-50 column chromatography, 437 fluorescence assay, 531 homogeneity, 538 hydroxyapatite column chromatography, 437-438 inhibitors, 540
SUBJECT INDEX isotope effects, 539 kinetic properties, 539 molecular weight, 539 Penicillium patulum preparation, 534-535, 536 properties, 538-540 purification, 536-538 radioactive assay, 531-532 Sephadex G-1O0 gel filtration, 437 specificity, 539 stability, 538 ultracentrifugation, 536 unit definition, 532 6-Methylsalicylic acid synthetase, 520-530 ammonium sulfate fractionation, 525 assay, 521-523 cell breakage, 524 DEAE-cellulose chromatography, 526-527 fluorometric assay, 521-523 homogeneity, 528 hydroxyapatite chromatography, 525-526 inhibitors, 528 kinetic properties, 528 PeniciUium patulum, preparation, 523 polyethylene glycol 1500 fractionation, 524-525 polyethylene glycol 6000 precipitation, 524 properties, 528-530 purification, 524-527 molecular weight, 528 reaction scheme, 529-530 Sepharose 6B, gel filtration, 526 shake culture, 524, 534 specificity, 529 stability, 528 unit definition, 523 Methyltransferase, see S-Adenosylmethionine: dedimethylamino-4aminoanhydrotetracycline N-methyltransferase, S-Adenosylmethionine :erythromycin C O-methyltransferase, S-Adenosylmethionine :indolepyruvate 3-methyltransferase Methymycin, 131 Micrococcin, 338
823
Micrococcus flavus, bacitracin synthetase
from, 551 Micrococcus roseus, penicillin acylase
from, 719 Micromonspora, N-Z amine-starch-glu-
cose medium from, 7 Micromonospora purpurea
aminoglycoside antibiotics from, 265 gentamicin from, media for, 21 Mithramycin, 127 Mitomycin, as curing agent, 52 Mitomycin C group, 152-153 Molecular conformation, chiroptical methods, 347-348 Molecular structure of antibiotics, see specific antibiotic proton magnetic resonance spectroscopy, 388-404 Moldicin B, 135 Monamycine, 338 Monazomycin, 338 Mono-N-acetylneomycin C, 227 Monoamidinated streptamines, 449 6-MSA, see 6-Methylsalicylic acid Mutagenic techniques, 29-36, see also Mutation, Mutagen-treated populations Mutation, 24-41, see also Mutagentreated populations alkylating agents, 32-35 assay, 26-27 fermentation, 25-26 ionizing radiation, 31-32 induced by combined action of mutagens, 36 induction techniques, 27-36 N-methyl-N'-nitro-N-nitrosoguanidine,
34-35 nitrogen mustard, 33-34 selection of colonies, 36-41 suspension preparation, 26 ultraviolet light irradiation, 29-31 Mutagen-treated populations, selection from, 36-41 auxotrophs, 38 morphological differences, 37 mutants producing one antibiotic entitity, 39-40
$24
SUBJECT INDEX
resistant to toxic analogs of precursors, 39 overlay techniques, 37-38 prototrophs, 38-39 random isolation, 37 Mycaminose, 134 L-Mycarose, 487 Mycarosyl erythronolide B, 495 Mycetins, 145 Mycobacillin, 338 Mycophenolic acid, 154 Myo-inositol 2-dehydrogenase, 433--434, 443 Myo-inositol : NAD ÷ 2-oxid0reductase, 433-439 assays, 434-438 aminodeoxy-scyllo-inositol (labeled), 434 extract preparations, 436 high-voltage paper electrophoresis, 436-437 mycelia growth, 435 myo-inositol (labeled), 434 myo-inositol (nonlabeled), 437--438 reagents, 434, 438 biological distribution, 438 properties, 438 purification, 439 reaction scheme, 433 specificity, 439
N NADP :tetracycline 5a(lla)dehydrogenase, 606-607 Nalidixic acid resistance (naF) as genetic marker, 44 Narbomycin, 198 Natural penicillins, 115 Neamine, 126, 338 Nebramycin, 271 Nectria, fungal penicillin acylase from, 721 Neomethymycin, 131 Neomycin N-acetylneomycins, relative mobilities of, 126 biosynthesis, 618, see also Aminoglycoside modifying enzymes
[laC] nuclear magnetic resonance studies, 409 gas-liquid chromatography, 220-228 apparatus, 222 bulk powder, 224 calculation, 226 column preparation, 222-223 freeze-dry procedure, 225 petrolatum-based ointments, 224 problems, 228-229 ion-exchange chromatography, 269 nonionic adsorption chromatography, 277 paper chromatography, 120 proton magnetic resonance spectroscopy, 391 solvent systems, 119-120, 338 Streptomyces ]raciae, media for, 22 structure, 614 thin-layer chromatography in pharmacokinetic studies, 211 Neomycin phosphotransferase assay, 616, 618 from Escherichia coli, 626 from Pseudomonas aeruginosa, 626 from Staphylococcus aureus, 627 Neomycin phosphotransferase I, 616 Neomycin phosphotransferase II, 616 Neomycin sulfate, 387 Neopterin, 520 Netropsin, 145-146 Neutralization analysis, ~-lactamase, 90-97, see also Immunological techniques, ~-lactamase Neutrapen, 642 Niddamycin, 131, 495 Ninhydrin, 117 Nisin fragments, 338 solvent systems, 338 structure, 330 Nitrogen-containing heterocyclic antibiotics, 152-154 Nitrogen mustard, 33-34 p-Nitrophenyl-a-D-mannopyroanoside, 638 p-Nitrophenylserinol, 735, 737 Nitrosated penicillin G potassium, 382 Nitrous acid as mutagen, 35 NMR, see nuclear magnetic resonance
SUBJECT INDEX species, penicillinacylase from, 720 Nojirimycin, 272 Nonionic adsorption chromatography, 275-280 destomycin, 277 kanamycin, 276-277 neomycin, 277 Nonpolyene glycosidic macrolide, 129-134 Novenamine, 304 Novobiocic acid synthetase, 502-508 3-amino-4,7-dihydroxy-8-methyl coumarin, 506 assay, 503-505 paper chromatographic, 504 thin-layer chromatographic, 504 cell-free extract, 507 hydrolysis of novobiocin, 505 4-hydroxy-3(3-methyl-2-butenyl)benzoic acid, 506 pH optimum, 508 specificity, 507 stability, 508 S l r e p t o m y c e s niveus, 502, 507 unit definition, 504 Novobiocin biosynthesis, 502-508, see also Novobiocic acid synthetase high-pressure liquid chromatography, 301-304 paper chromatography, 160-161, 163 structure, 502-503 Nuclear magnetic resonance, see Proton magnetic resonance spectroscopy Nuclear 0verhauser effect, 402, 407 Nueleoside antibiotics, 187 Nystatin, 135, 186 Nocardia
O Ochramycin, 136 Octaene, 136 Oleandomycin bioautography for quantitative estimation of, 186 paper chromatography, 131 spectropolarimetry, 356-359 circular dichroism spectrum, 357
825
optical rotatory dispersion spectrum, 358 thin-layer chromatography, 198, 211 Oligomycins, 137 Oligosaccharides with chromophore, 126-127 Olivomycin, 127 Optical rotatory dispersion, 347-373, see also Spectropolarimetry equations, 355 L-Ornithine, 445, 458 Oxolinic acid, 211-212 Oxygen-containing heterocyclic antibiotics, 154-157 Oxytetracyeline, 199, 318, 319 P P99 ~-lactamase, 678 P-125, 168 PA-114 A and B, 338 PA 312, 338 Pantetheine, 586 Paper chromatogram, see Paper chromatography Paper chromatography, 100-172, see also specific antibiotic alieyclic antibiotics, 156-159 amino acid antibiotics, 144-152 7-aminocephalosporanic acid, 116-118 aminoglycosidic antibiotics, 119-122 6-aminopenicillanic acid, 110-116 anthracyclines, 141-144 anthracyclinones, 141-144 aromatic antibiotics, 159-162 bioautography, 105-110 calculation, 104-t05 caphalosporin C family, 116-118 cephamycins, 116-118 chromatograms pH, 164-168 salting-out, 162-164 summarized, 168-171 classification of antibiotics by, 162-171 detection methods, 104 everninomicin group, 128 lincomycin group, 128 macrocyclic lactone antibiotics, 129-137
826
SUBJECT INDEX
oligosaccharides with chromophore, 126-127 penicillin, 110-116 peptide antibiotics; 144-152 polyene macrolide, 134-136 nitrogen-containing heterocyclic antibiotics, 152-154 quinone antibiotics, 137-144 RI values, 168-170 R m values, calculations, 104-105 samples, preparation of, 102-103 solvent systems, 103-104 streptothricin group, 123 tetracycline, 137-141 Paper electrophoresis, 280-286 Barton reagent for reducing properties, 286 bioautography, 282 classification of antibiotics using, 280 detection and results, 282-286 high-voltage electrophoresis, 281-282 ninhydrin reagent for amines, 286 relative mobilities of antibiotics to alanine, 282-285 Rydon Smith reagent for amides, 286 ultraviolet light detection, 283, 286 Pantetheine, 586 Paromomine, 126, 623 Paromomycin gas-liquid chromatography, 228 ion-exchange chromatography, 269 kanamycin acetylation, 623 structure, 614 Partition chromatography, 174-175 Patulin biosynthesis, see also specific enzyme m-hydroxybenzyalcohol dehydrogenase, 540-548 6-methylsalicylic acid decarboxylase, 530-550 paper chromatography, 154, 156 Peliomycin, 340 Penam antibiotics, 410 Penicillic acid, 154, 156 Penicillin, acylase, see Penicillin acylase biosynthesis, see also specific enzyme t-(a-aminodepyl) cysteinylvaline synthetase, 471-473
acyl-CoA:6-aminopenicillanic acid acyltransferase, 474-476 phenylacetyl:coenzyme A hydrolase, 482-487 penacyl:coenzyme A ligase, 476481 differential pulse polarography, 385-387 gas liquid chromatography, 248-251 high-pressure liquid chromatography, 309, 312-313, 319 ~-lactamase, 640 paper chromatography, 110-116 6-aminopenicillanic acid separation, 115-116 applications, 113-114 bioautography, 111 biosynthetic penicillins, 115 chemical detection methods, 111-113 labeled penicillins, 115 mobilities compared, 113 natural penicillins, 115 sampling, 110 solvent systems, 110-111 Penicillium chrysogenum, media for, 16 solvent systems for countercurrent distribution, 340 spectrofluorimetry for quantitative estimations of, 186 thin-layer chromatography, 204 Penicillin N, 409, 701 Penicillin V, 408-409, 418-425 Penicillin acylase, 485, 698-705 assay, 699-705 biochromatographic, 703-705 buffer, 700 chromatographic assay, 700 hydroxylamine assay, 669-700, 701-702 fl-lactamase as contaminant, 700-701 Bacillus subtilis preparation, 705 pH optimum, 700 substrate, 703 transformation of antibiotics, 206 from Acromobacter species, 719 from Alcaligenes ]aecalis, 718 from Bacillus megaterium, 711-721 culture, 711 deacylation, 715 fermentation, 711-712
SUBJECT INDEX immobilization of enzyme, 717, 721 inhibitors, 713 kinetic properties, 713, 714 molecular weight, 712 pH optimum, 713 properties, 712-721 purification, 712-713 specific activity, 712 substrate profile, 715-716 from Erwinia aroideae, 718 from Escherichia coli, 705-721 assay, 699-705 cell disruption, 708 cell harvest, 768 culture, 706 deacylation, 715 fermentation, 706-707 immobilization of enzyme, 717, 721 inhibitors, 713 kinetic properties, 713, 714 molecular weight, 712 pH optimum, 713 properties, 712-721 purification, 709-711 specific activity, 712 substrate profile hydrolytic direction, 715-716 synthetic direction, 716-717 fungal, 721-728, see also Penicillium chrysogenum acylase, Penicillium fusarium acylase assay, 699-728 specificity, 726-727 from Microccus roseus, 719 from Nocardia species, 720 from Proteus rettgeri, 718 from Pseudomonas melanogenum, 719 from Streptomyces lavendulae, 720 from Pleurotus ostreatus, 721 Penicillin acyltransferase, 476, 485 Penicillin amidase, see Penicillin acylase Penicillin amidohydrolase, 206 Penicillin deacylation, see Penicillin acylase Penicillin (cephalosporin) fl-lactam amidohydrolase, see fl-Lactamase Penicillinase assays, see ~-Lactamase physiological efficiency, 660
827
specific anti-~-lactamase sera for quantitative study, 86-100, see also Immunological techniques TEM-R factor, 684 thin-layer chromatography, 207 ~/-Penicillinase, 640 Penicillium chrysogenum acylase from, 722-723, see also Penicillium chrysogenum acylase 8- (a-aminoadipyl) cysteinylvaline, 471 acyl-CoA :6-aminopenicillanic acid acyltransferase, 475 for carbon molecular resonance spectroscopy, 414 media for, 16 phenacyl: coenzyme A ligase, 476-477, 478 phenylacetyl:coenzyme A hydrolase, 482 strain development program, 25 Penicillium chrysogenum acylase, 722-723 6-aminopenicillanic acid, 722 culture, 722-723 extraction, 723 specific activity, 723 unit definition, 723 PeniciUium ]usarium acylase, 723-725 activators, 725 chemical properties, 725 culture, Fusarium semitectum, 724 extraction, 724 fractionation, 724 inhibitors, 725 IRC-50 column chromatography, 725 kinetic properties, 726 molecular weight, 725 pH optimum, 725-726 phenoxymethylpenicillin hydrolysis rate, 726 properties, 725-728 stability, 725 Penicillium patulum media for, 15 6-methylsalicylic acid deearboxylase, 534-535 6-methylsalicylic acid synthetase from, 523-524 Penicillium species, media for, 15 Penicillium urticae, m-hydroxybenzyl alcohol dehydrogenase from, 540, 542
828
SUBJECT INDEX
Penicilloic acid, 71 Pentene, 135 Peptide antibiotics chiroptical studies, 352 paper chromatography, 144-152, see also Specific antibiotic thin-layer chromatography, 187 Peptide antibiotic-antimycin A, 253 Peptide antibiotic lactonase, 767-773 agar diffusion bioassay, 770-771 assay, 770-771 calcium phosphate gel chromatography, 771 DEAE-cellulose column chromatography, 771-772 inhibitors, 772 kinetic properties, 772 pH optimum, 772 properties, 772-773 purification, 771-772 Sephadex G-200 column chromatography, 772 specific activity, 772--773 specificity, 772 Peptidyl carrier protein, 595-602 Perimycin, 136 pH chromatogram, 164-168 Phage P1 kc in Escherichia coll, transduction with, 45-47 Phage P22 in S a l m o n e l l a t y p h i m u r i u m , transduction with, 47 Phenacetyl coenzyme A, 476 Phenacyl-coenzyme A ligase, 476-481 assay, 477-478 crude enzyme extract, 479 mycelium production, 478-479 P e n i c i l l i u m c h r y s o g e n u m preparation, 477, 478 pH optimum, 481 purification, 479-481 specificity, 481 stability, 481 1,10-Phenanthroline, 502 Phenoxyacetyl coenzyme A, 476, 486 Phenoxymethylpenicillin, 476 penicillin acylase assay, 699 substrate for fungal penicillin acylase, 722 Phenylacetic acid, 482-487, see also Phenylacetyl-coenzyme A hydrolase
Phenylacetyl coenzyme A, 486 Phenylacetyl-coenzyme A hydrolase, 482-487 ammonium sulfate precipitation, 485 assay, 482-483 buffer, 487 DEAE-cellulose column, 485 molecular weight, 486 P e n i c i l l i u m c h r y s o g e n u m preparation, 484 phenylacetic acid, activation, 432, 487 pH optimum, 486 properties, 485-486 purification, 484-485 Sephadex G-100 column, 485 shake cultures, 484 specificity, 485 thiol effect on assay, 486-487 unit definition, 484 [1-1~C]Phenylacetyl coenzyme A, 482, see also Phenylacetyl-coenzyme A hydrolase Phleomycin, 150 Phosphonomycin, 254 4'-Phosphopantetheine, 577 Phosphotransferase ATP :inosamine phosphotransferase, see Inosamine kinase clindamycin, see Clindamycin phosphotransferase dihydrostreptomycin-6-P 3'a-phosphotransferase, 634-637 Photodensitometry, 185-186 Photometric methods for dilution assay, 63-65 equations, 64 dose-response line, 64 interference, 63-64 Picromycin, 131 Pimaricin, 135 Piomycin, 153 Plasmid conjugative resistance, see Resistance plasmid resistance, see Resistance plasmid PMR spectroscopy, see Proton magnetic resonance spectroscopy Polyamino acids, 391 Polyclar AT (insoluble polyvinylpyrrolidone), 543, 545
SUBJECT INDEX Polyenes proton magnetic resonance spectroscopy, 391 thin-layer chromatography, 187 Polyene macrolides, 134-136 Polyfungins, 135 Polymyxin biosynthesis, 579-584, see also Polymyxin synthetase media for, 17-18 paper chromatography, 147 solvent systems for countercurrent distribution, 340 structure, 580 Polymyxin B, 622 Polymyxin B1, structure, 330 Polymyxin G, 200 Polymyxin synthetase :L-2,4-diaminobutyrate activating enzyme, 579-584 assay, 579-581 Bacillus polymyxa preparation, 589-581 crude extract preparation, 582-583 molecular weight, 583 properties, 583--584 purification, 581-583 stability, 583 Polyoxin, 153 Polypeptide antibiotics, 313-315, 335 Polypeptide hormones, 335, 337 Polypeptin, 340 Porfiromycin, 152, 153 Potassium benzylpenicillin, 360-362 Preparative thin-layer thromatography, 184-185, see also Thin-layer chromatography Primycin, 120 Programmed multiple development chromatography, 180 Protactinomycinolike antibiotic, 340 Protactinomycins, 340 Proteus mirabilis, resistance plasmids in, 45 Proteus rettgeri, penicillin acylase from, 718 Proteus valgaris, enzyme A from, 735 Protomycin, 158, 340 Proton magnetic resonance spectroscopy, 388-404 d6-acetone, 394 d:-acetonitrile, 393
829
acetylation, 403 anisotropic solvent, 393, 401 applicability, 390-392 area measurement, 394 aromatic coupling, 400 carbamation, 403 chemical shift measurement, 396 computer programs for molecular geometry, 402 coupling constants, 398-400 deuterium oxide, 393 deuterochloroform as solvent, 393 d~-dimethyl formamide, 393 d~-dimethyl sulfoxide, 393 geminal coupling, 398 hydrogen types, 397, 398 INDOR sweep, 396 instrumentation, 392 long-range couplings, 400 d,-methanol, 394 nuclear 0verhauser effect, 402 paramagnetic organometallic complex as shift reagent, 401-402 sample changing, 403-404 requirements, 392-394 shift interpretation, 397-398 sodium 4,4-dimethyl-4-silapentane-1sulfonate, 393 sodium 3-trimethylsilyltetradeuteriopropionate, 393 solvent, 393 spectral analysis, 395-396 spectrum changing, 400-402 deuterium, 400-410 interpretation, 394-396 structure proposal, 396-400 trifluoroacetic acid, 394 van der Waals effect, 400 vicinal coupling, 399 vin)4 coupling, 399-400 Prototroph, 38--39 Providencia, gentamicin acetyltransferase from, 624 Pseudomonas aeruginosa 3',4'-dideoxykanamycin B from, 271-272 IIIa f~-lactamase from, 673 gentamicin acetyltransferase from, 624-625
830
SUBJECT INDEX
kanamycin acetyltransferase from, 624 neomycin phosphotransferase from, 626 streptomycin phosphotransferase from, 627 Pseudomonas melanogenum, penicillin acylase from, 719 Pseudomonas salonaceum, bioautography with, 117 Purine nucleosides, 153-154 Puromycin biosynthesis, see also specific enzyme adenine, 515 2-amino-2-deoxy-D-lyxose 5-phosphate, 514 2-amino-2-deoxy-D-ribose 5-phosphate, 514 3-aminopentose, 514 O-demethylpuromycin, 515 N~,N6-dimethyladenine, 515 5'-phosphate esters, 515 puromycin S-adenosylmethionine :Odemethylpuromycin O-methyltransferase, 508-515 NS,Ns, O-tridemethylpuromycin, 514 Puromycin Soadenosylmethionine : Odemethylpuromycin O-methyltransferase, 508-515 S-adenosylmethionine, 509 assay, 509-511 cell-free extract, 512 DEAE-cellulose column chromatography, 512 O-demethylpuromycin, 509 pH optimum, 514 properties, 513-514 purification, 511-513 puromycin biosynthesis, scheme, 508, see also Puromycin, biosynthesis salt fractionation, 512 Sephadex G-200 gel filtration, 513 specific activity, 510-513 specificity, 514 stability, 514 unit definition, 510 Pyridoxal-P, 440, 442 Pyrimidine nueleoside, 152-153, 155 Pyrrolnitrin, 409
Q Quadrifidin, 340 Quadrilineatin, 163 Quantitative thin-layer chromatography, 185-186 Quinacrine as curing agent, 51 Quinocycline, 340 Quinomycin, 180, 203 Quinone antibiotics, 137-144
R
Racemomycin, 123 Reflectance spectroscopy, 181-182 Resistance determinant, 43 Resistance plasmid, 41-55 bacterial species with, 43 compatibility properties, 52--55 tests for, 54-55 conjugation, 43-45 curing agents, 49 determinants, 43 DNA, preparation of, 48 genetic marker, 44 genetic transfer, 43-49 fi character determination, 54 resistance characters associated with, 42 resistance transfer factor, 43 structure, 43 transduction, 45-48 with P1, 46 with P22, 47 transformation, 48-50 frequency, 49 kinetics of resistance phenotypes, 50 Reversed transfer factor, 43 Reversed-phase partition chromatography, 180 R factor, 41-55, see also Resistance plasmid R factor-containing strains are pathogens, 611=612 Rhodomycin, 340, 352 Rhodomycinone, 146 Riboflavin, 520 biosynthesis, 515-520, see also Guanosine triphosphate-8-formylhydro lase Ribostamycin, structure, 613
SUBJECT I N D E X
Rifamycin biosynthesis, 409 paper chromatography, 137, 138 solvent system for countercurrent distribution, 340 thin-layer chromatography, 200 Rifamycin resistance (rif a) as genetic marker, 44 Rifampin, 304-305, 319 Rifampin quinone, 305 Rimocidin, 135 Ristocetin, 127 Ristomycin, 127 R~ values in chromatographic structural .analyses, 183-184 Rolitetracycline, 385 Roseothricin, 280 Rotaventin, 340 RrE~,-mediated fl-lactamase, 678 Rubidin, 340 Rubidomycins, 145 Rubifiavin, 340 Rubradirin, 340 Rubromycin, 340
S Saccharomyces pastorianus, 110 Salmonella typhimurium
bacteriophage P22 with, 45 penicillin bioautography, 111 for transduction, 47 SAM, see S-Adenosylmethionine SAM: dedimethylamino-4-aminoanhydrotetracycline N-methyltransferase, see S-Adenosylmethionine: dedimethylamine-4-aminoanhydrotetracycline N-methyltransferase SAM :EaDM transmethylase, see S-Adenosylmethionine: erythromycin C O-methyltransferase SAM :indolepyruvate 3-methyltransferase, see S-Adenosylmethionine :indolepyruvate 3-methyltransferase Sangivamycin biosynthesis, 516 toyocamycin nitrile hydrolase, 759-762, see also Toyocamycin nitrile hydrolase
831
Sarcinea lutea
bioautography, 117 echinomycin assays, 771 penicillin bioautography, 111 Salting-out chromatograph, 162-164 Semisynthetic cephalosporin, 199 Semisynthetic penicillin, 207-208 Serratia marcescens, 6-aminopenicillanic acid assay, 700 Shigella flexneri, resistance plasmids in, 45 Showdomycin biosynthesis, 409 effect on guanosine triphosphate-8formylhydrolase, 517, 519 Siccanin, 185 Sideromycin, 146-148, 149 Silica gel chromatography, 291-296 adsorbent, 291-292 eluotropic series, 292-293 elution column, 295-296 monitoring column, 296 preparation of column, 293-295 sample application, 295 solvent system, 292-293, 294 Sisomicin, 623 Sodium azide resistance (aziR) as genetic marker, 44 Sodium dodecyl sulfate as curing agent, 51 Solvent systems aglycosidic nonpolyene macrolides, 136-137 7-aminocephalosporanic acid, 116 aminoglycosidic antibiotics, 119-120 6-aminopenicillanic acid, 110-111 cephalosporin, 116 cephamycin, 116 chloramphenicol, 159 countercurrent distribution, 331, 341 cycloheximide and other glutarimides, 156-157 everninomicin, 128 griseofulvin, 161 for hydrophilic substances, 104 lincomycin, 128 mycetin-rhodomycin-cinerubin group, 144 nonpolyene glycosidic macrolide, 129-130
832
SUBJECT INDEX
novobiocin, 160 oligosaccharide, 126 penicillin, 110-111 polyene macrolide, 134 purine nucleoside, 153 rifamycin, 136-137 sideromycin, 148 silica gel chromatography, 292-293, 294 steroid antibiotics, 158-159 streptothricin group, 123 tetracycline, 139-140 thin-layer chromatography, 188-193, 196-197, 200--201 Specific inhibitory coefficient, 66-78 Spectinomycin gas liquid chromatography, 215-217 proton magnetic resonance spectroscopy, 390 spectropolarimetry, 359--360 structure, 615 Spectropolarimetry, 347-373, see also specific antibiotic atmosphere, 370 biopolymer interaction with antibiotic, 367-369 blank, 372-373 chromophores, 350-351 circular dichroism, 347, 350 experimental parameters, 369 gain settings, 371 instrument calibration, 370-371 magnetic circular dichroism, 347 optical rotary dispersion, 347, 350 equations, 355 phenomenology of, 349-350 reproducibility, 373 sample concentration, 371-372 scanning speed, 371 slit widths, 371 solvent, 369 survey measurements, 372 temperature, 369-370 ultraviolet, 350 Spectroscopy, proton magnetic resonance, 388-404, see also Proton magnetic resonance spectroscopy Spermidine, 560 Spiramycin, 131, 198, 211 Staphylococcal chloramphenicol acetyltransferase, 751-752, see also Chlor-
amphenicol acetyltransferase, from Staphylococcus species
Staphylococcal fl-lactamase, 664-672, see also ~-Lactamase from Staphylococcus aureus
natural variants from studies on, 94-95 Staphylococcal penicillinase, 671 Staphylococcus aureus
bioautography for aminoglycosidic antibiotics, 120 for cephalosporins, 117 for penicillin, 111 everninomycin and lincomycin groups, 128 fl-lactamase from, 94, 664-672 neomycin phosphotransferase from, 627 resistance plasmids in, 45 Staphylomycin, 340 Staphylomycin S, 767, 773 Stendomycin Candida albicans, 771 peptide antibiotic lactonase, 767 solvent system for countercurrent distribution, 340 structure, 768 Steroid antibiotics, 158-159 Strain development, 24--41, see also Mutations Streptamine, 444-445 from dihydrostreptomycin, 448-449 monoamidinated, 449 Streptamine derivatives, 121 Streptidine biosynthesis, 429 from dihydrostreptomycin, 448-449 Streptidine-6-P, 629, 631 Streptidine moiety, 430, 444 Streptimidon, 158 Streptococcus ]aecalis, chloramphenicol acetyltransferase from, 738 Streptolidine, 123, 258, 262 Streptolidine sugar compounds, 261 Streptolydigin, 390 Streptomyces alboniger, puromycin S-adenosylmethionine: O-demethylpuromycin O-methyltransferase from, 508, 511, 514 Streptomyces aureolaciens
S-adenosylmethionine: dedimethylamino-4-aminoanhydrotetracycline
SUBJECT
N-methyltransferase from, 603, 605 chlortetracycline from, media, 19 NADP: tetracycline 5a(11a)dehydrogenase from, 606
Streptomyces bikiniensis L-alanine: 1D-1-guanidino-l-deoxy3-keto-scyUo-inositol aminotransferase assay, 462-465 L-glutamine :keto-scyllo-inositol aminotransferase, 442, 443 1-guanidino-l-deoxy-scyllo-inositol-4-P phosphohydrolase assay, 459 inosamine kinase, 445-446, 448 inosamine-P amidinotransferase, 452, 456 myo-inositol, 438 streptomycin 6-kinase, 631 streptomycin-6-P phosphohydrolase assay, 466, 468 Streptomyces clavuliger, fl-lactam antibiotics from, 117 Streptomyces coelicolor Miiller, clindamycin from, 756
Streptomyces erythreus erythromycin, media, 20 erythromycins A, B, and C from, 487, 496 fungal penicillin acylase from, 722
,~treptorngces #adiae neomycin, media, 22 streptomycin-6-P phosphohydrolase from, 469
Streptomyces galbus L-arginine :inosamine-P amidinotransferase, 457 streptomycin-6-P phosphohydrolase, 459
Slreptomyces gle'bosus, myo-inositol from, 438
Streptomyces griseoearneus L-arginine :inosamine-P amidinotransferase from, 457 streptomycin-6-P phosphohydrolase from, 469
Streptomyces griseus S-adenosylmethionine :indolepyruvate 3-methyltransferase, 498 L-alanine: 1D-l-guanidino-l-deoxy-3keto-scyllo-inositol aminotransferase, 464
833
INDEX
L-arginine: inosamine-P amidinotransferase, 457 L-glutamine :keto-~cyllo-inositol aminotransferase, 442 mannosidostreptomycin, 638 streptomycin, media, 23 streptomycin 3"-kinase, 633 streptomycin 6-kinase, 631 streptomycin-6-P phosphohydrolase, 465, 469 Streptomyces humidus, L-arginine:inosamine-P amidinotransferase, 457
Streptomyces hygroscopicus ]orma glebosus •-arginine :inosamine-P amidinotransferase assay, 452
1-guanidino-l-deoxy-scyllo-inositol-4-P phosphohydrolase assay, 461
keto-scgllo-inositol, 440 myo-inositol, 433, 434 Streptomyces kanamyceticus aminoglycoside antibiotics, 265 streptomycin-6-P phosphohydrolase, 469 Streptomyces fl-lactamase, see fl-Lactamase from Streptomyces albus Streptomyces lavendulae, penicillin ac3:lase from, 720-721 Streptornyces netropsis, fungal penicillin acylase, 266 Streptomyces niveus, novobiocic acid synthetase, 502 Streptomyces noursei, fungal penicillin acylase, 722
Streptornyces ornatus L-alanine : 1D-l-guanidino-l-deoxy-3keto-scyllo-inositol aminotransferase, 464 L-arginine:inosamine-P amidinotransferase, 457 L-glutamine :keto-scyllo-inositol aminotransferase, 442
,Streptomyce~ rimosus S-adenosylmethionine : dedimethylamino-4-aminoanhydrotetracycline N-methyltransferase, 603-604 guanosine triphosphate-8-formylhydrolase, 515-516 toyocamycin, 760 Streptorngces species 3022a, chloramphenicol, 735-737
834
SCB~ECT INDEX
Streptomyces venezuelae, chloramphenicol, media, 18 Streptomycin biosynthesis, see also specific enzyme aminoglycoside modifying enzymes, 616-618 dihydrostreptomycin-6-P 3'a-kinase, 634-637 L-glutamine :keto-scyUo-inositol aminotransferase, 439-443 guanidinated inositol moieties of streptomycin, 429-433
1-guanidino-l-deoxy-scyllo-inositol4-P phosphohydrolase, 459--461 inosamine kinase, 444--451 mannosidostreptomycin hydrolase, 637-640 myo-inositol :NAD ÷ 2-oxidoreductase, 433-439 streptidine-6-P, 629 streptomycin 3'-kinase, 632-634 streptomycin 6-kinase, 628-632 streptomycin-6-P phosphohydrolase, 465-470 countercurrent distribution, 333 derivatives, chromatographic behavior of, 635 paper chromatographic data, 121 relative mobilities of, 122 solvent systems for countercurrent distribution, 342 streptidine moiety, 430, 444 Streptomyces griseus, media, 23 structure, 614 thin-layer chromatography in pharmacokinetic studies, 211 Streptomycin-spectinomycin adenylyltransferase, 616, 625-626 Streptomycin adenylyltransferase, 618-619 Streptomycin 3"-kinase, 632-634 assay, 633, 634 biological distribution, 631 reaction scheme, 632 specificity, 634 stability, 633 Streptomycin 6-kinase, 628-632 assay, 629-631, 632 biological distribution, 631 properties, 631-632
reaction scheme, 628 specificity, 631 stability, 631 Streptomycin-6-P phosphatase, 460, 467-469 Strept omycin-6-P-phosphohydrolase, 465-470 assays alternative, 470 method I, [3'a2H]dihydrostreptomyein-6-P, 466-467 method II, streptomycin 6-P phosphatase, 467--468 method III, partially purified streptomycin-6-P phosphatase, 468 biological distribution, 469 [3'a-all] dihydrostreptomycin-6-P, 466 inhibitors, 470 purification, 460 reaction scheme, 465 specificity, 469 streptomycin-6-P phosphatase, 468 Streptomycin phosphotransferase, 616 assay, 618-619 from Escherichia coli, 627 from Pseudomonas aeruginosa, 627 Streptomycin sulfate, 382-384 Streptonigrin, 342 Streptothricin-like antibiotics ion-exchange chromatography, 256-263 structure, 257 Streptothricins chiroptical studies, 352 " detection, 123 paper chromatography, 123 structure, 257 Streptovaricin, 137, 390, 409 Streptovitacin, 158, 342 Streptozotocin, 342 Subtilins, 146, 330, 342 Succinimycin, 342 Sulfadimethoxine, 186 Suspensions, preparation for mutagenic treatment, 26-27 T
Terramycin, 334 Tetracycline bioautography, 45, 140
SUBJECT INDEX biosynthesis, see also specific enzyme 8-adenosylmethionine: dedimethylamino-4-aminoanhydrotetracycline N-methyltransferase, 603-606 chemical detection, 141 chiroptical studies, 352 gas liquid chromatography, 251-253 high-pressure liquid chromatography, 315-319 calculation, 316-317 NADP :tetracycline 5a(lla)dehydrogenase, 606-607 paper chromatography, 137-141, 142 bioautography, 140 detection, 140-141 solvent system, 139-140 photodensitometry, 185 solution spectrophotometry with thinlayer chromatography, 186 spectropolarimetry, 363-364 thin-layer chromatography, 199, 204, 211 5-OH, 7-CL Tetracycline, 342 Tetracycline hydrochloride circular dichroism spectrum, 364 differential pulse polarography, 382, 384-385 optical rotatory dispersion spectrum, 364 Tetraene, 135 Tetrameric chloramphenicol acetyltransferase, 755 Tetramycin, 135 Tetranitromethane, 662 Tetrin, 135 Thermoviridin, 342 Thiamphenicol, 254 Thin-layer chromatography, 172-213, see also specific antibiotic antifoam agents, 204-205 actinomycin, 187 adsorbents, 176 adsorption chromatography, 173-174 adsorption coefficient calculation, 174 alumina, 176, 187, 189 amidase, 207 antibiotics resistant to fl-lactamase or amidase, 208 basic water-soluble antibiotics, 187, 200 bioautography, 179, 186, 194-195, 198
$35
biosynthesis of antibiotics, 201-213 see also biotransformation biotransformation of antibiotics, 201-213 by single-enzyme, 206-209 in viv o, 209-213 buffers, 179 on cellulose 300 (MN), 200 chambers, 178 chromatoplate, 175-176, 179 circular thin-layer chromatography, 180 for classification of antibiotics, 186-201 for conjugation of antibiotics, 210 drum thin-layer chromatography, 178 Eastman chromatogram sheets, 191 extraction process, 210 evaporation of mobile phase, 178 fermentation, 201-206 antifoam agents, 204-205 chloroform, 205 hexane as solvent, 205 intracellular antibiotics, 205 pH, 205 sample preparation, 204 tetracycline, 204 water-soluble antibiotics, 205-206 fluorescent indicator, 178 gradient, 179-180 ion-exchange thin-layer chromatography, 180-181 Kieselguhr G, 176, 196-197 macrolide antibiotics, 187 mobile phase, 177-178 nucleoside antibiotics, 187 organic supports, 176-177 partition chromatography, 174-175 peptide antibiotics, 187 in pharmacokinetics, 209-213 polyene antibiotics, 187 preparative, 184-185 programmed multiple development chromatography, 180 quantitative, 185-186 bioautography, 186 photodensitometry, 185 with solution spectrophotometry, 186 spectrofluorimeter, 185 RI values, 192-193 reproducibility of, 182-183
836
SUBJECT INDEX
R~ values, 183-184 rapid-column chromatography, 182 reflectance spectroscopy, 181-182 reversed-phase partition chromatography, 180 samples preparation for spotting, 178 uniform application to plate, 177 screening antibiotics for antitumor activity, 203 separation of antibiotics, 186-201 on Sephadex G-15, 198-199 silica gel, 176, 180, 188 solid support, 176 solvent equilibrium, 182 solvent systems, 188, 189, 190-191 spotting, preparation of sample for, 178 spray reagent, 178 water-soluble basic antibiotics, 187, 200 Thin-layer electrophoresis, 286-287 5-Thio-2-nitrobenzoate, 742 Thiopeptin, 147 Thiostrepton chromatographic data, 146-147 peptide antibiotic lactonase, 767-773 Sarcina Iutea, 771 structure, 768 L-Threo-l-p-phenylphenyl-2-dichloroacetamido-l,3-propanediol, 366 Tobramycin, 612 Tobramycin acetyltransferase, 623 Tolypomycin, 137 Toyocamycin biosynthesis, 515-516, see also specific enzyme toyocamycin nitrile hydrolase, 759-762 paper chromatography, 154 Toyocamycin nitrile hydrolase, 759-762 activators, 762 ammonium sulfate fractionation, 760 assay, 759-760 heat effect, 762 hydroxyapatite adsorption, 762 inhibitors, 762 metal ions effect, 762 Michaelis constant, 762 pit optimum, 762 protamine sulfate treatment, 760
purification, 760-762 specific activity, 760 specificity, 762 Streptomyces rimosus preparation, 760
unit definition, 760 Transduction with phage P1 kc in Escherichia coli, 45-47 with phage P22 in Salmonella typhimurium, 47 Transformation, 48-50, see also Resistance plasmid Trehalosamine, 272 Triene, 135 Trichophyton mentagrophyta, fungal penicillin acylase from, 721 4-N [ (5'-Triphospho)-l'-ribosylamino]2,5-diamino-6-hydroxypyrimidine, 515, 52O Tryptone glucose yeast extract, 5 Tubercidin, 154, 156, 516 Turbidometric assay, see Photometric methods for dilution assay Tylosin, 131, 495 Tyrocidine biosynthesis, 560, see also Specific enzyme tyrocidine synthetase, 585 compared with gramicidin S, 579 compared with polymycin, 584 countercurrent distribution, 324-325 partition isotherms, 330 solvent systems for, 342 for structural studies, 330 structure, 586 Tyrocidine-synthesizing system, 586-595, see also Tyrocidine synthetase Tyrocidine synthetase, 585-602 amino acid activating subunits, 598 assay, 589-590 ATP-PP~ exchange, 588-589, 596 Bacillus brevis preparation, 587-588 carrier protein isolation from purified polyenzyme, 600-601 purification from crude extracts, 598-600 sodium dodecyl sulfate gel electrophoresis, 599, 601-602
SUBJECT INDEX dissociation of polyenzymes, 595-602 in crude extracts, 596-597 of purified polyenzymes, 597 enzymatic synthesis of, 588-589 extract preparation, 590 heavy enzyme dissociation of, 597-598 molecular weight, 595 purification, 592-595 intermediate enzyme molecular weight, 595 purification, 592, 594-595 light enzyme, purification, 591-592 ornithine-dependent PPI-ATP exchange, 588-589, 596 polyenzymes, dissociation products of, 595-602 purification, 590-595 Sephadex G-200 filtration, 590-591
UV, see Ultraviolet light, Ultraviolet light irradiation
V Validamycin, 254-256, 272-273 Valine, 411, 413 Vancomycin, 127, 204, 211 Vernamycin B peptide antibiotic lactonase, 767-773 structure, 768 Vibrio percolans, bioautography, 117 Vicinal coupling, 399 Vinyl coupling, 399-400 Viocin, 200 Violarin, 168 Viomycin, 120, 211 Virginiamycin, 305, 319
W
U U-12, 241 (rhodomycin-like), 342 U-12, 898, 342 U-13, 714, 342 Ultraviolet light, detection with paper electrophoresis, 283, 286 Ultraviolet light irradiation, mutation induction by, 29-31 caffeine as mutagen, 36 ethyleneimine as mutagen, 36 8-methoxypsoralen as mutagen, 36 Unamycin, 135 Usnic acid, 154
A B C D E F G H I J
5 6 7 8 9 0 1 2 3 4
837
Water-soluble antibiotics, 187, 200
X X-537 A, 352, 409 Xanthomycin, 342
Z
Zizanin, 342