Methods in Enzymology, Volume 4: Special Techniques for the Enzymologist

PREFACE The first three volumes of this series have dealt with the commonly employed methods for the preparation and assay of enzymes, coenzymes, and substrates. The present volume deals with certain special techniques which are considered to be of interest to those in the field of enzymology. Descriptions of many of these procedures are available in monographs dealing respectively with the physical chemistry of the proteins, with specialized methods for the measurement of metabolism, or with procedures for the synthesis, degradation, and measurement of isotopically labeled compounds. It nevertheless appeared worthwhile to have these descriptions written with the special problems of the enzymologist in mind and to have them brought together in a single volume. With the publication of this volume we bring to completion our original plans for a treatise on Methods in Enzymology. It has been our policy to select a recognized authority for the description of each laboratory procedure, with the aim of obtaining a reliable collection of reproducible methods. For the extent to which we have succeeded in this aim, ,we are grateful to all those investigators who have contributed. It would of course have been desirable that all procedures described in these four volumes be checked in an independent laboratory prior to publication. However, it is obvious that with such a system of checking, the publication of an organized treatise of this scope would have been impossible within any reasonable time period. Users of these volumes will undoubtedly find that certain articles are already in need of revision and that certain important new subjects are not covered at all. In an attempt to correct these unavoidable deficiencies, an organized single supplementary volume is planned, which will include material not heretofore covered, as well as revised procedures, to supplement all four of the published volumes. We welcome any suggestions concerning the content of this supplementary volume, which will be published approximately two years from this date.

Baltimore, June 1~, 1957

SIDNEY P. COLOWlCK NATHAN O. KAPLAN

Contributors to Volume IV Article numbers are

s h o w n in parentheses following the n a m e s Affiliations Listed are current.

S. ABRAHAM(22), University of California,

Berkeley, California I-I. S. ANKER (32), University of Chicago, Chicago, Illinois CAMILLO ARTOM (33), Bowman-Gray School of Medicine, Wake Forest College, Winston-Salem, North Carolina A. A. BENSON (36), Pennsylvania State University, State College, Pennsylvania I. A. BERNSTEIN (23), University of Michigan, Ann Arbor, Michigan M. BIER (5), Fordham University, New York, New York ](. BLOCH (29), Harvard University, Cambridge, Massachusetts R. It. BVRRIS (16), University of Wisconsin, Madison, Wisconsin M. CALVIN (36), University of California, Berkeley, California I. L. CHAIKOFF (35), University of California, Berkeley, California BRITTON CHANCE (12), University of Pennsylvania, Philadelphia, Pennsylvania MILDRED COHN (37), Washington University, St. Louis, Missouri S. P. COLOWICX (34), The Johns Hopkins University, Baltimore, Maryland F. H. C. CRICK (4), Cambridge University, Cambridge, England H. FRAENKEL-CONRAT (ll), University of California, Berkeley, California GEORGE GOMORI* (18), Palo Alto Medical Research Foundation, Palo Alto, California ])AVID M. GREENBERG (28), University of California, Berkeley, California W. Z. HASSID (22), University of California, Berkeley, California ROGER ~ . ]-~ERRIOTT (8), The Johns Hopkins University, Baltimore, Maryland N. O ]~-APLAN (34), Brandeis University, Waltham, Massachusetts

of contributors.

EDWARD D. KORN (26), National Insti-

tutes of Health, Bethesda, Maryland A. HELGE F. LAURELL (1A), University of

Uppsala, Uppsala, Sweden DONALD J. R. LAURENCE (7), Chester

Beatty Research Institute, London, England M. LEvY (10), New York University, New York, New York KATHARINE F. LEWIS (25), Lankenau Research Institute, Philadelphia, Pennsylvania OLIVER H. LOWRY (17), Washington University, St. Louis, Missouri WILFRIED F. H. M. MOMMAERTS (6),

School of Medicine, University of California, Los Angeles, California R. R. PORTER (9), The National Institute for Medical Research, London, England J. H. QUASTEL (13, 14), McGill-Montreal General Hospital Research Institute, Montreal, Canada MORTON ROTHSTEIN (28), University of California, Berkeley, California ANTHONY SAN PIETRO (21), The Johns Hopkins University, Baltimore, Maryland ]~. K. SCHACHMAN (2}, University of California, Berkeley, California P. G. SCHOLEFIELD (14), McGill-Montreal General Hospital Research Institute, Montreal, Canada DAVID SHEMIN (27), Columbia University, New York, New York FRITIOF S. SJOSTRAND (19), Karolinska Institute, Stockholm, Sweden DANIEL STEINBERG (20), National Institutes of Health, Bethesda, Maryland J. A. STEKOL (30), Lankenau Hospital Research Institute, Philadelphia, Pennsylvania It. E. SwIM (24), Western Reserve University, Cleveland, Ohio

* Deceased. vii

° . .

Vlll

CONTRIBUTORS TO VOLUME IV

University of California, Berkeley, California ALVIN TAUROG (35), University of California, Berkeley, California ARNE TISELIUS (1), University of Uppsala, Uppsala, Sweden SIDNEY UDENFRIEND (20), National Institutes of Health, Bethesda, Maryland M. F. UTTER (24), Western Reserve University, Cleveland, Ohio HAROLD TARVER (31),

WOLF VISHNIAC (15), Yale University,

New Haven, Connecticut Lankenau Research Institute, Philadelphia, Pennsylvania P. W. WILSON (16), University of Wisconsin, Madison, Wisconsin D. L. WOOD (3), Bell Telephone Laboratories Inc., Murray Hill, New Jersey HARLAND G. WOOD (23), Western Reserve University, Cleveland, Ohio SIDNEY WEINHOUSE (25),

Outline

of Volumes

I, II, III,

VOLUME PREPARATION Section I. General Preparative

AND

IV, and V

I

ASSAY

OF ENZYMES

Procedures

A. TissueSliceTechnique. B. TissueHomogenates.

C. Fractionation of Cellular Components. D. Methods of Extraction of Enzymes. E. Protein Fractionation. F. Preparation of Buffers.

Section II. Enzymes of Carbohydrate

Metabolism

A. Polysaccharide Cleavage and Synthesis. B. Disaccharide, Hexozide, and Glucuronide Metabolism. C. Metabolism of Hexoses. D. Metabolism of Pentoses. E. Metabolism of Three-Carbon Compounds. F. Reactions of Two-Carbon Compounds. G. Reactions of Formate.

Section III. Enzymes of Lipid Metabolism A. Fatty Acid Oxidation. B. Acyl Activation D. Phospholipid and Steroid Enzymes.

and Transfer. C. fipases and E&erases.

Section IV. Enzymes of Citric Acid Cycle VOLUME PREPARATION

AND

II

ASSAY

OF ENZYMES

Section I. Enzymes of Protein Metabolism A. Protein Hydrolyzing Enzymes. B. Enzymes in Amino Acid Metabolism (General). C. Specific Amino Acid Enzymes. D. Peptide Bond Synthesis. E. Enzymes in Urea Synthesis. F. Ammonia Liberating Enzymes. G. Nitrate Metabolism.

Section II. Enzymes of Nucleic Acid Metabolism A. Nucleases. B. Nucleosidases. C. Deaminazes. D. Oxidases. E. Nucleotide Synthesis.

Section III. Enzymes in Phosphate Metabolism A. Phozphomonoesterases. B. Phosphodiesteraaes. C. Inorganic Pyro- and Poly-phosphatazes. D. ATPases. E. Phosphate-Transferring Systems.

Section IV. Enzymes in Coenzyme and Vitamin

Metabolism

A. Synthesis and Degradation of Vitamins. B. Phozphorylation zyme Synthesis and Breakdown.

Section V. Respiratory

of Vitamins. C. Coen-

Enzymes

A. Pyridine Nucleotide-Linked, Enzymes. D. Unclassified.

Including Flavoproteina. B. Iron-Porphyrins. xi

C. Copper

xii

OUTLINE

OF

VOLUMES

I,

II,

VOLUME

PREPARATION AND

III,

IV,

AND

V

III

ASSAY

OF SUBSTRATES

Section I. Carbohydrates A. Polysaccharides. B. Monosaccharides. C. Sugar Phosphates and Related Compounds. D. Unphosphorylated Intermediates and Products of Fermentation and Respiration.

Section II. Lipids and Steroids A, Isolation and Determination of Lipids and Higher Fatty Acids. B. Preparation and Analysis of Phospholipids and Derivatives. C. Fractionation Procedures for Higher and Lower Fatty Acids. D. Preparation and Assay of Cholesterol and Ergosterol.

Section III.

Citric Acid Cycle Components

A. Chromatographic Compounds.

Analyses of Organic Acids. B. Specific Procedures for Individual

Section IV. Proteins and Derivatives A. General Procedures for Determination of Proteins and Amino Acids. B. General Procedures for Preparation of Peptides and Amino Acids. C. Specific Procedures for Isolation and Determination of Individual Amino Acids.

Section V. Nucleic Acids and Derivatives A. Determination, Isolation, and Characterization of Nucleic Acids. B. Determination, Isolation, Characterization, and Synthesis of Nucleotides and Nucleosides.

Section VI. Coenzymes and Related Phosphate Compounds A. General Procedures for Isolation, Determination, and Characterization of Phosphorus Compounds. B. Specific Procedures for N-Phosphates and Individual Coenzymes.

Section VII. Determination

of Inorganic Compounds

VOLUME SPECIAL

TECHNIQUES

IV

FOR THE

ENZYMOLOGIST

Section I. Techniques for Characterization of Proteins (Procedures and Interpretations) A. Electrophoresis; Macro and Micro. B. Ultracentrifugation and Related Techniques (Diffusion, Viscosity) for Molecular Size and Shape. C. Infra-red Spectrophotometry. D. X-ray Diffraction. E. Light Scattering Measurements. F. Flow Birefringence. G. Fluorescence Polarization and Other Fluorescence Techniques. H. The Soluhility Method for Protein Purity. I. Determination of Amino Acid Sequence in Proteins. J. Determination of Essential Groups for Enzyme Activity.

OUTLINE

OF

VOLUMES

Section II. Techniques for Metabolic

I,

II,

III,

IV,

AND

V

... x111

Studies

A. Measurement of Rapid Reaction Rates; Techniques and Applications, Including Determination of Spectra of Cytochromes and Other Electron Carriers in Respiring Cells. B. Use of Artificial Electron Acceptors in the Study of Dehydrogenases. C. Use of Percolation Technique for the Study of the Metabolism of Soil Microorganisms. D. Methods for Study of the Hill Reaction. E. Methods for Measurement of Nitrogen Fixation. F. Cytochemistry.

Section III. Techniques for Isotope Studies A. The Measurement of Isotopes. B. The Synthesis and Degradation of Labeled Compounds (Including Application to Metabolic Studies) : Monosaccharides and Polysaccharides; Citric Acid Cycle Intermediates; Glycolic, Glyoxylic and Oxalic Acids; Purines and Pyrimidines; Porphyrins; Amino Acids and Proteins; Steroids; Methylated Compounds and Derivatives; Sulfur Compounds; Fatty Acids; Phospholipids; &enzymes; Iodinated Compounds; Intermediates of Photosynthesis; On-Labeled Phosphorus Compounds.

VOLUME PREPARATION

Section I. General Preparative

AND

V

ASSAY

OF ENZYMES

Procedures

A. Column Chromatography of Proteins. B. Preparative Electrophoresis. C. Preparation and Solubilization of Particles (Bacterial, Mammalian, and Higher plant). D. Mammalian Cell Culture. E. Protoplasm.

Section II. Enzymes of Carbohydrate

Metabolism

A. Polysaccharide Cleavage and Synthesis. B. Disaccharide, Hexoside and Glucuronide Metabolism. C. Metabolism of Hexoses, Pentoses and 3-Carbon Compounds. D. Hexosamine and Sialic Acid Metabolism. E. Aromatic Ring Synthesis.

Section III. Enzymes of Lipid Metabolism A. Fatty Acid Synthesis and Breakdown. B. Acid Activating Enzymes. C. Phospholipid Synthesis and Breakdown. D. Steroid Metabolism.

Section IV. Enzymes of Citric Acid Cycle A. Krebs Cycle. B. Krebs-Kornberg Cycle. C. Related Enzymes.

Section V. Enzymes of Protein Metabolism Proteolytic Enzymes. B. Amino Acid Dehydrogenases and Transaminases. C. Amino Acid Activating Enzymes. D. Other Enzymes of Amino Acid Breakdown and Synthesis. E. Enzymes of Sulfur Metabolism. A.

xiv

Erratum P. 140, line 3: should read “19.21

for Volume I

g.” instead of “21.01

Erratum

g.”

for Volume II

P. 432, line 2: should read “6.0 ml.” instead of “60 ml.”

Errata for Volume III P. P. P. P.

663, line 3: should read “0.35 y” instead of “35 7.” 843, line 2 from bottom: should read “KH2POr” instead of “K2HPOd.” 948, line 6: should read “31” instead of “0.031.” 1055, entry in author index: Abraham, S., 64.

[1]

ELECTROPHORESIS

[1]

3

Electrophoresis

By ARNE TISELIUS Enzymes, as well as proteins in general, are ampholytes, and therefore their electrochemical properties in relation to the pH of the medium are useful for their characterization. The mobility in an electric field at defined pH values and particularly the pH value at which the migration is zero (the isoelectric point) are widely used for this purpose, as these properties can be determined accurately and conveniently with the methods now available. The name electrophoresis for such phenomena used to refer chiefly to colloids and substances of large molecular weight. However, as the methods gradually have been extended also to low molecular weight material, the new term of "ionophoresis" has been introduced for this case. As it is very difficult to draw a limit here, and as the phenomena involved and the experimental methods applied are practically the same, the author has preferred to use the term electrophoresis throughout this paper. A particularly interesting and valuable feature of electrophoresis is that the differences in rates of migration for the various constituents of a mixture lead to a certain separation, each component migrating at its own characteristic rate. Thus an electrophoretic analysis is possible which can give valuable information concerning the composition of complex mixtures in solution and the homogeneity or purity of a preparation. Another advantage is that this separation is such a gentle procedure, as it involves only the migration in a medium of approximately constant composition. For preparative purposes this may be very important, especially the recent developments in this field which have provided some very useful methods with many possible applications in enzymology as well in protein chemistry in general. A considerable experience collected during the past twenty years shows that the electrophoretic mobility is a highly specific property, and thus even closely similar proteins often may be differentiated by electrophoresis. This is true, however, only if a not too narrow range of pH is investigated, as in certain pH regions mobilities of different substances may differ very little. It should also be noted that the method, on account of its gentleness, will not resolve complexes of different substances unless they are extensively dissociated in solution under the conditions of the experiment. Many cases are known in which enzymes or other proteins, even after repeated crystallization, give inhomogeneous electrophoretic diagrams. Protein crystals tend to adsorb foreign material, but this phenomenon is probably less marked in electro-

4

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[1]

phoretic separation where all components are in solution. Thus positively and negatively charged proteins may migrate almost independently of each other in buffered solutions. This is by no means universally true, however, and cases are known where interaction occurs. 1-3 Fairly stable complexes may be formed between dyes and proteins in solution, and these are not separated by electrophoresis. The demonstration of the existence of such complexes may, however, be of great interest from an entirely different point of view, if they are found in extracts of biological material which have not been subject to drastic operations. They give an indication of the presence of such associates in the original material which for many problems in enzyme chemistry may be significant. Complex formation may also be utilized to aid in the separation. The bestknown example is perhaps the electrophoretic fractionation of uncharged carbohydrates which have been complexed with boric acid (Consden and Stanier4). No doubt there are many interesting possibilities of increasing the specificity of electrophoretic analysis by such methods (see also Cann et al.4~). With methods which depend on differBOUNDARY ZONE ential migration through a medium it is SEPARATION SEPARATION convenient to distinguish between boundFIG. 1. Boundary and z o n e ary methods and zone methods, as entirely separation. different techniques are used (Fig. 1). In boundary separation the migration of the boundaries is observed, and the experiment is usually performed in a U-shaped tube where the protein solution is layered under a buffer solution. Thus the system is stabilized by the density differences, and the migration can take place in free solution without the addition of a stabilizing medium. In this case, however, only the fastest and slowest components can be isolated in substance. Intermediate components overlap, but although their separation is observed it cannot easily be utilized for a real isolation. In this respect zone separation is superior. A zone of the sample will, during its migration, split up into as many different zones as it contains differently migrating components, each zone consisting of a single component, which is easily isolated. As the zones have a higher density than the medium, however, 1L. G. Longsworthand D. A. MaeInnes, J. Gen. Physiol. 25, 507 (1942). W. Grassmann, in "The Chemical Structure of Proteins" (Wolstenholme and Cameron, eds.), pp. 55, 195. Churchill, London, 1953. s IV[.Lautout and M. de Serge, Compt. rend. 240, 1282 (1955). R. Consden and W. M. Stanier, Nature 169, 783 (1952). ~"J. R. Cann, J. G. Kirkwood, R. A. Brown, and O. J. Plescia, J. Am. Chem. Soc. 71, 1603_(1949).

/[:

[1]

ELECTROPHORESIS

5

they are not stable, and the important advantage of zone methods has to be gained at the price of stabilizing the system by using a suitable filling material, such as cellulose, starch powder, filter paper, or a density gradient. This introduces a new factor which may influence mobilities and isoelectric points in a way which is difficult to predict. But if the separation is the main purpose, these methods are very useful and have found many applications during the last years. Paper strip electrophoresis has become particularly popular as a micro-method and will be discussed in the following section by Dr. H. Laurell (Vol. IV [1A]). Zone electrophoresis in packed columns has recently been developed to a high degree of perfection. It bears the same relation to paper strip electrophoresis as column chromatography does to paper chromatography, and it allows the separation of considerable quantities of material. This method will therefore also be discussed in some detail. There are some special modifications of electrophoretic analysis without a clear-cut zone or boundary separation which have gained preparative importance. This is particularly true of the electrophoresis convection method and of methods depending on electrophoresis through membranes. The literature on electrophoresis is very extensive. A selection of references to books or articles of a more general interest is given below. 1. General principles and boundary methods. 5-~9 2. Theory. ~4,2° 5 H. A. Abramson, L. S. Moyer, and M. H. Gorin, "Electrophoresis of Proteins." Reinhold Publishing Corp., New York, 1942. 5 H. A. Abramson, E. J. Cohn, B. D. Davis, F. L. Horsfall, L. G. Longsworth, D. A. MacInnes, H. Mueller, and K. Stein, Ann. N . Y . Acad. Sei. 39, 105 (1939). 7 H. J. Antweiler, " D i e quantitative Elektrophorese in der Medizin." SpringerVerlag, Berlin, 1952. 8 D. R. Briggs, in "Biophysical Research M e t h o d s " (F. M. Uber, ed.), pp. 271-300. Interscience Publishers, New York, 1950. 9 H. B. Bull, "Physical Biochemistry," 2nd ed., pp. 161-190. John Wiley & Sons, New York, 1951. 10 L. G. Longsworth and D. A. MacInnes, Chem. Revs. 24, 271 (1939). n L. G. Longsworth, Chem. Revs. 30, 323 (1942). 1, j . A. Luetscher, Physiol. Revs. 27, 621 (1947). 13 K. G. Stern and M. Reiner, Yale J. Biol. and Med. 19, 67 (1946). 14 It. Svensson, Arkiv Kemi Mineral. Geol. 22A (1946). 15 E. Wiedemann, Scientia Pharm. 17, 45 (1949). 16 A. Tiselius, Scientia 45, 163 (1951). 17 A. Tiselius, Naturwissenschaften 37, 25 (1950). Is A. Tiselius, Trans. Faraday Soc. 35, 524 (1937). 19 A. Tiselius, Nova Acta Regiae Soc. Sci. Upsaliensis [4]7, No. 4 (1930). 25 L. G. Longsworth and D. A. MacInnes, J. Am. Chem. Soc. 62, 705 (1940).

6

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[l]

3. Zone methods in general and preparative electrophoresis. 21-25 4. Paper electrophoresis (see Vol. IV [1A]).

Boundary Electrophoresis For boundary electrophoresis experiments a U-shaped tube is generally used in which the sample, dissolved in a suitable buffer solution, is allowed to form a layer under a buffer solution of the same composition, so that the boundaries will migrate in a medium of approximately constant composition. The electric current is introduced with reversible electrodes, separated from the boundaries by a sufficient volume of buffer solution to prevent changes in the composition produced at the electrodes to reach the boundaries. The boundaries are stabilized by the differences in density, and thus no special stabilizing medium is required as in zone electrophoresis. Consequently the " f r e e " electrophoretic migration of the components can be measured, and the true mobilities can be calculated directly from the experiments. To secure a sufficient stability the concentrations should not be too low, and it is generally difficult to obtain reliable results at concentrations below 0.01% (protein) unless special precautions are taken (low current). The heat produced by the current may cause convections, and when studying systems of high conductivity (e.g., serum) or low protein concentration it is essential to minimize this disturbance by running the experiment at a temperature just above 0 °. 18As water has a density maximum around -~4 °, the density differences produced by temperature changes in this region are very much smaller than at room temperature, for example. As low temperature is of advantage for other reasons in protein and enzyme work, most experiments of this type are nowadays being performed in refrigerated baths. To facilitate optical observation of the boundaries by the methods described below, the electrophoresis cell is given a rectangular cross section. The U-tube is assembled by one or several such cells, mounted with plane-parallel plates which are greased so that they can slide horizontally a sufficient distance to cut the contents of the tube into a number of fractions. This sliding arrangement is also used when the boundaries 21 H. G. Kunkel, in " M e t h o d s of Biochemical Analysis," Vol. I, pp. 141-170. Interscience Publishers, New York, 1954. 22 H. J. McDonald, "Ionography. Electrophoresis in Stabilized Media." Year Book

Publishers, Chicago, 1955. 23H. Svensson, Advances in Protein Chem. 4, 252 (1948). ~4R. L. M. Synge, in "General Methods of Separation: Electrical Transport Methods in Modern Methods of Plant Analysis, I" (Paech and Tracey, eds.), pp. 55-65. Springer-Verlag, Berlin, 1956. 25A. Tiselius and P. Flodin, Advances in Protein Chem. 8~ 461 (1955).

[1]

ELECTROPHORESIS

7

are to be formed at the beginning of the experiment. Most modern equipment of this type is provided with a "compensation device" which allows a slow and accurately controlled injection of buffer solution into one or the other of the electrode tubes during the experiment. Thus the boundaries may be displaced to any desired position in the tube, and fast-moving boundaries may be kept in the observation field much longer than would otherwise be possible.

FIG. 2. Schematic drawing of an apparatus (manufactured by LKB-Produkter Fabriks AB, Stockholm) for boundary electrophoresis, showing the composite U-tube and the electrodes. Cells 2, 3, and 4 are filled with the sample, and ceils 1 and 5 as well as the electrode vessels with buffer solution. Figure 2 shows a diagrammatic representation of a boundary electrophoresis apparatus with a composite U-tube. Although optical observation of the boundary migration is usually preferred in this type of work, it should be noted that electrophoretic mobilities may also be determined analytically in this apparatus ("transference method"). If cells 2, 3, and 4 in Fig. 2 at the start contain the sample, of known concentration (for example, nitrogen, or any enzymatic activity), and cells 1 and 5 contain only buffer, the experiment is allowed to run until cell 1 is partially filled with migrating material and cell 4 partially emptied. If the bottom cell (cell 3) composition does not change, the increase on one side must

8

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[1]

be equal to the decrease on the other. If this difference is Am, the original concentration is c (in the same unit), and the cross-section area of the tube is q, the distance of migration is hm/cq. The quantity Am is determined by taking out the contents from each cell and analyzing at the end of the experiment. It should be noted that this method is applicable also when c and m refer to enzymatic activities and thus may be useful when the actual enzyme concentration in the sample is too low to make optical observation possible. In complicated systems valuable information may also be obtained by combining this procedure with optical observation of the boundaries, and it may be possible this way to determine which of the observed boundaries refers to the enzyme or other material with measurable biological activity which is being studied. Direct optical observation (by photography) of the migration of the boundaries is most commonly used, however, and has the great advantage that it offers a convenient way of studying the heterogeneity of the sample, as each differently migrating component usually will give rise to a separate boundary. The optical methods also allow a quantitative estimation of the relative amount of the different components present (electrophoretic analysis). As substances to be studied generally are colorless, refractive index or ultraviolet light absorption methods are usually preferred. One may distinguish between concentration and concentration-gradient methods, the latter being most suitable for heterogeneity studies. In the author's early experiments 19 the ultraviolet absorption method introduced by Svedberg and Nichols :6 for ultracentrifugation measurements was used. The electrophoresis U-tube is made of quartz and is photographed in transmitted ultraviolet light from a quartz mercury vapor lamp, the light being filtered through quartz cells containing bromine and chlorine. In later work when this method has been used for studying protein chromatography we have found that the somewhat troublesome chlorine-bromine filters may be replaced by a solution of cobalt and nickel sulfate, according to Kasha. 2~ As this method is not sensitive enough to analyze composite boundaries, the author 28 in 1937 introduced refractive index observation, based on the well-known Toepler schlieren method, which depends on gradients rather than on concentrations. The ultraviolet method has the advantage of being more specific than refractive index methods, however, and may perhaps deserve renewed attention, even though it requires the introduction of an all quartz system. Interferometric observation also records concentrations and has been introduced lately by several authors (see, ~6 T. Svedberg and J. B. Nichols, J. Am. Chem. Soc. 48, 3081 (1926). ~ M. Kasha, J. Opt. Soc. Amer. 88, 929 (1948). 28 A. Tiselius, Trans. Faraday Soc. 35, 524 (1937).

[1]

ELECTROPHORESIS

9

for example, Svensson 29 and Svensson and Forsberg~°). These methods are highly sensitive and are often useful, but the evaluation of the diagrams is perhaps somewhat more laborious than with the gradient methods. The former are particularly useful for determinations of relative amounts of the components, the latter for high resolution of boundary composition. A combination of both is probably most valuable. So far most results published have been obtained by gradient methods, and these will therefore be discussed in more detail. They are based on the deviation which a light beam suffers if it passes through a refractive index (that is, a concentration) gradient. Thus if the beam is made to pass horizontally through an electrophoresis tube containing a boundary, it will be deviated downward in those parts of the tube where the boundary is located. If a camera is focused on the tube and if the camera lens is provided with a horizontal edge diaphragm, light from all parts of the tube will be able to pass into the camera except from those levels where the boundary is situated, the light from these parts being deviated below the diaphragm edge. Thus on the image the boundary will come out as a black band. Each setting of the diaphragm corresponds to a certain deviation--that is, to a certain value of the refractive index gradient. As Longsworth ~1 first showed, a complete recording of the gradient throughout the boundary region as a function of the vertical distance in the electrophoresis tube may be obtained by coupling the movement of the edge with a movement of the photographic plate. The same result may also be obtained by a purely optical arrangement introduced by Philpot 32 and further developed by Svensson, ~3 where no movable parts are required and the recording is achieved by the aid of a cylindrical lens combined with a special diaphragm (Fig. 3). For details the reader is referred to the literature recommended in footnotes 5 to 15. The diagrams obtained give the refractive index gradient, d n / d x , as a function of the distance of migrating in the cell. Thus, by integration, the total n v a l u e - - t h a t is, the concentration--can be computed for each sufficiently well-separated component. Typical gradient diagrams are shown in Fig. 4. Both these procedures work satisfactorily and have found extensive use. In addition, mention should be made of the highly accurate "scale m e t h o d " by Lamm 34 which has found wide application especially in 59H. Svensson, Acta Chem. Scan& 3, 1170 (1949). 30H. Svensson and R. Forsberg, J. Opt. Soc. Amer. 44, 414 (1954). al L. G. Longsworth, J. Am. Chem. Soc. 61, 529 (1939). 3~j. S. L. Philpot, Nature 141, 283 (1938). a3H. Svensson, Kolloid-Z. 87, 181 (1939); see also ref. 14. 34O. Lamm, Z. physik. Chem. A138, 313 (1928).

10

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

Ill

ultracentrifugation and diffusion work but does not seem to have come into general use in electrophoresis, probably because the evaluation of the diagrams is somewhat laborious. Detailed instructions for working with the different types of commercially available electrophoresis apparatus are provided with the instruments and will not be discussed further here. Some general remarks about the procedure may, however, be useful to readers who have no previous experience in electrophoresis work. Boundary electrophoresis, including transference measurements (see p. 7), is so far the most accurate and Op//CO/ S~/s/ern

A

B

C

D

E

f

G

H

Fro. 3. Optical system for boundary observation in eleetrophoresis. The dashed beams . . . . and pass through the cell, C, outside the boundary region and are therefore not deviated but are focused at the upper end of the inclined slit, E. They pass through the cylindrical lens, G, without horizontal deviation and give rise to the "base line" in the curve on the film, H. The beam passes through the boundary in t h e cell, C, and is deviated to the lower end of the inclined slit, E, which gives rise to a horizontal deviation in the cylindric lens, G, resulting in an image point near the peak of the curve on the film, H. [From H. Svensson, Tek. Tidskr, $7, 1952.]

least objectionable method of determining mobilities and isoelectric points for proteins and other large molecular weight substances with a pronounced electrochemical character. It should always be observed that, even if these characteristics are sharply defined and very useful for the characterization of such substances, they depend markedly on other ions present, so that the buffer used and its concentration always should be stated. The other application is the study of homogeneity, and electrophoretic analysis is often used as a sensitive criterion on purity. One must remember, however, that differences may exist which are not reflected in the electrophoretic behavior, and under all conditions the analysis should be made at a number of different pH values before any conclusions are drawn. The possibility of complexes which are too weakly dissociated to be separated into their constituents by electrophoresis should also be kept in mind (cf. p. 4). There are some complications inherent in the method which often

[1]

ET.ECTI~OPHORESm

11

A scend;n9

J

j

~

AIo.

Descer~/n 9

t . 6lob. A

B FIG. 4. Typical boundary electrophoresis diagrams, as obtained by the apparatus and optical arrangement in Figs. 2 and 3. (A) Human pathological serum, showing the main protein components: albumin and a-, f~-, and -y-globulins. (B) Crystalline lactic dehydrogenase, containing two components. Descending pattern in phosphate buffer, pH 5.7, ionic strength 0.1. [According to J. B. Neilands, S c i e n c e 115, 143 (1952).]

12

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[1]

puzzle research workers in this field and which have been given the common name of "boundary anomalies." Thus, one finds frequently that descending and ascending boundaries of the same substance and in the same experiment move at somewhat different rates. "False" boundaries, which are due to discontinuities in the buffer medium and thus do not represent new components, are sometimes observed, especially in concentrated solutions, as the well-known ~-boundary in serum experiments. As a rule these disturbances are much reduced if the protein concentration is lowered. The anomalies are more marked, however, with substances of lower molecular weight, and they make it generally impossible to study by this method the migration of amino acids, for example. The general problem of the migration boundaries in electrolyte solutions is a very complex one, but the theory has been worked out in detail (see, for example, Longsworth and MacInnes, 2° Dole, 35 and Svensson~4). Moving boundary electrophoresis of proteins and similar substances is to be regarded as a limiting case, in which one component (the protein) contributes relatively little to the electrolytic conductivity of the system, although it is present in an appreciable concentration. Thus the migration of the protein does not change appreciably the distribution of the potential gradient, and it moves in a medium of practically constant composition and therefore in a constant electric field. This is generally only approximately true, however. If substances are studied for which the ratio conductivity:weight is higher and approaches that value for the buffer ions used, the anomalies dominate the phenomenon and make the interpretation of the results very difficult The decisive factor is thus not the molecular size but the conductivity per unit weight of the substance to be studied The methods of zone electrophoresis to be described below have the advantage of showing a much greater latitude toward boundary anomalies and thus permit the study of substances which previously could not be subject to electrophoresis.

Zone Electrophoresis in Packed Columns and Similar Media (Except Paper Strip Electrophoresis) The development in this field has been particularly rapid during the last few years, and no attempt will be made here to present the historical development. The author has preferred to review the present situation with particular emphasis on procedures of which he has had personal experience. The apparatus required for zone electrophoresis is comparatively simple. Optical observation is not necessary (although in some cases it a~ V. P. Dole, J. Am. Chem. Soc. 67, 1119 (1945).

[l]

ELECTROPtIORESIS

13

would no doubt be of advantage). The zones are localized by analytical or by staining methods. The crucial problem is the filling material necessary for the stabilization of the zones. Proteins and other substances to be studied are often adsorbed to finely divided powders. This tends to give rise to tailing phenomena which greatly interfere with the separation. Thus sometimes the mobility may be reduced to practically zero. The stabilizer should be hydrophilic but insoluble. If it is a powder it should be easy to pack into homogeneous columns which contain a sufficient amount of the solvent medium. There will always be some electrical transport of the solvent through the stabilizer during the experiment (electroendosmosis). This will not interfere with the separation as it only moves the system of zones in the direction of electroosmotic flow, but experience shows that it is important to keep this flow as low as possible. Among powdered materials cellulose and starch have been most widely used so far. In the author's laboratory Flodin and Kupke 3~have found that cellulose powder prepared from cotton by alcoholysis is superior to ordinary cellulose, and this material is now preferred in all our zone electrophoresis investigations. It is advisable, however, to test the material for its adsorption properties on the system to be studied by running a zone through a small column without c u r r e n t - - t h a t is a chromatographic experiment. Gels are often permeable enough also to large protein molecules to make them suitable as stabilizing media for zone electrophoresis. They have the advantage of giving sufficient stabilization at low dry weight, and thus adsorption phenomena are less disturbing. They often give beautiful zones which may be observed optically if the gel is translucent. Silica gel was used by Consden et al.'~7 in their " i o n o p h o r e t i c " separations of peptides and amino acids, but this gel appears to be less suitable for proteins. Agar gel has been used with success by Gordon et al., 3~ and recently Grabar et al. '39 have made extensive electrophoretic investigations by this technique, also combining it with the Oudin serological precipitation method which makes possible a very sensitive identification of different antigens (immuno-clectrophoresis). Smithies t° has obtained very beautiful separations of proteins in starch gels. A disadvantage of the gels is that it is difficult to elute the zones quantitatively without contamination. In this respect powdered stabi36p. Flodin and D. W. Kupke, Biochim. et Biophys. Acta 21, 368 (1956). 37R. Consden, A. H. Gordon, and A. J. P. Martin, Bioehem. J. 40, 33 (1946). a8A. H. Gordon, B. Keil,' and K. Sebesta, Nature 164, 418 (1949). ~9p. Grabar, C. A. Williams, Jr., and J. Courcon, Biochim. et Biophys. Acta 17, 67 (1955). 400. Smithies, Biochem. J. 61, 629 (1955).

[1]

ELECTROPHORESIS

15

-:-4 D4

---2 -_w

-----

--A,

z----.--

----AJ

~_w_

0

_.--__ -

--2-

~.-

--_--.

--_--.

Fu~. 5. Large t y p e of zone c o l u m n electrophoresis a p p a r a t u s according to J. P o r a t h

[Biochim. et Biophys. Acta 22~ 151 (1956)]. Details in the text.

16

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[1]

.c. .j

~5

!1 ~o

2b

~o

~0

~

60

7o

'

~o

9o

Tube r~m~er

~o

A Fro. 6A. Mixture of "neutral" amino acids in I N acetic acid resolved by 61 hours of electrophoresis at 30 ma. (2300 v.) in a column 2 X 250 cm. packed with cellulose powder. E is the extinction as determined by the ninhydrin reaction. Each amino acid, 2 mg. [J. Porath, Biochim. et Biophys. Acta 22, 151 (1956).]

.~

~70

~,a.~

~

0

,~ I I

~

~ 11

~0

~00

I,/

//

thO

"~"

I1

H

200 250 [itlcft~e, rd vol~jrn~,ml

B FTO. 6B. Mixture of vitamin B~ components in acetate buffer, p H 5,1, ionic strength 0.05, resolved by 14 hours of electrophoresisat 18 ma. in a column 3 X 50 cm. packed with starch. Each component 2.6 mg. [From N. Siliprandi, D. Siliprandi, and H. Lis, Biochim. et Biophys. Acta 14, 212 (1954).]

[1]

ELECTROPHORESIS

17

l a r g e c o l u m n s t h e c a p a c i t y is c o n s i d e r a b l e . T h u s on c o l u m n s of 150-cm. l e n g t h a n d 6.5-cm. d i a m e t e r as m u c h as 20 to 30 g. of p r o t e i n c a n be s e p a r a t e d a n d s e r u m s a m p l e s c o r r e s p o n d i n g t o 200 ml. u n d i l u t e d s e r u m h a v e b e e n f r a c t i o n a t e d . P e p t i d e s , a m i n o acids, a n d o t h e r low m o l e c u l a r w e i g h t m a t e r i a l l e n d t h e m s e l v e s v e r y well to t h i s k i n d of s e p a r a t i o n . F i g u r e s 6 A - C s h o w s o m e e x a m p l e s of r e s u l t s o b t a i n e d w i t h y e a s t enolase, 2.0

2.0

o

~I,5

1.5

2

~ I.0

1.0

O

~0.5

0.5 5"

20

30

40

FRACTION NUMBER

C Fro. 6C. Cellulose column electrophoresis of yeast enolase. Column dimensions: 1.8 cm., diameter; 70 cm., length. Buffer: 0.0154 M NaH~P04, 0.0115 M Na:B4OT, pH 8.2, # = 0.05. Current: 15 ma. Time: 45 hr. The experiment was performed in a cold room (4°). The protein concentration (O) of the fractions was determined by measuring the optical density at 280 m# in a Beckman DU spectrophotometer using 1-cm. cells, 1 rag. per milliliter giving a value of 0.895. The activity (A) was estimated as the change in optical density at 240 m~ per minute (&D.~40/min.) at 22 ° when 10 p]. of a fraction was added to I ml. of substrate solution (2.4 X 10-3 M DL-2-phosphoglycerate and l0 -3 M mg. ++ in tris(hydroxymethyl)aminomethane-HC1 buffer, pH 7.4, ~ = 0.05) in a 1-cm. cell. [Experiments by Dr. B. Malmstr6m, Uppsala, to be published.[ t h e d a t a b e i n g g i v e n in t h e figure legend. W h e n s e p a r a t i n g low m o l e c u l a r w e i g h t s u b s t a n c e s v o l a t i l e buffers as r e c o m m e n d e d b y P o r a t h 48 a r e v e r y useful, as d i a l y s i s c a n n o t be u s e d in t h i s case to g e t r i d of t h e e l e c t r o l y t e s c o n t a i n e d in t h e f r a c t i o n s i s o l a t e d . P r e c i s e m o b i l i t y m e a s u r e m e n t s c a n n o t be m a d e in zone m e t h o d s w h e r e s t a b i l i z i n g m e d i a a r e used. I t a p p e a r s , h o w e v e r , t h a t t h e d i f f e r e n t c o m p o n e n t s e m e r g e f r o m t h e c o l u m n in t h e o r d e r t o be e x p e c t e d f r o m t h e i r m o b i l i t i e s in free s o l u t i o n . K o l i n , 49 S v e n s s o n a n d V a l m e t , 5° a n d 48j. Porath, Nature 174, 478 (1955). 49 A. Kolin, Proc. Natl. Acad. Sci. U.S. 41, 101 (1955). 5o H. Svensson and E. Vahnet, Science Tools 2, 11 (1955).

[1]

ELECTROPHORESIS

19

vessel and will therefore flow as a narrow vertical zone through the medium and leave the cell through one or a few of the bottom outlets. If a horizontal electric field is applied, however, the zone will split up as shown in the figure, and separated fractions may be collected at suitable outlets. In principle, it should be possible to fractionate very large quantities by such procedures, and the method may even gain industrial application. Maintaining constant conditions of flow and a constant current is very essential for obtaining good resolution. Brattsten 56-5~ has recently published a detailed study of the method and has described its application to serum protein fractionation. For smaller quantities sheets of filter paper may be used which simplifies the apparatus. Further information about this method may also be found in a review by Durrum et al. 59 So far, the capacity does not seem to surpass what can be conveniently handled in a zone column apparatus of the type described above (pp. 14-15), which also has the advantage of simpler construction and more easily controlled manipulation. For still larger capacities the continuous zone electrophoresis no doubt offers promise.

Other Methods of Preparative Electrophoretic Separation The above methods have in common that they allow a control of the separation by observation of boundaries or zones either in the electrophoresis tube itself or in the elution diagrams from the columns. There are some other methods where this is not possible (at least not directly) but which have proved to be quite useful in preparative work. This is particularly true of the "electrophoresis convection" method first proposed by Kirkwood 6° and experimentally realized by Nielsen and Kirkwood. 6~ A description of the apparatus is given in papers by Cann el al. 4~ and Timasheff et al. 82 The latter paper describes a semicontimlou~ modification of the method and gives further references. The method is based on the layering phenomenon sometimes observed in electrodialysis of proteins between vertical membranes. The proteins are concentrated by migration toward one membrane and tend to form a layer, which on account of its higher density will be transported by 5~ I. Brattsten, Arkiv Kemi 4~ 503 (1952). 57 I. Brattsten, Arkiv Kemi 8, 205, 227, 347 (1955). 5a I. Brattsten, Continuous Zone Electrophoresis by Crossed Velocity Fields in a Supporting Medium, Dissertation, Uppsala, 1955. ~ R. J. Block, E. L. Durrum, and G. Zweig, " A Manual of Paper Chromatography and Paper Electrophoresis," pp. 380-390. Academic Press, New York, 1955. s 0 j. E. Kirkwood, J. Chem. Phys. 9, 878 (1941). e~L. E. Nielsen and J. G. Kirkwood, J. Am. Chem. Soc. 68, 181 (1946). 6~ S. N. Timasheff, J. B. Shumaker, Jr., and J. G. Kirkwood, Arch. Biochem. and Biophys. 47, 455 (1953).

20

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[l]

convection to the bottom of the cell. Several sharp horizontal boundaries, separating layers of different composition, may thus often be obtained. The Kirkwood apparatus consists essentially of a flat rectangular cell, separated by large surface membranes from the electrode compartments, through which a suitable buffer solution is circulated. The membranes should be semipermeable, thus allowing the buffer ions but not the protein material to pass freely. In the course of the experiment the fastest migrating substances are collected toward the bottom and the slowest at the top. The method is in principle analogous to the well-known Clusius thermodiffusion method for separation of gases. It has been applied with notable success to the fractionation of serum proteins and insulin, for example, and should be easy to adopt for large-scale work. Multimembrane apparatus where the material to be separated is migrating through the membranes has found some use for the electrophoretic separation of peptides and amino acids. With suitable membranes proteins and other large molecular weight material also may be fractionated. The apparatus required is of the same type as a common electrodialyzer and thus quite simple. The membranes serve to keep the separated components apart, but also have a very marked effect on the course of the separation. The result depends largely on the rates of migration within the membranes, which may differ quite considerably both absolutely and relatively from m0bilities in free solution, as the membrane charge and other specific effects have a decisive influence. Thus it is usually necessary to combine membranes of different kinds. Specific effects can naturally be utilized to improve the separation. A detailed discussion of methods of this type has been given by Svensson. 28 Mould and Synge 63 have had some success in attempts to separate substances according to molecular size by migrating them in collodion strips with the aid of electroosmotic transport of the solvent (electrokinetic ultrafiltration). In part the effects obtained seem to be due to adsorption , but the method appears to have interesting possibilities, also in zone separation. t3 D. L. Mould and R. L. M. Synge, Biochem. J. 58, 571 (1954).

32

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

[2] Ultracentrifugation, Diffusion, and Viscometry B y H. K. SCHACHMAN

I. Introduction Theoretical and experimental advances which have been made in recent years in ultraeentrifugation, diffusion, and viscometry have resulted in the availability of powerful methods for the determination both of the degree of purity of a given preparation of a protein and of the size and shape of the macromolecules. It is of importance, therefore, that enzymologists be aware of these methods, their potentialities, and their limitations. In addition to considerations of purity and molecular size we will also discuss in this chapter some applications of diffusion and sedimentation methods which possess special appeal to the enzymologist. Techniques are available, although infrequently used, whereby the sedimentation and diffusion of a biologically active material can be studied, even in impure preparations and with only microgram quantities of material, as long as there is a sensitive and accurate biological assay specific for the entity under investigation. With these methods, reasonably accurate values of the molecular weight of an enzyme can be obtained even before purification of the enzyme is achieved. Some of the methods we will discuss are rapid, and the techniques can be performed at low temperature so that labile enzymes can be studied without danger of inactivation. In most cases, a fairly complete study can be performed with as little as 10 to 20 mg. of material, and in special circumstances microgram quantities will suffice. The same methods are equally applicable to the examination of molecules smaller than most proteins and can be used therefore in studies of coenzymes and substrates or the products of proteolytic enzymes or nueleases. In this way, these physical methods can be used to study the mechanism of enzymatic action on large substrate molecules. Ultracentrifugation, in particular, is of use in the study of interaction of enzyme molecules with themselves as in association equilibria or in interaction of enzyme molecules with other molecules such as DPN. For all the work described in this chapter there is now available commercial apparatus which, though expensive and complicated, is generally installed by the manufacturer ready for operation. The detailed description and explanation of the working parts of this equipment will be omitted from our discussion as will be those operating procedures generally supplied by the manufacturer in technical manuals. Only special

[9.]

ULTRACENTRIFUG&TION'~ DIFFUSION, AND VISCOMETRY

33

parts of the experimental procedures common to all different makes of apparatus and requiring techniques developed through experience will be elaborated upon in this discussion.

II. Ultracentrifugation Introduction Ultracentrifuges can be used either to study the velocity of sedimentation of macromolecules through a solution under the influence of a centrifugal field or to study the distribution of the macromolecules in a centrifuge cell which is rotating at much lower speeds so that the molecules obtain equilibrium positions throughout the cell. The former method is known as the sedimentation velocity method and has to date been the more widely used. In a sedimentation velocity experiment the ultracentrifuge is operated at speeds up to 60,000 r.p.m. so that the solute molecules which initially were uniformly distributed throughout the solution in the ultracentrifuge cell are caused to settle toward the periphery of the cell. This migration of the solute molecules leaves a region containing only solvent molecules (if the solute molecules are more dense than the solvent, then the newly created solvent region will be near the meniscus; for solutes which are less dense than the solvent, such as lipoproteins in concentrated salt solutions, the solute will float, leading to a region of pure solvent at the bottom of the cell) in addition to the region in the cell where the concentration of solute is uniform. Between the supernatant and the solution of uniform concentration, known as the plateau region, there is a transition zone in which the co~centration varies with distance from the axis of rotation. This transition zone is called the boundary; and the sedimentation velocity method is based on observations, by optical methods, of the movement of the boundary which in turn is a measure of the movement of the solute molecules in the plateau region. Figure 1A shows schematic drawings of the concentration of solute as a function of distance in the cell at different times. Since the more commonly used optical systems do not record concentration, but rather the change in concentration with distance, the corresponding curves are drawn in Fig. lB. It is such diagrams, as illustrated in Fig. 1B, which are used to study the purity of a given preparation and to determine the sedimentation coefficient, s, of the sedimenting material. It should be noted that the sedimentation velocity method permits the direct measurement of sedimentation coefficients which are related to the size and shape of the sedimenting molecules, but other independent data are needed for the evaluation of molecular weights. In the second application of the ultracentrifuge, known as the sedimen-

34

T E C H N I Q U E S FOR CHARACTERIZATION OF P R O T E I N S

[2]

tation equilibrium method, the ultracentrifuge is operated at relatively low speeds (about 8000 r.p.m, for a protein with a molecular weight of 69,000). Under these conditions the transport of solute in a centrifugal direction due to sedimentation is sufficiently slow as to be counterbalanced by transport in a centripetal direction by the diffusion resulting FROM M E N I S C U S ( C M )

DISTANCE 0

I

I00 Z 0

oi8

I

I

I

,.2

S

1-.IZ w ¢_) z

o ¢3

oi4

50-

J z (.9 ¢e o

/ 0 '

I

B

I-Z tl.I

£9, Z 0 I.n,F.z hi (...) Z 0 U I

I

6.O 6,4 6~8 L2 DISTANCE FROM AXIS OF ROTATION (CM)

Fro. 1. Plot of concentration (A) and concentration gradient (B) versus distance from the axis of rotation during a sedimentation velocity experiment. Sedimentation is to the right.

from the concentration gradient created by the partial sedimentation of the macromolecules. During the first stages of a sedimentation equilibrium run, the concentration decreases at the meniscus and increases at the bottom of the cell, owing to sedimentation. As a consequence of back diffusion, however, a region devoid of solute is not created as in the sedimentation velocity method. Instead the concentration will remain finite at the meniscus as long as the centrifuge is not operated at too high a speed. Under ideal circumstances the concentration at the meniscus

[9.]

ULTRA.CENTRIFUGATION',

DIFFUSION,

AND

VISCOMETRY

35

will approach a value about one-half its initial value. Similarly, sedimentation plus back diffusion at the bottom of the cell leads to a region in which the concentration is higher than the original concentration. In the sedimentation velocity method, where the role of diffusion is much less important, this region of high concentration is restricted to a very thill

O z

DISTANCE FROMMENISCUS(CM) 0.4 0.8 1.2 i ~

o_ I-< n-I-z ~A Z 0°

IO0

Z 0 ~

A

kz hi

B

Z

_o i.n,i-.z

~

4j

Z 0u

g i

i

6.0 6.4 6.8 7.2 DISTANCE FROMA X I S O F R O T A T I O N ( C M ) Fla. 2. Plot of concentration (A) and concentration gradient (B) versus distance from the axis of rotation during a sedimentation equilibrium experiment. Centrifugal direction is toward the right. layer on the cell bottom where the sedimented material is packed as a gel-like pellet. In the center of the cell, during the early stages of a sedimentation equilibrium study, the concentration is independent of position in the cell and practically the same as the initial concentration. As the run proceeds, the plateau region disappears and there is only one position in the cell having a concentration equal to the initial concentration. Finally, after a considerable period of time an equilibrium state is reached after which no further changes in concentration occur with time. These changes in concentration with distance as a function of time are

36

TECHNIQUES FOR CHARA.CTERIZA.TION" OF PROTEIN'S

[2]

visualized in Fig. 2A, and the corresponding gradient curves are illustrated in Fig. 2B. In cont-rast to the sedimentation velocity method, measurements of the concentration distribution at sedimentation equilibrium give directly the molecular weight of the sedimenting macromolecules (both methods require knowledge of the partial specific volume of the macromolecule, and this will be discussed later). Despite this obvious advantage and the additional factor that the theoretical foundations for the equilibrium method are more firm than those for the sedimentation velocity method, there are few recorded examples of the application of the sedimentation equilibrium method to the study of enzymes. This seemingly anomalous situation has arisen both because of the lack of apparatus capable of sustained, continuous use for the long periods (days in the case of proteins) required before equilibrium is reached, and because many proteins are not sufficiently stable to withstand such experiments. A commercial apparatus is now available which is capable of continuous operation even at low temperatures, but it is of greater importance to the enzymologist that considerable theoretical advances have been made which enable us now to determine molecular weights in times as short as one hour. Owing largely to the theoretical work of Archibald I and certain technical improvements especially with regard to optical methods, it seems safe to predict that measurements during the approach to sedimentation equilibrium will become as routine as those by the currently more popular sedimentation velocity method. Because of the importance of these developments we will present in this chapter the method involving the approach to sedimentation equilibrium despite the knowledge that the method is still in its infancy and further refinements both in theory and technique may render the discussion obsolete. We are here using the term "approach to sedimentation equilibrium" rather loosely, since this title leads to the implication that the experiments are conducted for times almost sufficient to attain complete equilibrium. Actually for the use of the equations of Archibald there are no restrictions that the system be near equilibrium. There might be serious objection then to our inclusion of this method under the section entitled "sedimentation equilibrium," especially since this method is clearly not a thermodynamic one. To some extent our classification is arbitrary although it can be justified operationally. If the experiments are designed so as to measure most accurately the velocity of transport of material through the cell as by the proper choice of the centrifugal field for the system under investigation, then the experiments can be classified as sedimentation velocity studies. If, alternatively, the experimental condi1 W. J. Archibald, J. Phys. & Colloid Chem. 51, 1204 (1947).

[9.]

ULTRACENTRIFUGATION, DIFFUSION~ AND VISCOMETRY

37

tions are selected so as to measure precisely the distribution of matter throughout the cell, then the experiments are of the sedimentation equilibrium type. Most ultracentrifuge work done at present is performed in the Model E ultracentrifuge manufactured by the Spinco Division, Beckman Instruments, Inc., Belmont, California. This instrument is completely equipped with the necessary accessories for a variety of ultracentrifuge studies. The majority of the work is performed with the schlieren optical system for viewing and photographing the movement of sedimenting materials. Some special applications, however, can be better performed by absorption techniques using either ultraviolet light or visible light of the appropriate wavelength. A brief description of applications using light absorption techniques will be presented in a later section. A variety of ultracentrifuge cells in optical thicknesses from 1.5 ram. to 30 ram. are available, and the choice of cell depends to some extent on the type of study. For very dilute solutions the cells with the longer optical path are desirable, even though they require larger volumes of solution and even a special rotor. The smaller cells require even less than 0.2 ml. of solution; but, of course, optical registration of the boundary suffers unless high concentrations are used. Cells can be either 4 ° sectors or 2 ° sectors; the latter require longer exposure times and more care in alignment of the cell in the rotor, but they have the decided advantage of requiring smaller volumes of solution. Cells should always be cleaned and dried at the conclusion of the run because salt solutions can cause etching of the cells. Filling needles should have their tips ground flat so as to remove sharp points, and special caution is needed to avoid scratching of the cells. For alkaline or acid solutions, cells with a plastic centerpiece made of Kel-F or epoxy are recommended, and the quality of the latter is so high that they may prove to be the cell of greatest practicality and economy. With plastic cells, no gaskets are required between the centerpiece and the windows, so that assembly of the cells is facilitated. Doublesector cells are available in which one section can be filled with the solution and the other with the solvent. Although the patterns are not so distinct with these double cells, they furnish a base line superimposed on the pattern containing the sedimenting protein, and they can be of use in sedimentation velocity studies. Also special optical selectors facilitate the simultaneous recording of more than one ultracentrifugal analysis in a special rotor containing two or more cells. For equilibrium runs the special one-third speed attachment for the ultracentrifuge is necessary. The optical systems have for years had a bar as the schlieren diaphragm, but it is clear that a phase plate, now commercially available,

38

T E C H N I Q U E S FOR CHARkCTERIZATION OF PROTEINS

[2]

gives much better results and is strongly recommended for all types of work and especially for sedimentation equilibrium. The phase plate can be mounted on the optical track in place of the bar. Since it has been found in recent years that the rotor cools about 0.8 ° on stretching, owing to the high speeds, there has been a growing need for a continuously reading indicator of the temperature of the rotor during the run. This is now available, and the same unit can also control the temperature by activating a heater in the vacuum chamber. For long runs, especially at low temperatures, this is a valuable addition to the ultracentrifuge. Most of the patterns are photographed with Kodak spectroscopic plates, Type I-D(2), with a yellow filter in the optical system, but higher resolution photographs can be obtained with either Kodak M or Kodak Metallographic plates. These are slower plates and longer exposure times are needed, but this disadvantage is largely offset by the replacement of the half-surfaced viewing mirror with the newly developed swing-out mirror. For colored materials like hemoglobin, the yellow filter is replaced by a red filter, and red-sensitive I-N plates are used.

Sedimentation Equilibrium Principle. Although the time required for attainment of equilibrium may be as long as 4 days for a protein of molecular weight about 60,000, Archibald has shown how molecular weights can be calculated from data obtained during the early stages of the run. He pointed out that solute does not leave the centrifuge cell either at the meniscus or the bottom of the cell and that, therefore, the conditions for equilibrium (transport of solute by sedimentation equals transport by diffusion) are fulfilled at these two places in the cell at all times of the run. The equation describing this condition is identical to that which holds elsewhere in the cell when equilibrium is attained, and we can write for a homogeneous material M -

RT (1 -

?p)~2

(dc/dx)m _ x~cm

RT (1 -

~p)~2

(dc/dX)b

(1)

XbCb

In equation 1, M and I~ are the anhydrous molecular weight and the partial specific volume of the solute, R is the gas constant, 8.314 X 107 ergs/mole/degree, T is the absolute temperature, p is the density of the solution, (o is the angular velocity of the centrifuge rotor in radians per second, (r.p.m.) (27r)/60, and cm and (dc/dx)m are the concentration and concentration gradient at the position x~, which is the distance from the axis of rotation to the meniscus. The corresponding quantities with the subscript b refer to the bottom of the cell. The procedure requires knowledge of the concentration gradients, (dc/dx)m and (dc/dX)b, and the concentrations, c~ and Cb. The former can

[2]

ULTRACENTRIFUGATION, DIFFUSION~ AND VISCOMETRY

39

be obtained directly from the photographic plates after suitable corrections for optical constants or with the aid of an auxiliary sedimentation run, data from which obviate measurement of the optical constants both of the apparatus and the solution. As shown in Fig. 2A, cm and cb are, respectively, less and greater than Co, the initial concentration; if the measurements are made early in the run, cm and cb are almost equal to Co. Corrections for the change in concentration at the meniscus and bottom of the cell are made with the equations derived by Klainer and Kegeles: ~ em ~

Co

- -m 2 X

and cb = co d- xb ~1

x2(dc/dx)dx

//

(2) x2(de/dx)d x

where X refers to a position in the plateau region ( d c / d x equals zero). It should be emphasized that the method as presented here, employing equation 2, is that proposed by Klainer and Kegeles, and it requires that the ultracentrifuge run be of limited duration such that there is still a region in the cell in which the concentration is independent of position, i.e., some plateau region still exists. This is clearly evident from the photographic plates, since the plateau region produces the fiat portion of the curve (usually known as the base line) in the optical system commonly employed. If the run is continued for longer times so that there is no longer a fiat portion of the curve seen on the photographic plates, then equation 2 is not applicable and cm and Cb must be evaluated in a manner different from that proposed by Klainer and Kegeles. Since accurate results can be obtained in the shorter runs, we will emphasize in our discussion the evaluation of molecular weights from equations 1 and 2. When the greatest accuracy is desired and experimental factors such as the lability of the enzyme do not preclude runs of long duration, it is advantageous to continue the ultracentrifuge runs for longer times, even after the plateau region is lost. The calculations then become laborious unless complete equilibrium throughout the cell is attained, in which case there is a minimum of calculations. In order to demonstrate the procedures, a sample calculation is included. Whenever such long runs are contemplated it is advisable to fill the ultracentrifuge cell to only about one third of its normal capacity. This reduction in the height of the liquid column, in a radial direction, will materially reduce the length of time required for attainment of equilibrium. E x p e r i m e n t a l Procedure. About 0.8 ml. of solution of enzyme at a concentration between 0.5 and 1.0% is required for the measurements. 2 S. M, Klainer and G, Kegeles, J. Phys. Chem. 59, 952 (1955).

40

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

Knowledge of the exact concentration of the protein is not necessary despite the fact that co appears in equation 2. If the protein is available in a dried form devoid of impurities, then dissolution of the protein in the desired buffer will suffice; otherwise it is necessary to dialyze the protein solution against the buffer to equilibrate the two liquids. An additional small amount of the dialyzate or buffer used to dissolve the enzyme is also necessary. Buffers with ionic strengths of 0.1 are desired in order to minimize electrostatic effects caused by the sedimentation of charged macromolecules. Two and possibly three ultracentrifuge runs are required, and these will now be discussed in the order in which they are customarily performed. For the first, the sedimentation equilibrium run, a conventional 12-mm. ultracentrifuge cell is required. In general, 4 ° sector centerpieces are used, but if material is in short supply a 2° sector cell will work well and some saving of material can be effected by its use. Before filling the cell with the solution, 0.1 ml. of Dow-Corning No. 555 silicone fluid is introduced into the cell by means of a 0.25-ml. syringe with a No. 22 needle. The solution of protein is then added to the cell, which will hold about 0.6 ml. if the 4 ° centerpiece is used. The layer of silicone fluid serves a double purpose. First, it creates a base for the aqueous solution which has the contour of an arc drawn from the center of rotation. This eliminates the convective disturbances which result when the aqueous solution is placed directly in the cell, the bottom of which does not have the proper shape. Second, it provides a dense, transparent medium for the bottom so that the bottom of the aqueous phase is clearly visible on the photographic plate. Thickening of the line between the two phases during the run is a good indication of the presence of aggregated or denatured material. Without this dense, transparent layer in the bottom of the cell, the values of xb, cb, and (dc/dx)b are not so readily measurable because of the arrangement of the optical system in existing equipment and the deviation of light due to the concentration gradient at the bottom of the cell. The use of silicone fluids or any noninteracting dense liquid effects a considerable saving in calculations plus increased precision in the evaluation of cb and (dc/dx)b. After the cell is sealed in the conventional manner and aligned properly in the rotor, the rotor is fastened onto the coupling of the drive shaft and the temperature of the rotor is measured by means of the external thermocouple supplied with the instrument. The vacuum chamber is then closed, the vacuum pumps turned on, and after a vacuum of about 1.0 ~ is reached the centrifuge is accelerated to the desired speed. For satisfactory work the ultracentrifuge rotor should be operated at a speed such that the concentration at the bottom of the cell, at equilibrium, is about five times that at the meniscus. If the

[9.]

ULTRACENTRIFUGATION, DIFFUSION, AND VISCOMETRY

41

molecular weight is known approximately, then a satisfactory speed in revolutions per minute can be calculated from [5 X 1 0 n / M ( 1 - Vp)]~, but it should be emphasized t h a t good results can be obtained even at speeds quite far removed from such calculated speeds. In the case of proteins varying in molecular weight from 10,000 to 200,000, speeds from 15,000 to 3000 r.p.m, will be ideal. As soon as the centrifuge rotor attains the desired operating speed, pictures are taken at various angles of the schlieren diaphragm. If the concentration of enzyme is in the neighborhood of 1%, then angles of 80 °, 70 °, and 60 ° are recommended. For more dilute solutions, 70 °, 60 °, and 50 ° are preferred; and for very dilute solutions an early picture at 40 ° is desirable. The optical system of the ultracentrifuge requires only the modification t h a t the bar in the standard instrument be replaced b y a phase plate. This replacement of the bar by a phase plate effects a considerable sharpening of the curve of concentration gradient versus distance on the photographic plates. As a consequence, values proportional to dc/dx can be obtained with high precision. The early photographs are obtained so as to have available a base line for later measurements. After experience with the method is gained, some of the initial photographs at various angles (optical sensitivity) can be omitted, and only those taken which correspond to the angles used later in the run. Pictures during the run should be taken every 8 to 16 minutes at angles of the schlieren diaphragm such that, at the meniscus and b o t t o m of the cell, the deviation of the curve above the base line is 2 to 3 ram. on the photographic plate. Deviations much greater than that are difficult to measure because of the steepness of the curves at the meniscus and b o t t o m of the cell; deviations much less than about 2 ram. are too small for precise measurements (unless plates are to be read on a microcomparator). Operation of the ultracentrifuge for 1 or 2 hours (five to ten pictures at 16-minute intervals) will yield sufficient data for the precise determination of molecular weights. If the material is unstable, however, the length of the run can be reduced. F r o m longer runs additional data can be obtained which will yield greater precision especially in the detection of inhomogeneity. After the requisite number of pictures are taken, the centrifuge rotor is decelerated, the vacuum chamber opened, and the temperature measured. The contents of the cell can be recovered by means of a syringe, and the cell should then be dismantled and cleaned with detergent solution, rinsed with distilled water, dried, and reassembled for subsequent runs. The second ultracentrifuge run performed in a synthetic boundary cell 3 provides the value of co, in equation 2, in arbitrary units, and it also 3 E. G. Pickels, W. F. Harrington, and H. K. Schachman, Proc. Natl. Acad. Sci. U.S. 38, 943 (1952).

42

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

furnishes a calibration of the optical system so that the values of (dc/dx) can be determined readily. Cells of various designs and sector angles (2 ° , 3° or 4 °) can be used for this run, but the optical path or centerpiece thickness must be the same as in the cell used for the sedimentation equilibrium run. Almost invariably this will be 12 ram. Another basic requirement is that there should be no mixing as the contents from the upper reservoir or cup empty into the sectoral cavity to form the boundary. About 0.3 ml. of the same enzyme solution used in the previous run is introduced into the sectoral cavity. This is the bottom solution. If the synthetic boundary cell manufactured by the Spinco Division, Beckman Instruments, Inc., is used, the cup is placed into the cell with an orientation such that the pinhole opening in the bottom of the cup is superimposed over the rubber plug which is in the hole drilled into the shoulder of the centerpiece. Then the trap door gasket is placed around the cup. By means of a 1-ml. pipet or a syringe, about 0.2 ml. of solvent is placed in the cup, and then the trap door is placed on the cell which is then ready for insertion into the rotor. If the cell designed by Kegeles4 is used, the solvent is placed into each of the upper reservoir holes before the window is placed on the centerpiece. Then the cell is assembled in the conventional way, and the enzyme solution (bottom solution) is introduced with a syringe as in the conventional cell. The cell is then sealed just as in the sedimentation equilibrium run. After the cell is placed in the rotor and the rotor coupled to the drive shaft, the vacuum chamber is closed without measurement of temperature which is unnecessary for this run. It is advisable, especially in the case of the synthetic boundary cell containing a cup, to start the rotor spinning at low speed (with about 3 to 6 amperes of driving current) without waiting for the chamber to be evacuated. This forces the solution to the bottom of the cell and minimizes possibilities of mixing of solution and solvent prior to the actual formation of the boundary. After the rotor is spun for about 5 minutes with the low driving current, the drive voltage is raised and maintained at a value necessary to provide about 6 to 8 amperes of drive current. This produces a slow acceleration of the rotor, leading to sharp, symmetrical boundaries. In the synthetic boundary cell with the cup, the contents of the cup begin to empty at about 5000 r.p.m., but slight variations in this break-through speed do occur, depending on the age of the rubber plug. The rotor should be accelerated slowly with the drive current raised to about 10 amperes. Frequently, the cup is not entirely emptied before reaching a speed equal to that utilized in the equilibrium run, and it is therefore advisable to use a higher speed setting so that the braking mechanism will not be brought into operation. 4 G. Kegeles, J. Am. Chem. Soc. 74, 5532 (1952).

[9.1

ULTRACENTRIFUGATION, DIFFUSION, AND VISCOMETRY

43

It is not necessary that this run be at the exact speed of the equilibrium run, since the run is used only to provide area measurements which serve as a calibration of the optical system and a measure of the concentration and refractive index increment of the solution. After the boundary has spread sufficiently so t ha t the entire curve is visible, which requires about 5 minutes for proteins, pictures are taken at different angles of the schlieren diaphragm corresponding to those used in the sedimentation equilibrium run. Caution should be exercised that the angles in the second run reproduce faithfully those of the first run. To avoid errors due to backlash in the remote control mechanism for adjusting the angle of the schlieren diaphragm, the desired angular setting should always be approached from the same direction, i.e., clockwise or counterclockwise. This run with the synthetic boundary cell should only co~lsume a shor~ time, sufficient for photographs at each angle used in the sedimentatioH equilibrium run. A period of 20 minutes allows the boundary to spread sufficiently by diffusion for accurate measurements of the area. The centrifuge can then be stopped and the sample recovered, if desired. These two runs will generally suffice for the determination of the molecular weight of enzymes, but for small molecules in salt solutions it is necessary to perform still a third run. Before describing this run, it should be pointed out that the second run (in the synthetic boundary cell) may be omitted if it is certain that the sedimenting material is homogeneous with respect to molecular weight. This will be illustrated later when the calculations are presented. The third run performed with the pure solvent provides the base line necessary for the calculations made from the sedimentation equilibrium run. If one is dealing with a coenzyme, a substrate, or even a protein of molecular weight much below 10,000, then the centrifugal force required for the equilibrium study will be sufficient to cause some redistribution of the buffer ions in the solution. Thus the curvature of the (dc/dx) versus x curve in the sedimentation equilibrium run would be due in part to changes in the salt concentration, and corrections must be made for this effect. At speeds of about 10,000 r.p.m, the sedimentation of the salt is so little and its diffusion so great that the salt concentration is uniform throughout the cell. Under those conditions the base line is obtained as already indicated in the discussion of the sedimentation equilibrimn run. If the centrifuge is run at higher speeds, then a separate run of the solvent (or dialyzate) is necessary, and this should be performed in a manner identical to that used for the solution. This requires not only that the speed and bar angles of the pictures be the same, but also that the same cell should be used with extra precautions to ensure filling to the same extent. Reproducing the volumes in the cell can be performed most conveniently by weighing

[9.]

ULTRACENTRIFUGATION, DIFFUSIOST, AND VISCOMETRY

45

run in the ultracentrifuge and in the calibration run with the synthetic boundary cell. The sample was ribonuclease at a concentration of 1%, and the centrifuge was operating at 11,150 r.p.m. Shortly after reaching speed, the first picture was taken in order to give a base line. The second, third and fourth pictures show how the picture changes during the first 3 hours. In order to show the pattern after the base line (plateau region) is lost, the last pictures were taken after 7 and 26 hours of operation. These pictures are photographic prints of the original centrifuge plates, with the white vertical bands at each side of the pictures corresponding to the holes in the reference cell; thus on each photograph there are two points whose distances from the axis of rotation are readily determined from the construction of the rotor and reference cell. The right-hand

~: ~

i

.... :i

FIG. 4. Ultracentrifuge patterns from a run in the synthetic boundary cell for the determination of the concentration required for a study during the approach to equilibrium.

edge of the black band on the left part of each photograph from most ultraeentrifuges lies at a distance 5.70 em. from the axis of rotation. This should be determined for each instrument. Acceleration of the rotor produces some stretching, and this line then corresponds to a distance 5.72 em. from the axis of rotation at speeds of 60,000 r.p.m. Once this position is known, all measurements on the photographic plate, or an enlargement of it, can be related directly to distances from the axis of rotation. The photographic plates are placed in an enlarger, emulsion side up, and the. curves such as those shown in Fig. 3 for ribonuclease are traced at a magnification of about fivefold. It has been found that tracing the curves on thin, unruled paper is most desirable. To facilitate tracing the necessary parts of the photograph, the vertical lines corresponding to the inner edges of the two reference cell holes, the air-solution meniscus, and the solution-silicone fluid interface are drawn first with the aid of a ruler. Then the horizontal lines through the reference holes, the lines through the air phase and the silicone solution, and finally that through the solution are drawn. In drawing the curved line corresponding to the concentration gradient near the meniscus and the bottom of the cell, it is advisable to draw those regions as if the vertical lines were not present, almost as if one could imagine those curves pro-

46

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

jected a slight distance beyond each of the vertical lines. This represents the most difficult part of the tracing, and it is especially difficult when low angles of the schlieren diaphragm are used so that the curves are very steep at the ends of the cell. It is for that reason that angles of 80 ° and 70 ° are preferred, as long as there is sufficient gradient to allow measurements. As the run proceeds, the curvature extends over a greater region in the cell and, as a consequence, tracing is somewhat easier. After the curves are drawn, the tracings are placed on a light box, and graph paper with centimeter and millimeter divisions is superimposed on the original tracings in such a manner that the vertical line corresponding to the air-solution meniscus lies on one of the heavy centimeter divisions. If possible, the flat part of the curve corresponding to the plateau region should also be placed under one of the heavy ruled divisions of the graph paper. In this way, the tracing can be transferred to graph paper. For experiments in which the centrifugal force is sufficiently low that no redistribution of electrolyte occurs and a separate ultracentrifuge run of the buffer can be neglected, it is sufficient to continue the straightline portion of the curve in both directions (toward top and bottom of the cell) until it intersects the vertical lines corresponding to the liquidair and solution-silicone fluid interfaces. This is illustrated in Fig. 5, which also shows some of the symbols used in the detailed calculations. For runs in which the speed is so great that salt redistribution occurs, then the corrections must be made as discussed later. At millimeter intervals starting with the meniscus, called n = 0, the vertical distances between the curved and the extrapolated straight-line portions are recorded. These distances, labeled z~, are proportional to dc/dx with the proportionality constant including optical constants of the apparatus, the concentration of the solute, the refractive index increment of the solute, and the magnification factor of the enlarger. In view of the fact that the same factors are involved in the last picture of Fig. 4, which is a representative photograph from the ultracentrifuge run with the synthetic boundary cell, their evaluation is unnecessary. Tables IA and IB show a sample calculation from the sedimentation equilibrium run. In column 1 are listed the values of n, i.e., the number of the interval. Column 2, labeled R~, gives the distance in centimeters from the reference mark to the interval in question. This distance is next converted into a real distance within the centrifuge cell, and the values are listed in column 3 under R,,/F, where F is the magnification factor relating distances in the cell to distances on the enlargement. This is readily evaluated, since the tracing includes the inner edges of the reference holes in the counterbalance cell. These edges are 1.60 cm. apart in the cell, and F is, therefore, RR/1.60. In the next column the distances R,,/F are con-

[2]

ULTRKCEINTTRIFUGATION~ DIFFUSION~ AND VISCOMETRY

47

verted to values of x,, which are the distances from the axis of rotation (xn = R , / F + 5.70). Column 5 contains the square of these values, and column 6 contains the z~ values. In column 7 are listed the values of .~.r,~z~, and the sum of these values, at the b o t t o m of the column, gives a

RTR

B

M

RBR

iJ-'F m"*l

x\\

Rb \\\

\\\

Rn In: tOO)

x\\

\\\

\\\

(n:lOOl /Z b

Z n (n:3)

\\\

Zn " ~ - ~

m~/

9'0 1001104

,\\\

I \\\

RTR RBR

,k\\ REE AIR REE SILICONE HOLE LAYER LAYER HOLE FIG. 5. Schematic diagram for illustrating the calculations of molecular weight from run during the approach to equilibrium. This corresponds to a print of the original ultracentrifuge pattern. The cross hatched regions and all black lines would be white on the original plates. Also on the original plate would be two horizontal lines corresponding to the image of the phase plate border at the reference holes. These are omitted in the above sketch but should be used in tracing of the curves and in the microcomparator since they furnish a good reference for all measurements in the vertical direction. measure of the integrals in equation 2. To obtain the exact value of the integral in the proper units, this sum must be multiplied by O.1/F, which is the horizontal width of each segment of the summation. The first part of the table contains the data obtained at the top of the cell and, according to equation 2, the value of the summation must be multiplied

48

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

b y 1 / x , , 2. T h i s n u m b e r is t h e n s u b t r a c t e d f r o m co t o g i v e cm. T a b l e I B g i v e s t h e d a t a f r o m t h e b o t t o m r e g i o n of t h e cell; t h e s u m m a t i o n h e r e is m u l t i p l i e d b y 1 / x b ~, a n d t h e r e s u l t a n t is a d d e d t o co t o g i v e cb. T h e v a l u e T A B L E IA SEDIMENTATION EQUILIBRIUM OF RIBONUCLEASE

Calculations at the Meniscus (Time = 38 minutes, F = 12.19, 17 = 0.709 cc./g., T = 298.9 °, ~2 = 1.3633 X 106) 1

2

3

4

5

n

R~ cm.

R, F cm.

x~ (5.70 + R , , / F ) cm.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.0

0.181 0.189 0.197 0.205 0.213 0.222 0.230 0.238 0.246 0.254 0.263 0.271 0.279 0.287 0.295 0.304 0.312 0.320 0.328

5.881 5.889 5.897 5.905 5.913 5.922 5.930 5.938 5.946 5.954 5.963 5.971 5.979 5.987 5.995 6.004 6.012 6.020 6.028

6

7

x,, "~

z,~

x,,2z,~

cm. ~

em.

em. 8

1.41 1.30 1.20 1.08 0.99 0.88 0.77 0.66 0.57 0.50 0.42 0.35 0~28 0.19 0.13 0.10 0.08 0.04 0.02

48.766 45.084 41.730 37.659 34.614 30.862 27.077 23.272 20.152 17.725 14.934 12.479 10.009 6.810 4.672 3.605 2.892 1.450 0.727

34.586 34.680 34.775 34.869 34.964 35.070 35.165 35.260 35.355 35.450 35.557 35.653 35.748 35.844 35.940 36.048 36.144 36.240 36.337

384.519 nx

cm = co

= 18

x,,, ~ F

x"~zn;

"dx ,, =

1.41

n0~0

1 0.1 c~ = 1.2167 -- 34.58~ ' 19.5/1.6 (384.519) = 1.1255 Mr, _

RT

(1

--

F'p)~

(dc/dx),~

_

6.3636 • 104 ' (0 lz~)"-.---"-.~---" ~ l ) 1.41 (l

13,600

x,,,c,,,

of Co is o b t a i n e d f r o m a n e n l a r g e m e n t of t h e p a t t e r n o b t a i n e d i n t h e r u n u t i l i z i n g t h e s y n t h e t i ~ b o u n d a r y cell. T h e m a g n i f i c a t i o n of t h e e n l a r g e r m u s t , of c o u r s e , b e t h e s a m e i n b o t h t r a c i n g s . F o r t h e c o m p u t a t i o n of co t h e b a s e l i n e is d r a w n u n d e r t h e p e a k f r o m t h e s t r a i g h t - l i n e r e g i o n s o n

[2]

49

ULTRACENTRIFUGATION'j DIFFUSION~ AND VISCOMETR¥

e i t h e r side of t h e p e a k a n d t h e a r e a e v a l u a t e d . T h i s c a n b e d o n e e i t h e r b y m e a n s of a p l a n i m e t e r g i v i n g r e s u l t s in s q u a r e c e n t i m e t e r s or b y s u m m i n g t h e o r d i n a t e s , z~, a t 0.1-cm. i n t e r v a l s a n d t h e n m u l t i p l y i n g t h e r e s u l t a n t b y 0.1. I t h a s b e e n f o u n d t h a t t h e p l a n i m e t e r o p e r a t i o n is less tedious, but both methods give comparable accuracy. The value obtained TABLE IB SEDIMENTATION EQUILIBRUM OF RIBONUCLEASE

Calculations at the Cell Bottom 1

2

3

4

Rn

xn

n

R, cm.

F cm.

(5.70 -t- R,,/F) cm.

116 118 120 122 124 126 128 130 132 134 136 138 140 142

13.8 14.0 14.2 14.4 14.6 14.8 15.0 15.2 15.4 15.6 15.8 16.0 16.2 16.4

1.132 1.149 1.165 1.182 1.198 1.214 1.231 1.247 1.264 1.280 1.296 1.313 1.329 1.346

6.832 6.849 6.865 6.882 6.898 6.914 6.931 6.947 6.964 6.980 6.996 7.013 7.029 7.046

5

6

7

x,~2

z,~

x,,2z,,

cm. ~

cm.

cm?

46.676 4fi.909 47.128 47.362 47.582 47.803 48.039 48.261 48.497 48.720 48.944 49.182 49.407 49.646

0.01 0.03 0.06 0.09 0.12 0.18 0.26 0.42 0.60 0.82 1.05 1.35 1.68 2.06

0.467 1.407 2.828 4.263 5.710 8.605 12.490 20.270 29.098 39.950 51.391 66.396 83.004 102.271 428.150

nb= 142 cb = co + zb---1 2 dX.F ~

x,Yz.; ( d=e2)"b0 6 d x x

nx=ll6 1 0.2 cb = 1.2167 + 49.64-~6 " 19.5/1.6 (428.150) = 1.3582

Mb -

I?T

(1 - ~p)~2

(dc/dx)b _ 6.3636. 104. 2.06 Xbcb (7.046)(1.3582)

13,700

b y e i t h e r of t h e s e t w o m e t h o d s is t h e n m u l t i p l i e d b y 1 / F t o c o r r e c t for m a g n i f i c a t i o n in t h e x d i r e c t i o n , a n d t h e r e s u l t a n t v a l u e is Co. A t t h e b o t t o m of T a b l e s I A a n d I B a r e s h o w n t h e final c a l c u l a t i o n s . Since t h e t e m p e r a t u r e of t h e , r o t o r is k n o w n , i t is c o n v e r t e d i n t o a b s o l u t e t e m p e r a ture, a n d t h e m o l e c u l a r w e i g h t is c a l c u l a t e d f r o m e q u a t i o n 1. W h e n one is d e a l i n g w i t h s m a l l e r m o l e c u l e s a n d u t i l i z i n g h i g h e r c e n t r i f u g a l fields, t h e s o l v e n t is r u n s e p a r a t e l y a n d p i c t u r e s a t t i m e s a n d

[2]

51

ULTRACENTRIFUGATION~ DIFFUSION~ AND VISCOMETRY

T A B L E II SEDIMENTATION EQUILIBRIUM OF RIBONUCLEASE Calculations throughout the entire cell after loss of plateau region* (Time = 63 hours, F = 1.882, V = 0.0709 cc./g., 7' = 296 °, o~2 = 1.3633 × 10 ~) 1

2

n

n ~°

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88 92 96 100 104 105.7

3

5

4

R,~"°

x,~~

cm.

cm.

6

(z,~2)~'o cm.

2

z,, ~ ° ell1.

2 6 10 14

1.447 1.487 1.527 1.567

6.469 6.490 6.512 61533

41.8415 42.1200 42.3995 42.6735

0.0774 0.0794 0.0817 0.0838

18 22 26 30 34 38 42 46 50 54 58 62 66 70 74 78 82 86 90 94 98 102 104.8

1.607 1.647 1.687 1.727 1.767 1.807 1.847 1.887 1.927 1.967 2.007 2.047 2.087 2.127 2.167 2.207 2.247 2.287 2.327 2.367 2.407 2.447 2.475

6.554 6.576 6.597 6.618 6.639 6.661 6.682 6.703 6.724 6.746 6.767 6.788 6.809 6.831 6.852 6.873 6.894 6.916 6.937 6.958 6.979 7.001 7.016

42.9550 43.2370 43.5135 43.7910 44.0765 44.3625 44.6425 44.9235 45.2120 45.5020 45.7860 46.0705 46.3625 46.6555 46.9430 47.2315 47.5275 47.8240 48.1150 48.4070 48.7065 49.0070 49.2240

0.0861 0.0889 0.092l 0.0957 0.0997 0.1039 0.1080 0.1121 0.1164 0.1209 0.1259 0.1307 0.1354 0.1410 0.1471 0.1527 0.1584 0.1646 0.1706 0.1771 0.1836 0.1901 0.1959

c,a =

Co -~" Xb 2

-- X,n~ L J x ~

1

Ff

dx

xz dc

7

8

c~ cm.

x 2

cHI.

9

z,,

10 I dc

xc dx

cm.

0.05158 0.05313 0.05491

6.458 6.479 6.501

0.0766 0.230 0.0782 0.227 0.0806 0.226

0.05665 0.05843 0.06026 0.06215 0.06411 0.06614 0.06826 0.07047 0.07277 0.07515 0.07762 0.08019 0.08287 0.0~565 0.08853 0.09153 0.09465 0.09790 0.1013 0.1048 0.1084 0.1122 0.1161 0.1201 0.1219

6.522 6.543 6.565 6.586 6.607 6.628 6.650 6.671 6.692 6.713 6.735 6.756 6.777 6.798 6.820 6.841 6.862 6.883 6.905 6.926 6.947 6.968 6.990 7.011 7.020

0.0828 0.0847 0.0874 0.0904 0.0938 0.0976 0.1018 0.1060 0.1100 0.1141 0.1186 0.1232 0.1286 0.1328 0.1380 0.1440 0.1502 0.1552 0.1616 0.1676 0.1736 0.1806 0.1866 0.1936 0.1981

-zb2

x~ dc

fx

m dxx

0.223 0.222 0.221 0.221 0.222 0.223 0.224 0.226 0.226 0.226 0.227 0.227 0.229 0.228 0.229 0.230 0.231 0.230 0.231 0.231 0.231 0.231 0.230 0.230 0.231

dx]

n = 105.7

fxz~ dC dx = dx m dxx ~-

~ n=O

z~ -- (2.126 × 10-2)(3.223) + (0.925 × 10-2)(0.1959) = 7.033 X 10 -2

n = 105.7

~xb m

dc _ dx x ~ --,- clx =

~

xn2z,, = (2.126 X 10-2)(148.19) -~ (0.925 X 10-2)(9.641) = 3.240 (3.240 -- (49.280)(7.033 X 10 -2) c,, = 0.08148 -b 49.280 -- 41.706 = 0.05158 * The centrifuge plate for this calculation was measured with a microcomparator, and the n intervals are in t e n t h s of millimeters. ax

n=O

52

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

indeed a plot of this data as log ( 1 / x ) ( d c / d x ) pletely straight.

[2]

versus x 2 is almost com-

Sedimentation Velocity I n t r o d u c t i o n . The primary function of this method is the determina* tion of the sedimentation rate of molecules, but it should be stressed that another powerful application of the method lies in its ability to furnish, often in a simple experiment of short duration, information about the purity of the material under investigation. In this latter regard, sedimentation velocity studies are like electrophoresis experiments, since each of the techniques causes the partial separation of different types of molecules, depending on their physical chemical properties. As a result several boundaries may be observed, or one broad boundary which clearly indicates inhomogeneity of the particles. Thus the sedimentation velocity method can indicate, within limits, of course, whether one or more molecular species is present in solution, provided these different molecules show variations in molecular size and shape. With sufficient research, precise information can be obtained about the amount of each species present in a solution, and thus the ultracentrifuge is an extremely valuable guide in studies aimed at the isolation and purification of an enzyme. Solute molecules in a solvent of different density are caused to move under the influence of a high centrifugal field, and the rate at which they move is a function both of the molecular weight of the solute and of the frictional resistance which the molecules or particles experience as they move through the solvent. Sedimentation velocity measurements of themselves do not yield molecular weights without recourse to other data, such as diffusion coefficients. The equation describing the relationship between the sedimentation rate and the properties of the molecule is

M(1 s -

Nf

Cp)

(4)

where s is the sedimentation coefficient, M is the anhydrous molecular weight of the sedimenting substance, l? is the partial specific volume of the solute, p is the density of the solution, N is Avogadro's number, 6.02 X 1038, and f is the frictional factor which is a function of the size, shape, and hydration of the sedimenting molecules. Movement of individual molecules is followed by examination of the boundary between solvent and solution, as illustrated in Fig. 1. For most purposes, if the boundary is symmetrical, movement of the maximum ordinate of the peak is a sufficiently accurate indicator of the movement of individual molecules. Actually it is the second moment of the gradient curve which

[2]

ULTRACENTRIFUGATION, DIFFUSION, AND VISCOMETRY

53

gives a true picture of the movement of the molecules, but calculations of this second moment are very laborious and often unnecessary. The sedimentation coefficient is defined as the velocity of the sedimenting molecules per unit field, as shown in equation 5, s

-

1 dx ~ x dt

(5)

where x is the distance of the boundary in centimeters from the axis of rotation, t is the time in seconds, and ~ is the angular velocity in radians per second. Generally, sedimentation coefficients lie in the range of 0.25 to 500 X 10-13 cm./sec./dyne/g., or 0.25 to 500 X 10-'a second, and the unit 1 X 10-'3 second has been termed 1 S. where S. is the Svedberg. Determination of Sedimentation Coe~icients. For proteins of molecular weight below about 250,000 the ultracentrifuge should be operated at the top speed of 59,780 r.p.m. ; and for larger molecules the speed should be adjusted so that the boundary moves across the cell in a period of several hours. Photographs are taken at periodic intervals, depending oil the rate of movement of the boundary. It is always advisable to examine the pattern through the viewing screen during the acceleration of the rotor in case material present in the solution sediments across the cell before the rotor attains the desired operating speed. If the substance under investigation has a sedimentation coefficient below 3 S., the use of the synthetic boundary cell is recommended. To damp out charge effects with proteins, ultracentrifuge runs are generally made in salt solutions at an ionic strength of 0.1 or greater. The temperature before and after the run is recorded if the external contact thermocouple is still in use; if the new temperature indicator is available, then the temperature can be recorded during the run. It should be stressed, for the former case, that the temperature of the rotor during a run at 60,000 r.p.m, is about 0.8 ° less than the average of the temperatures obtained from the contact thermocouple. Photographs can be read in a variety of ways, depending on the accuracy desired. For routine work with materials which sediment as relatively sharp boundaries, we have employed a simple light box containing a calibration plate which permits direct reading of boundary positions in centimeters from the axis of rotation. The calibration plate is made by photographing the ruled glass disk supplied by the manufacturer for the determination of the magnification factor of the different lenses. The lines on the resulting photograph will be from 1.8 to 2.2 mm. apart, depending on the particular instrument. With this plate fastened onto the frosted-glass top of a light box, a grid is available whose lines

[9.]

ULTRACENTRIFUGATION, DIFFUSION, AND VISCOMETRY

55

sedimentation coefficients for each time interval and correct them indep e n d e n t l y for temperature, assuming a linear change in temperature. For m a x i m u m use of the data, the averaging of the resulting sedimentation coefficients should be performed in the manner suggested b y Kegeles and G u t t e r ! Alternatively, the times between photographs can be corrected for the variation of the viscosity of water with temperature, as suggested b y Oncley, 9 and then log x can be plotted against the corrected times. log x

x (cml~

~t

Temo:23.7" c,( 20) :0 0.82600

(-~o)

6.700

=1.0065, ~] =0.709CC

solv

/ 0.81800

o/° D/" S = 2.503d

Iogx/dt (60)( ,,02)

6.580 =

(slope)(2.3 0 3 / 6 0 [( 2"rr)(, ,j59780 . . ~ - - - ~]2

)

S = 1.98 x 10"13sec. = 1.98 S 6.520 t , I t o t6 52 Time in m{nutes FIG. 6. D e t e r m i n a t i o n of sedimentation coefficient of ribonuelease from a plot of log x (x is the distance of b o u n d a r y to axis of rotation) versus t (t is the time in minutes). 0.81400 .....

,

Sedimentation coefficients are generally reported as s20.~, the value the material would have in a solvent with the density and viscosity of water at 20 ° . Corrections of the observed sedimentation coefficient, sob,., to this standard state are made according to the equation

where (nt/n20) is the principal correction factor corresponding to the viscosity of water at t ° relative to t h a t at 20 ° , (n/no) is the relative viscosity of the solvent to t h a t of water, and p20.~ and pt are the densities of water at 20 ° and the solvent at t °, respectively. The term pt can be calculated readily from (P/Po)Pt,w. I t is implicitly assumed t h a t V in the reference solvent is the same as in the actual solvent used in the experi9 j . L. Oncley, Ann. N. Y. Acad. ~ci. 41, 121 (1941).

56

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[9.]

ment. For the experimental d a t a presented in Fig. 6, calculation of s20.w according to equation 6 leads to the result 820.~ = (1.98)(0.9147)(1.024)(1.0139) = 1.88 S. We have used p, as the density of the solvent, although some workers recommend use of the density of the solution. I t is clear t h a t the density of the solution is correct for sedimentation equilibrium; but there is no corresponding theoretical basis for the density t e r m in sedimentation velocity. In a n y case the difference between solution and solvent density is generally very small, and further, data should be extrapolated to infinite dilution so t h a t no significant error can result from the use of either density. For slowly sedimenting materials in high salt concentrations, formation of the b o u n d a r y between solvent and solution near the center of the cell is especially useful, since it virtually eliminates the need for a reference run of solvent alone. This run is ordinarily necessary as a " b a s e l i n e " run, since the sedimentation of salts causes refractive index gradients at the meniscus and b o t t o m of the cell. The observed p a t t e r n would then be the sum of t h a t due to the material of interest plus t h a t due to the salt. If a conventional cell is used, then a reference run of solvent is made under identical conditions, and the patterns from this run are subtracted from the corresponding patterns in the run of the solution. When the synthetic b o u n d a r y cell is used, the rate of acceleration of the rotor should not be so great as to cause convection which leads to poor boundaries. After the cup containing the solvent is completely emptied, the drive current should be adjusted to about 10 amperes until the operating speed is achieved. For m a n y substances the sedimentation coefficient depends on concentration, and runs at different concentrations are necessary to enable extrapolation of the data to infinite dilution. T h e data can be plotted in a variety of ways, but it is preferable to plot 1Is versus concentration. If viscosity data for the solutions at different concentrations are available, then a plot of s versus c and (v/~o)s versus c (c is concentration) on the same scale provides two curves which should extrapolate to the same value. This greatly facilitates the determination of s at infinite dilution. Measurement of Concentration. The area under the curves shown in Fig. 1 gives a measure of the concentration of the sedimenting material, and the m e t h o d of evaluating the area from synthetic b o u n d a r y cell runs has already been discussed. With a microcomparator and patterns obtained with the phase plate as the schlieren diaphragm these areas can be reproduced with a precision of b e t t e r t h a n 1%. Areas from runs at high speeds in a conventional cell cannot be obtained so accurately. For the

[2]

ULTR&CENTRIFUGATION, DIFFUSION, AND VISCOMETRY

57

latter, it is essential that a "base-line" run be made with pure solvent. This corrects not only for the gradients of refractive index due to sedimentation of salts but also for cell distortion. If possible, this "base-line" run should be made without taking the cell apart, and the volume of solvent in the cell should be the same as in the run with solution. The plastic, double sector ultracentrifuge cell permits the superposition on the photographic plate of the pattern due to the solution and the solvent. With this cell both solvent and solution are run simultaneously and the area of a sedimenting boundary can be determined with great precision since the superposition of the base line on the pattern of the moving boundary eliminates the uncertainties in construction of the base line from separate ultracentrifuge runs. Small amounts of both more rapidly and more slowly sedimenting material can be readily detected by the deviation of the solvent pattern from that of the solution in the region of the cell in front of or behind the principal boundary. In using this cell special efforts should be made to fill both halves of the cell equally so that the menisci are at the same distance from the axis of rotation. The syringe, 0.5 ml., which is first used for transferring the solution into the cell can then be rinsed and used for the solvent. Before using the cell for concentration determinations it should be tested by running solvent in both halves to be certain that cell distortion does not preclude superposition of the base lines from each sector. Since there is some loss of contrast on the photographic plates due to overlapping of the patterns from the two halves of the cell, high contrast photographic plates, such as the 5~etallographic plates, should be used. Whenever concentration determinations are to be made, either on purified components or in the analysis of mixtures, it is recommended that the double sector cell be employed. The solvent and solution patterns should be superimposed either from tracings of enlarged patterns (5X) made with a photographic enlarger or arithmetically if the areas are obtained by summing ordinates measured with a two-dimensional comparator. For this superposition of the patterns, use is made of the images corresponding to the reference holes in the counterbalance. After the base line is drawn under the peak, the area can be measured. The concentration of sedimenting material ill per cent, co, is obtained from the equation Atan~

(x) 2

co = a b m x m ~ A n E ~

where A is the measured area, a is the cell thickness in centimeters (along the optical path), b is the optical lever arm in centimeters (this value is supplied by the manufacturer and is equal to the focal length of the upper

58

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[9.]

collimating lens or the distance from this lens to the plane of the schlieren diaphragm), Ois the angle of the schlieren diaphragm as read directly on the ultracentrifuge, mx and mc are the magnification factors of the camera and cylindrical lenses, respectively (these are evaluated according to the manufacturer's instructions with the aid of the ruled glass disk supplied with the ultracentrifuge), and E is the magnification of the enlarger (this is unity if a comparator is used). The term (X/Xo) 2, where x0 is the position of the boundary at zero time and x is the position at a time corresponding to the pattern subject to analysis, is the correction factor to account for the dilution of the contents of the plateau region as a result of the sector shape of the cell and the variation of centrifugal field with distance. To obtain the concentration in weight per cent, An, which is the specific refractive increment, must also correspond to weight per cent. It is equal to the difference in refractive index between a solvent and a solution at a concentration of 1%. For many proteins, An is about 0.00186. If a pure protein is available, the synthetic boundary cell can be used to calibrate the optical system by forming a boundary between solvent and a solution of known concentration. Also, there is available from the manufacturer a special calibration cell which is very useful for checking or determining the optical constants of the ultracentrifuge. Determination of Homogeneity. The minimum requirement for homogeneity of a given sedimenting substance is that the material sediment across the cell as a single, symmetrical boundary without too much broadening of the boundary. This is, of course, a qualitative statement; detailed quantitative measurements can be and, indeed, must be made if homogeneity is to be demonstrated. It should be emphasized that it is extremely dangerous and often incorrect to conclude that a material is homogeneous just because the boundary remains symmetrical and sharp. Frequently boundaries are artificially sharp because of the dependence of sedimentation coefficient on concentration, and tests for homogeneity should be made on dilute solutions and, if possible, as a function of concentration to enable extrapolation to infinite dilution. This is also important in the analyses of mixtures in terms of the relative concentrations of the different components, because of the ultracentrifuge anomaly which causes the slower component to appear to be present in greater concentration than the true concentration. 1°,11 For materials with a sedimentation coefficient below about 1 S., the boundary cannot traverse the whole cell becatlse of the present limitations in rotor speed. Thus the great potential of the ultracentrifuge in resolving different molecular 10 j. p. Johnston and A. G. Peston, Trans. Faraday Soc. 42, 789 (1946). 11 W. F. Harrington and H. K. Schachman, J. Am. Chem. Soc. 75, 3533 (1953).

60

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

employing conventional optical techniques, and recourse is made to the so-called analytical method which is based on the determination of the quantity of substance sedimenting across a fixed plane in a centrifuge cell. This method requires a special ultracentrifuge cell called the separation or partition cell which is so constructed as to effect a separation of the contents of the cell into two parts at the conclusion of the run. The amount sedimenting across the partition is determined by the difference between the total amount of material originally in the cell and that which is left above (or centripetal to) the separating plate at the conclusion of the ultracentrifuge experiment. Also required for the method is, of course, a specific and sensitive analysis for the material in question. This may be a bioassay, a chemical analysis, or a physical-chemical measurement such as radioactivity or ultraviolet absorption. As will be seen shortly, the analyses need be only relative so that measurement at the conclusion of the run of the number of units of activity in the upper solution relative to the number in the original solution gives a value of the sedimentation coefficient. The separation cell affords still another important advantage to biochemists interested in a specific biologically active substance. Frequently in the isolation and purification of a given substance two or more boundaries are observed optically in the ultracentrifuge, and it is important to determine which, if either of these corresponds to the material of interest. As long as there is a specific biological test available, the sedimentation coefficient of the substance can be determined even in the presence of much larger quantities of other components. Even when only one component is observed optically and the material is thought to be purified, it may be profitable to measure the sedimentation rate by activity measurements to see if there is a correlation between the physical property of the bulk constituent and the component with the biological activity. For some time there has been available only the partition cell of Tiselius et al., 12 but very recently a cell designed from quite a different point of view has been introduced by Yphantis and Waugh. 1~ Both of these cells are now available commercially from the Spinco Division, Beckman Instruments Inc., Belmont, California, and they are both made by modification of standard 12-mm. 4 ° centerpieces. In the cell of the type proposed by Tiselius et al., slots are made in the walls of the centerpiece at a position approximately two-thirds of the distance from the top. Into these slots fits a thin perforated plate containing about twenty three holes drilled through it, each hole being about 0.28 mm. in 1, A. Tiselius, K. O. Pedersen, and T. Svedberg, Nature 140, 848 (1937). 13 D. A. Yphantis and D. F. Waugh, J. Phys. Chem. 60, 623, 630 (1956).

[2]

ULTRACENTRIFUGATION, DIFF~ISION, AND VISCOMETRY

(}l

diameter. In operation, a piece of Whatman No. 50 filter paper, 12 mm. long and about 0.5 mm. narrower than the porous plate, is overlaid on the plate and they are inserted into the centerpiece. It is implicitly assumed that neither the filter paper nor the perforated plate interferes with the process of sedimentation and, further, that the sedimentation process is convection-free as in the standard ultracentrifuge cells. The function of the barrier is to prevent mixing of the upper and lower solutions at the conclusion of the experiment. In the cell designed by Yphantis and Waugh the separating plate is actually movable and rests on two thin synthetic rubber strips which serve as a spring. Since the plate and rubber are more dense than the liquids customarily employed for analysis, the plate sinks to the bottom of the cell during acceleration of the rotor and the cell simulates a conventional ultracentrifuge cell. On deceleration, at 2000 to 3000 r.p.m., the centrifugal force on the plate is no longer sufficient to counteract the lifting action of the rubber springs, and the plate rises very slowly until it rests against specially constructed stops in the centerpiece. If the cell and movable partition are made and assembled correctly so that there is a precise fit when the plate is in the rest position, the solutions in the two compartments can be effectively separated from each other with little risk of mixing. Surprising as it may seem, the slow movement of the partition from the bottom of the cell to its rest position causes little or no generalized stirring of the contents of the cell. This has been the subject of careful experimentation by Yphantis and Waugh, 13 who, in experiments in which the rotor was decelerated and accelerated, showed that the movement of the partition back and forth through a boundary caused only a little distortion of the boundary. This is true even with solid partitions, so that the liquid must flow around the partition when it is Caused to move in the cell. The success of this technique depends, as in the case of the synthetic boundary cell, on the stability resulting from magnification of small density gradients under the influence of a centrifugal field. Whereas stirring of stratified layers of solution differing in density by only 0.001 g./ml, will occur readily at one gravity, such stirring will not take place in a centrifugal field in which the force on the liquid is many hundreds of times that of gravity. Static experiments with a dye indicator present in the centrifugal compartment and absent in the centripetal part showed that there was little mixing of the liquids when the cell was handled in a fashion simulating a typical ultracentrifuge experiment. Thus the two separation cells functioning in quite different ways achieve, to more or less the desired extent, the same purpose. There is not as yet sufficient experience with the movable partition cell for a detailed comparison with the older cell, but it appears that the

62

TECHNIQUES FOR CHARACTERIZATION OF PROTEIN'S

[2]

newer cell will function better for proteins and low molecular weight materials, whereas the partition cell of Tiselius et al. does interfere with sedimentation and there is a partial disintegration of the sedimenting boundary as it approaches the partition from above. Furthermore, a second boundary forms just centrifugal to the barrier. This probably results from a temporary pile-up of some sedimenting material on the filter paper. Whatever the cause, convection does occur and this may lead to erroneous results. Epstein and Lauffer14 circumvented this effect of the barrier by adding a small amount of sucrose to the solution below the barrier, thereby causing this solution, even if there is some removal of solute by sedimentation, to be heavier than the solution centripetal to the partition. In this manner the disturbance in the upper liquid can be effectively eliminated. In terms of operating procedure there are only minor differences, depending on which cell is used. With the old cell, complete filling in one step is almost impossible. The top portion of the cell is filled by means of a 1-ml. syringe equipped with a No. 22 stainless steel needle the tip of which is ground fiat. This liquid is forced into the bottom compartment by centrifuging at a speed of about 2000 to 3000 r.p.m, for about 1 minute. Then the upper compartment can be filled. This intermediate step can be eliminated in the use of the Yphantis-Waugh cell, since the tip of the needle can be used to depress the platform during the filling of the entire cell. It is a good idea to weigh the filled cell before the ultracentrifuge run and then again at the conclusion of the run, before and after removing the contents of the upper compartment. This is useful, since small leaks in the cell can be detected, and also the weight of the sample removed is determined readily. For this weighing, a small plastic block with a hemicylindrical cavity in the top serves as a stand for proper orientation of the cell with the filling hole pointing upward. Unlike most ultracentrifuge studies, the exact time of the run is important, and it is therefore necessary to record the speed of the rotor at different times on the way up to speed and then at the conclusion of the run during the deceleration period. Generally it will be found that acceleration of the rotor under the influence of 12 to 14 amperes will be linear and, to a very good approximation, the time of acceleration to the desired speed is equivalent to one-third of that period at the final operating speed. Thus one-third of the acceleration and deceleration times must be added to the time that the rotor is at speed for the computation of the sedimentation time. Special precautions are needed in the deceleration of the rotor, since rapid deceleration leads to convective disturbances, as evidenced by the formation of hypersharp spurious boundaries. Yphantis 14 H. T. Epstein and M. A. Lauffer, Arch. Biochem. and Biophys. 35, 371 (1952).

64

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

Several runs should be made for different periods of time such that

ct/co varies from about 0.8 to about 0.1. For most enzymes, runs of a duration sufficient to produce a lowering of the average concentration in the upper solution to about 10% of the original will take only 2 or 3 hours at top speed, and the equation presented above is valid. If the molecules being studied are smaller, say about 2000 in molecular weight, then the plateau region will vanish in a 3-hour period and equation 9 is no longer applicable. Yphantis and Waugh 13 have shown, however, that it is possible to obtain both sedimentation coefficients and diffusion coefficients from experiments in which the plateau region is lost and the reader is directed to their papers for this important new development. Application of Absorption Optics to Ultracentrifugation. Many substances of interest to enzymologists have such high extinction coefficients either in a particular region of the visible spectrum or in the ultraviolet that the sedimentation of these materials is more readily examined by absorption techniques than by customary schlieren methods. For example, the amount of ultraviolet light transmitted through a solution of deoxyribonucleic acid at a concentration of 0.001% is only about half the light transmission of the solvent. This difference is readily recorded as variations in the degree of blackening of photographic plates. In contrast, the difference in refractive index between such a solution and the solvent is so small as to be virtually indetectahle by present schlieren methods. Thus ultracentrifugal analysis of materials at such great dilutions is dependent on the availability of absorption methods. Also many substances, the main features of whose absorption spectra are known, such as the cytochromes or hemoglobin, can be studied specifically, even in crude mixtures, by the use of light of the appropriate wavelength. Therefore, it seems likely that there will be a return to the absorption method originally developed and employed by Svedberg and his co-workers. 15 Techniques at present are still in a state of flux, and only very general comments will be made here. A special ultraviolet absorption optical system is now available commercially and can be incorporated in existing ultracentrifuges. This employs light of 254 m~ and can be used for materials which, at that wavelength, have an optical density of 0.2 (in a 1-cm. cuvette). For absorption work at wavelengths in the visible, the schlieren system can be used as an absorption system through the use of appropriate filters to isolate the desired wavelengths. Interference filters are very useful for this purpose. If the absorption method is to give accurate information concerning 15T. Svedberg and K. O. Pedersen, " T h e Ultracentrifuge," Oxford University Press, New York, 1940.

[9.]

ULTRACENTRIFUGATION', DIFFUSION, AN'D VISCOMETRY

65

the change in concentration of the absorbing material as a function of the position in the ultracentrifuge cell, the blackening of the photographic plate must give a faithful record of the intensity of light striking it. Thus, the blackening of the plate must be inversely related to the optical density of the solution in the cell. Further, this blackening must be measured in some type of photodensitometer. For this purpose use is made of the modified Analytrol, manufactured by the Spinco Division, Beckman Instruments, Inc. With suitable precautions and some calculations this instrument in conjunction with the photographic plates gives a curve relating concentration of the absorbing substance as a function of distance from the axis of rotation. This will be recognized as the integral of the patterns given by schlieren methods. From these curves at different times, sedimentation coefficients are readily calculated. Also analysis of the boundary spreading to determine the diffusion coefficient and polydispersity of the sedimenting material is readily achieved by the use of absorption patterns. For strongly absorbing materials like cytochromes or nucleic acids, a detailed analysis can be performed, with the aid of the synthetic boundary cell, on only about 10 ~ of material.

Partial Specific Volume The use of ultracentrifuge data from either sedimentation equilibrium or sedimentation velocity studies requires the evaluation of the term (1 - Vp). Since the partial specific volume, V, of proteins is generally between 0.70 and 0.75 ml./g., and the density, p, is normally close to unity, errors in the determination of 17 are essentially multiplied by a factor of 3 in the final calculation of molecular weights. As a consequence it is imperative that accurate values of V be obtained, and it is just in this area that detailed experimental studies are lacking. The classical method for determining partial specific volumes involves density measurements both on solutions of known concentrations and oll the solvent. From each solution density, p, paired with the density of the solvent, p0, an apparent specific volume is calculated. It has been found almost invariably for proteins that the apparent specific volume is independent of concentration of the protein, and thus the average value for different concentrations is equal to the partial specific volume. In addition to the classical method of determining partial specific volumes there are two other methods which give a reasonably reliable value for 17, and a brief discussion of them will follow a treatment of the more popular method. There are a variety of procedures for determining densities of solutions, and all of them have been used at one time or another. The most

[9.]

ULTRACENTRIFUGATION~ DIFFUSION, AND VISCOMETRY

67

pycnometers of only 2-ml. capacity can be used although greater precision is attainable with larger volumes. Greater precision in measuring the density of solutions can be obtained either through the use of the density gradient column of Linderstr0mLang and Lanz 16 or by means of the magnetic float method developed by MacInnes et al. 17 The former method requires only very small volumes of solution, whereas the latter requires volumes that in many cases are almost prohibitively large. For the use of the magnetic float method which requires apparatus that is not as yet commercially available, the reader should consult the paper by MacInnes and co-workers, is Density measurements with the gradient column require only simple equipment consisting of a constant-temperature bath, a column of liquid in which the density increases, preferably in a linear manner, from top to bottom and in which aqueous solutions are insoluble, a capillary pipet for the delivery of small drops of protein solution into the column, a method for the precise measurement of the height of the drops in the column, and, finally, a set of reference liquids whose densities are accurately known. In the use of the gradient column, mixtures of c.p. bromobenzene and rectified white kerosene are employed. Two mixtures of these liquids are prepared so that they differ in density by about 0.02 g./ml., and they encompass the density range of the solvent and protein solutions. Each of the bromobenzene-kerosene solutions must be saturated with water by shaking with a dilute solution (about 0.2 M) of potassium bromide fo}lowed by storage overnight. This saturation of the liquids for the gradient column is necessary so that there will be no evaporation of water from the drops of protein solution; otherwise, there would be an increase in concentration of protein in the drops, leading to erroneous results. After separation of the individual solutions from the potassium bromide solution, the gradient column is prepared by the method of Linderstr0m-Lang and Lanz. 16 This involves careful layering of the lighter solution over the heavier one in a long cylindrical column held in the well-controlled constant-temperature bath. The two solutions are then mixed partially by gentle stirring so as to produce a gradual change in composition, and hence in density, from the top to the bottom of the column. The stirring operation is an art, and it takes considerable practice before this operation can be performed in a satisfactory manner. 1~K. LinderstrOm-Langand H. Lanz, Compt. rend. trav. lab. Carlsberg S$r. chim. 21, 315 (1938). 17D. A. MacInnes, M. O. Dayhoff, and B. R. Ray, Rev. Sci. Instr. 9.2, 642 (1951). is M. O. Dayhoff, G. E. Perlmann, and D. A. MacInnes, J. Ant. Chem. Soc. 74, 2515 (1952).

68

TECHNIQUES FOR CHARACTERIZ.~TION OF PROTEINS

[2]

Too little stirring leads to a density gradient of only limited extent with uniform regions above and below the actual density gradient, whereas too much stirring produces a column equally unsatisfactory, since the column is almost uniform in density. The density gradient column is allowed to stand undisturbed for a period of 24 hours so that diffusion can smooth out any abrupt density gradients. Then the appropriate standards made from solutions of analytical-grade recrystallized potassium chloride are added to check the gradient column. If the positions of the drops show that the density gradient is satisfactory, then the column is ready for use. Drops are added by means of a capillary pipet delivering about 0.2 mm. 3, and there should be sufficient standard drops of known density to bracket closely the solutions whose densities are to be determined. Several drops can be measured along with several standards almost simultaneously, and the drops should be added with the most dense being first, then the next dense, and so on. The positions of the drops after they reach equilibrium positions, which requires only about 15 minutes, are most readily determined by a cathetometer, although a scale mounted alongside of the column may suffice, depending on the accuracy desired. The density of a given unknown solution, d, is calculated by interpolation according to the equation h - h, (d2 - dl) d = dl T h~ hi

(10)

where h, hi, and h~ are, respectively, the positions measured from some reference mark near the top of the column to the center of the unknown drop, the lower density standard, dl, and the higher density standard, d2. After each experiment the drops are removed from the column which is then ready for use after several hours of standing. Removal of the drops is achieved either by touching the drops with a thin glass rod onto which the drops will adhere so that they can be transferred to the wall of the column at the top of the liquid or by pouring a small amount of sea sand (obtainable from Merck) through the column. Density measurements can also be achieved by hydrostatic weighing in a commercial apparatus such as a Westphal balance or in a modified analytical balance. Accurate densities can also be achieved by differential density measurements using a pair of similar pycnometers one of which is filled with solution and the other with solvent, and then the solutions are exchanged. 19 After the densities of the solutions and solvent are determined, the 19 For a review see chapter on determination of density by N. Bauer, in "Physical Methods of Organic C h e m i s t r y " (Weissberger, ed.), 2nd ed., Part I, p. 253. Interscience Publishers, New York, 1949.

[9"]

ULTRACENTRIFUGATION, DIFFUSION, AND VISCOMETRY

69

apparent specific volume is determined by substituting in the equations Va,p. = 100/d - (100 - n)/do

(11a)

n

or 1

V~,,. = d0

1 (~odO) x

(llb)

where d and do are the densities of solution and solvent, respectively, n is the percentage concentration of solute by weight, and x is the concentration of protein in grams per milliliter of solution. The concentration (tan be determined in a variety of ways. If the protein is in distilled water, then a weighed aliquot is lyophilized followed by drying in vacuo over P~05 at about 110° until constant weight is achieved. Alternatively, the dry weight can be determined by micro-Kjeldahl analysis for nitrogen if the conversion factor relating nitrogen to protein is known. For proteins which must be maintained in a buffer, it is important to perform a dialysis and then the composition can be determined by the difference in dry weights between solution and dialyzate. Frequently the amount of protein is so small as to preclude measurements both of concentration and of density, and recourse can be made to a method employing the ultracentrifuge. As shown in equation 4, the sedimentation coefficient is proportional to (1 - ITp). Thus determination of the density of a solution in which the sedimentation coefficient is equal to zero gives a measure of l?. Accordingly the sedimentation rate of the protein is measured in a series of solutions of increasing density and the resulting data plotted in a manner to enable extrapolation to the value of p corresponding to zero sedimentation rate. This value for the density of the solution is equal to l/i?. Since the equation is restricted to two component systems, it is not valid to employ any material such as sucrose to increase the density of the solution. It appears, however, that mixtures of D~O and H20 act as a one-component solvent and therefore they can be used for these experiments. Unfortunately, D20 solutions are not sufficiently dense to reduce the sedimentation rate of proteins to zero, and a long extrapolation of the data is necessary. With accurate data, however, this can be performed to yield reliable results, and the method has, therefore, much to offer. The data are plotted as ~s versus p, where v is the viscosity of the solvent in poises, i.e., including the buffer contribution and the D20, s is the measured sedimentation coefficient, and p is the density of the solvent. The best-fitting straight line is obtained by the least squares method, and the equation of this line is then used to calculate the value of p corresponding to zero sedimentation rates. Even if the data are obtained with high precision, there is some

70

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[9.]

u n c e r t a i n t y i n t h e final result, n o t o n l y b e c a u s e of t h e h a z a r d o u s e x t r a p o l a t i o n , b u t also b e c a u s e of e x c h a n g e of some of t h e h y d r o g e n s of t h e p r o t e i n m o l e c u l e w i t h d e u t e r i u m i n t h e water. T h i s l a t t e r effect is, however, likely to be a s m a l l one a n d for t h e p r e s e n t c a n be neglected. TABLE I I I CALCULATION OF THE APPARENT SPECIFIC VOLUME OF RIBONUCLEASE FROM ITS AMINO ACID COMPOSITION

Amino acid residue

Number of residues/ molecule~

W~

(% by weight a of residue)

Vi

ViWi

(specific volume of residue)

(% by volume of residue)

Aspartic acid Asparagineb

7 9b

5.69 7.31 b

0.59 0.60

3.36 4.39

Glutamie acid Glutamineb

4 8b

3.63 7.27 b

0.66 0.67

2.40 4.87

1.25 6.12 6.34 1.74 2.30 9.44 7.56 5.95 3.52 3.32 3.13 6.84 3.73 9.22 4.43

0.64 0.74 0.86 0.90 0.90 0.63 0.70 0.63 0.75 0.76 0.77 0.71 0.67 0.82 0.70

0.80 4.53 5.45 1.57 2.07 5.95 5.29 3.75 2.64 2.52 2.41 4.86 2.50 7.56 3.10

Glycine Alanine Valine Leucine Isoleucine Serine Threonine Half cystine Methionine Proline Phenylalanine Tyrosine Histidine Lysine Arginine

3 12 9 2 3 15 10 8 4 5 3 6 4 10 4

Total = 126

NW~ = 98.79

~,W~VI = 70.02

Y,W~V~/F,W~ = V~ = 0.709 cc./g.

a The number of each amino acid residue per molecule of ribonuclease, which gives a molecular weight of 13,895 for this enzyme, was obtained from the data of C. H. W. HiTs, W. H. Stein, and S. Moore, J. Biol. Chem. 211, 94i (1954). b To take into account the seventeen amide groups per molecule of RNase, it was assumed that these were approximately equally distributed between the glutamic and aspartic residues. A p p a r e n t specific v o l u m e s of p r o t e i n s c a n be e s t i m a t e d q u i t e r e l i a b l y f r o m t h e a m i n o acid c o m p o s i t i o n , since it has b e e n f o u n d t h a t t h e v o l u m e s of t h e m a c r o m o l e c u l e s are closely e q u a l t o t h e s u m of t h e i n d i v i d u a l a m i n o acid residues. T h i s p o i n t has b e e n e m p h a s i z e d b y M c M e e k i n a n d

72

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

concept, formulated by Fick in 1855, can be written dc

dm = - D A ~ dt

(12)

where dm is the mass of material transported in the time, dr, and A is the cross-sectional area of the diffusion cell. The negative sign is present in this equation because the transport of solute is in the direction of decreasing concentration. Thus, if we think of a vertical cell in which the bottom solution is the more concentrated and the positive direction is downward, then mass is transported in the negative direction. Diffusion coefficients can be measured, by direct application of equation 12, from determinations of the amount of material diffusing across a porous membrane separating a pure solvent from a homogeneous solution of some solute. Such diffusion studies, referred to as diffusion through a porous disk, are dependent on the establishment of a steady state in which the amount of solute diffusing per unit time is constant. This method is particularly useful for biological materials for which a specific bioassay is available. Though the method does not give absolute values for the diffusion coefficient, it is particularly valuable for the enzymologist who can use the method for studies of enzymes which have not been purified. The second, more popular method of studying diffusion, known as the free diffusion method, is based on another equation derived by Fick. This relates the rate of change of concentration with time to the rate at which the concentration gradient is changing with position, x, in the cell, as shown in equation 13. dc d2c d--{ = D ~dx (13) In performing measurements by this method, a sharp boundary is created between the solvent and the solution, and the change in concentration of the solute as a function both of distance and of time is followed by any one of a variety of optical procedures. With modern procedures and in apparatus now commercially available it is possible to obtain absolute diffusion coefficients with an accuracy exceeding 1%. Measurements by this method are also sensitive in the detection of impurities either much larger or smaller than the main component or in the determination of the homogeneity with respect to size of the principal diffusing species. In the free diffusion method great care is required in the design and use of the apparatus to ensure the formation of almost infinitely sharp boundaries and to eliminate any vibrations and resultant convection stemming from inadequacies in the mechanical construction or temperature control. For porous disk diffusion experiments, however, most of

[2]

ULTRACENTRIFUGATION, DIFFUSION, AND VISCOMETRY

73

these difficulties are nonexistent because the diaphragm with its extremely fine pores effectively prevents convective transport of liquid from the upper solution to the lower. Meanwhile independent stirring above and below the diaphragm can be used to maintain the homogeneity of each of the two solutions. Solute molecules in a homogeneous solution are in constant motion due to thermal energy, and such motion, known as Brownian movement, has been related to diffusion by Einstein and Smoluchowski. In fact, if we were able to measure precisely the random movements of a given macromolecule in solution over a long period of time (or, alternatively, the motions of many particles at a specific time), we could calculate its diffusion coefficient from an equation derived by these workers. The driving force for such movement is the thermal energy, kT, where k is the Boltzmann constant, 1.38 X 10- ~ erg/degree, and T is the absolute temperature. These randomly moving macromolecules experience a resistance to motion caused by the viscosity of the medium, and this resistance is characterized by a frictional coefficient, f, which is related to the size and shape of the kinetic unit. For particles of any shape which are large compared to the solvent molecules, D-

kT f

(14)

and therefore determination of the diffusion coefficient gives a direct measure of the frictional coefficient which also appears in the equation relating the sedimentation coefficient to molecular weight. Diffusion Through a Porous Disk

This method, introduced by Northrup and Anson 2~ and applied by them and others to the measurement of the diffusion of enzymes, is elegantly simple. Despite numerous limitations it should be employed whenever approximate results are desired for a specific enzyme, whether the enzyme is pure or not. The method is ideal in the latter situation as long as a sensitive and specific assay is available. Figure 7 shows a bell-shaped glass diffusion cell fitted with a stopcock at the top and with a fiat sintered-glass disk (or diaphragm) at the base. In practice, the cell is filled with the solution containing the diffusing substance and is carefully lowered, by means of an adjustable suspension on a rack and pinion device, until the lower edge of the sintered-glass disk just contacts the upper surface of the solvent contained in the outer vessel. Special efforts are necessary so that the glass diaphragm touches the lower liquid over its entire area. Also, caution should be exercised so ~2j. H. Northrop and M. L. Anson, J. Gen. Physiol. 12, 543 (1929).

74

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

that the diaphragm is not below the surface of the outer liquid; otherwise a stagnant layer will develop. The technique then consists in measuring the amount of solute appearing in the outer liquid as a function of time. This gives dm/dt of equation 12. Initially the difference in concentration between the inner and outer solutions is known, and for most experiments the time of diffusion is so short that there is no significant change in these concentrations. For the use of equation 12 we must know the concentration gradient which involves dx, the effective thickness of the diaphragm, and al~o A, the cross-sectional area of the diaphragm. Neither of these can be determined readily, since the diameter and length of the pores in the diaphragm no doubt vary from region to region. This

1

-I---"SOLUTION

FIG. 7. Porous disk diffusioncell. difficulty can be obviated through the determination of the "cell cons t a n t " by studies with a substance of known diffusion coefficient. As the technique was originally devised by Northrup and Anson, the solution, or more dense liquid, is on the top, but the pores of the diaphragm are sufficiently narrow to prevent mass flow of liquid despite the obvious gravitational instability. Thus diffusion of solute molecules through the pores of the sintered-glass disk into the solvent below creates a region just below the diaphragm which is more dense than the solvent further down in the cell. Convective stirring results, and the heavier liquid sinks to the bottom being replaced by fresh solvent. Similarly, just above the diaphragm there is a temporary diminution in concentration which also produces instability so that stirring results. The working basis of the method then rests on the hypothesis that the concentration above the membrane remains the same as the original concentration and that in the lower solution is similarly equal to the original concentration of that liquid. Of cours% after a long period of diffusion both of these concentra-

7(]

TECHNIQUES FOR CttARACTERIZATION OF PROTEINS

[2]

open or single cell to a double cell in which the volumes of both compartments are fixed and neither of the solutions is in contact with air, thereby minimizing difficulties due to evaporation. Several different cells have been suggested, and one which seems to be simple to use and free of some of the disadvantages of the open cell is shown in Fig. 8. 23 This cell also has provision for mechanical stirring of the liquid on either side of the porous membrane, since evidence has been presented showing that the density stirring in the Northrup-Anson cell is not completely effective and that stagnant layers exist in the vicinity of the diaphragm. In the

J

~.

ISOLVENT

--SINTERED GLASS

~

--SOLUTION

FIG. 8. Closed porous disk diffusioncell. cell shown in Fig. 8, the more dense solution is placed in the bottom so that gravity is a stabilizing rather than a disturbing force, thereby lessening bulk flow of liquid because of either a faulty membrane or gravitational instability when high concentrations were used in the upper solution. The cell is made of Pyrex glass with each end having a volume of about 50 ml. The diaphragms are Corning medium-grade or Jena G-4, fritted-glass disks (average pore size 15 #), of about 5-cm. diameter and 2- or 3-mm. thickness. In each compartment is placed a glass-enclosed iron wire about 3 mm. in diameter and of length slightly less than the diameter of the diaphragm. These glass stirrers are so constructed with respect to length of wire and thickness of glass that the one in the upper compartment sinks in the liquid and that in the lower one floats. The stirrers can be made to rotate by surrounding the cell with a rotating ~a R. H. Stokes, J. Am. Chem. Soc. 72, 763, 2243 (1950).

[2]

ULTR/kCElgTRIFUGATIOI% DIFFUSION~ AND VISCOMETRY

77

permanent magnet. With a stirring rate of about 20 r.p.m, it is found that no stagnant layers exist and that the solutions in each compartment are uniform in composition. To reduce difficulties from contamination of the porous disk by stopcock grease, Stokes uses rubber stoppers to seal each compartment. The rubber stoppers are equipped with small capillary tubes so that the liquid in the diaphragm is not disturbed when the stoppers are inserted or withdrawn. The capillary in the upper rubber stopper is open to the air to allow for volume changes during diffusion, and the lower capillary is fused to a small glass stopcock which can be opened prior to removal of the stopper. Although the stopcock is greased lightly, the grease does not readily contaminate the cell if care is exercised. As with the Northrup-Anson cell, the solution and solvent must be degassed and special precautions must be taken that no air is trapped in the pores of the glass disk during the filling operation. First the entire cell and diaphragm are filled with solution, then the solution in the upper compartment is removed by suction, and after several rinses solvent is added to this cell. Diffusion is allowed to occur with stirring for several hours. Again the upper compartment is emptied by suction; rinsing is performed and the liquid replaced by fresh solvent previously brought to the same temperature as the diffusion cell. Stirring is then resumed and the time recorded. Depending on the sensitivity of the assay procedure, one may have to allow a diffusion time of many hours. At an appropriate time the upper compartment is drained, the cell inverted, and finally the lower compartment is drained. The necessary determinations are made, and the diffusion coefficient is then calculated from equation 15. The cell is immediately refilled and the experiment repeated. If the newly determined diffusion coefficient agrees with the first one, then it can be assumed that a steady state had been reached and tl~e experimental values are valid. Thermostats are generally maintained at the desired temperature with variations no greater than _0.01 °. With the cell shown in Fig. 8, there appears to be little, if any, bulk flow of liquid through the diaphragm, and special precautions about leveling of the diaphragm are unnecessary. For calibration purposes it has been found that potassium chloride is an excellent standard. If the diffusion experiment is performed at 25 ° with 0.1 M potassium chloride diffusing into an equal volume of distilled water for a period such that the final concentrations are 0.075 M and 0.025 M for the solution and solvent, respectively, then the diffusion coefficient is 1.867 X 10-5 cm.2/sec. The volumes of the individual compartments of the cell are usually determined by weighing the cell empty and filled with water and then by converting the weight of the water into volume through use of the known density of water.

78

TECHNIQUES FOR CHARACTERIZATION OF PROTEII~S

[2]

This method gives satisfactory results with enzymes, although one must, of course, be certain that the enzyme molecules are not adsorbed on the surface of the diaphragm or that the pore size is not so great as to allow bulk flow. In view of the simplicity of the technique it is advisable to run experiments with different cells so that irregularities or deficiencies in an individual cell are readily detected. Free Diffusion Introduction. In the past ten years, major modifications have occurred in our techniques for the study of free diffusion with the result that today such diffusion studies can be performed routinely, and in commercially available apparatus, with an accuracy greater than that obtainable in the determination of sedimentation coefficients. This tremendous advance in experimental tech~fique was due largely to the rediscovery of interferometric optical methods and their adaptation to diffusion studies, As a consequence of these developments the older, more common procedures used in diffusion measurements of protein are now obsolete. We will therefore omit discussion of the older methods of handling the data obtained through the use of the familiar schlieren optical systems. This seemingly arbitrary decision is justifiable because the newer methods are neither more difficult nor more time consuming than the older methods, and further the accuracy of newer techniques is of the order of 10 times that of the latter. A thorough discussion of diffusion measurements involving schlieren optical systems is presented by Lundgren and Ward, 24 and the research worker who does not have available newer commercial apparatus equipped with interferometric optical systems should turn to that article for a detailed description of the different methods of handling the data. There are now available in this country three different instruments (and in Europe several more), each of which is completely equipped for precise diffusiometry by interferometric methods. Two of these, Model B, American Instrument Company, Inc., Silver Spring, Maryland, and Model H, Spinco Division, Beckman Instruments, Inc., Belmont, California, are versatile in that they possess two different interferometric optical systems in addition to the usual schlieren optical systems. The apparatus, Model 38A, made by the Perkin-Elmer Corporation, Norwalk, Connecticut, possesses one interferometric optical system along with the schlieren system. After many years of experimentation which led to a variety of ingenious designs for diffusion cells, there now seems to be widespread ~4 H. P. Lundgren and W. H. Ward, in "Amino Acids and Proteins" (Greenberg, ed.), Chapter 6. Charles C Thomas~ Springfield, Ill., 1951.

80

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

mercial models both the Rayleigh and the Gouy interferometric optical systems, and diffusion coefficients of comparable precision can be obtained by either method. A decision as to which is the method of choice in studying proteins is, at best, somewhat arbitrary; and the use in this laboratory of the Rayleigh method is based largely on convenience and ease of handling the data coupled with the ready availability of tables of probability functions necessary for the calculation of diffusion coefficients. For the Gouy method such detailed tables of the necessary mathematical functions are not yet generally available, and laborious, Enzyme Buffer

U

U

! 2

3

4

FIG. 9. Filling of Tiselius electrophoresis cell for a diffusion experiment. time-consuming interpolations within regions of the existing published tables are necessary. The Gouy method offers an important advantage, since it is very sensitive during the early stages of the diffusion experiment when the boundary is very sharp and the consequent interference fringe pattern is greatly spread. Early in a diffusion experiment the Rayleigh pattern is greatly compressed with a resulting loss in resolution; but the resolution of this method improves as the boundary broadens with time, whereas the resolution of the Gouy method decreases. This difference between the two methods can be decisive if one is dealing with a labile enzyme, since the length of a diffusion experiment using the Gouy method can be appreciably less than that with the Rayleigh method. It should be noted, however, that a long period for dialysis of the protein

[2]

ULTRACENTRIFUGATION, DIFFUSION, AND VISCOMETRY

81

solution against a buffer is required for either method, and the duration of the diffusion experiment itself may not be an overpowering factor. The Rayleigh method requires a reference optical limb with a refractive index close to that of the solvent, whereas the Gouy method requires no reference optical path. With present designs of diffusion cells, this constitutes an advantage for the Gouy method for solvents other than dilute salt solutions. In view of the foregoing brief, comparative comments about the two methods, the decision of the author to emphasize in this discussion the Rayleigh method should not be construed as an indication that it is a superior method but rather that it has been found in this laboratory more

F~G. I0. A representative schlieren and Rayleigh pattern from a diffusionexperiment on ribonuclease. convenient and simpler for work dealing with proteins. Where there is a desire for accuracy of the type desired by workers primarily interested in the theory of diffusion, the worker should examine the elegant studies of Gosting and his collaborators 28 employing the Gouy method and those of Longsworth 2~ with the Rayleigh method. The Rayleigh method can be used as well for electrophoresis, whereas the Gouy method cannot yield information about the position and number of boundaries. It is therefore important that enzymologists presently familiar with schlieren methods should acquaint themselves with the Rayleigh method because of its broad utility. In this regard most of the commercial equipment now gives simultaneous photographs of the schlieren and Rayleigh patterns as shown in Fig. 10 for a peptic digest of ribonuclease. E x p e r i m e n t a l Procedure. It is desirable to perform diffusion experiments at the isoelectric point of the protein, since charge effects there are 2~D. F. Akeley and L. J. Gosting, J. Am. Chem. Soc. 75, 5685 (1953). :7 L. G. Longsworth, J. Am. Chem. Soc. 74, 4155 (1952).

82

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

minimal. Frequently other factors such as insolubility or loss of activity preclude this practice, and experiments at other pH values can be performed satisfactorily if there is sufficient electrolyte to damp out charge effects which otherwise would lead to high values for the diffusion coefficient. Well-buffered solutions at an ionic strength of 0.1 suffice for this purpose. For the microcell in use in this laboratory, 3 ml. of protein solution at a concentration of 0.5 g. per 100 ml. and a volume of 300 ml. of solvent are ample. After the protein is dissolved in an aliquot of the buffer, it is dialyzed in the cold for a period of about 24 hours. The number of changes of buffer surrounding the dialysis bag and the length of the dialysis period depend largely on the purity and on other factors such as the stability of the enzyme. In certain cases, such as a study of the peptic digestion of ribonuclease, dialysis of the solution under investigation is precluded because of the permeability of the dialysis tubing to some of the products of the reaction, and diffusion experiments are conducted without dialysis as a means of equilibrating the protein solution against the buffer. For such situations special precautions are taken to subject the buffer to the same treatment as the protein solution so that the content of electrolyte in solution and solvent are as nearly the same as possible. Dialysis is conveniently performed by hanging the dialysis bag containing the solution in an Erlenmeyer flask in which is placed a glass-enclosed magnet and the appropriate solvent. The entire flask can be placed on a magnetic stirrer and stored in a cold room until equilibration is achieved. Dialysis tubing (obtainable from the Visking Corp., 6733 West 65th St., Chicago 38, Illinois) can be obtained in various diameters, and we find that 3~-inch tubing serves well for the small volumes used in this work, since ,it leads to a large surface relative to the volume of the protein solution, thereby ensuring a rapid equilibration. Before filling the diffusion cell it is advisable to centrifuge the solution and solvent for a few minutes at about 3000 r.p.m, to remove lint and any insoluble material such as denatured protein. It is important, before assembly of the cell to form a continuous U-tube as shown in Fig. 9, to lubricate each of the glass end plates of the three sections which slide relative to one another. For this purpose a grease made of two parts of petroleum jelly (Vaseline) and one part of mineral oil proves ideal for most work. The lubricant is readily mixed over a gently boiling water bath, and care should be taken to exclude lint from the grease after it is prepared. Some workers prefer as a lubricant a Dow-Corning silicone fluid, No. 200, of viscosity 7500 to 10,000 centistokes. This is satisfactory, but its removal from the cell at the completion of the experiment is difficult. In lubricating the glass plates, precautions should be observed to prevent the grease from entering the

84

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

due to thermal gradients is lower at temperatures near the maximum density of the solvent (ca. 1°). This is not nearly so serious as in electrophoresis, and if the material is stable at higher temperatures then diffusion experiments should be conducted at higher temperatures, since diffusion rates are much higher at 25 ° than at 1°. A 5-ml. syringe equipped with a 14-inch hollow stainless steel needle, No. 17 gage, with a blunt tip is used to transfer the protein solution to the cell. In filling the cell, the rack is tipped to the side so that the bottom section of the U-tube is at an angle and there is little tendency to trap air bubbles in the cell as the liquid is added. In this laboratory, observations of the diffusion process are generally conducted in the left-hand limb of the cell. Accordingly, the rack is tipped toward the right and the syringe needle is placed in the right-hand limb to avoid any contact of the needle with the windows of the diffusion channel. Sufficient solution is added so that the level of the solution in both limbs is about half to two-thirds the way up in the center section. The rack is righted and the needle withdrawn. At this point the cell looks like that illustrated by Fig. 9-1. By means of one of the knobs on the cell rack the bottom section is slid to the right, thereby disconnecting the left- and right-hand limbs of the cell as shown in Fig. 9-2. The syringe needle is again placed in the right-hand limb of the cell, and sufficient protein solution is added to raise the level in that limb just above the glass end plates of the center and top section. Less than 2 ml. of protein solution is involved in filling half of the left-hand section, the bottom section, and the entire right-hand section of the microcell. With the rack in place in the water bath, buffer is very carefully and slowly added down one of the side windows of the left-hand channel by means of a motor-driven syringe with a needle containing a very fine tip. This addition is continued until the buffer level in the lefthand limb corresponds roughly with that in the right-hand limb, as in Fig. 9-2. After a little practice this layering of solvent over solution can be done readily and reproducibly with the formation of rather sharp boundaries between the two liquids. In the microcell with a channel width of only 2 mm. there is little tendency for mixing if the operations are performed with care. The needle is then withdrawn and the center section is moved to the left, dragging with it the bottom section so that the cell sections are arranged as shown in Fig. 9-3. After rinsing of the upper, right-hand section free of protein, solvent is now added to both limbs slowly by means of a 25-ml. pipet whose tip rests against the wall of the extension tubes..This addition is repeated until the level of liquid is above the side arms of the extension tubes. Because of the connection between the two tubes, the level in the two vessels automatically reaches the same value; and there is consequently no danger of flow of liquid

[9.]

ULTRACENTRIFUGATION, DIFFUSION, AND VISCOMETRY

85

when the center section of the cell is moved to the right to form a continuous U-tube, as shown in Fig. 9-4. During the few minutes that have elapsed after the layering of solvent over the protein solution, some diffusion of the protein occurs. Furthermore, the layering itself and the subsequent handling of the cell produce distorted and relatively blurred boundaries so that we now create essentially a new boundary by the siphoning procedure of Kahn and Polson. ~5 This boundary sharpening technique can be performed in a variety of ways with equal success, and the method described here has its origin in the experiences of other workers. ~8 Owing to short supply of some of the diffusing substances, we have imposed the important additional restriction that the method require only a small amount of solution even if there is some sacrifice in the quality and sharpness of the initial boundary. A long, stainless steel needle with a special tip ground to an angle of 45 ° is filled with solvent and lowered into the left limb by means of a rack and pinion until the tip is at the level of the boundary. Care should be taken during this operation in order to prevent contact of the needle with either of the optical windows of the diffusion cell. The needle is 14 inches long, No. 17 gage, and onto its end is soldered a 2-inch length of tubing with inner diameter of 0.012 inch and wall thickness of 0.010 inch. The upper end of the needle is connected to a three-way stopcock either directly or by means of a short length of thick-walled, narrow-bore rubber tubing. If the needle and stopcock are rigidly coupled, extra caution is required in mounting the stopcock on the rack so that the mere turning of the stopcock doesn't impart any movement onto the needle. If there is a short length of flexible tubing between needle and stopcock, then a tubing should be selected which doesn't collapse when suction is applied; also the needle and stopcock must move in unison so that there is no bending of the tubing when the needle is raised or lowered. One end of the stopcock is connected by tubing to a motor-driven syringe of 5-ml. capacity. This tubing, like the other, should have a narrow bore and heavy wall thickness. A length of about 15 inches of Tygon tubing is connected to the third arm of the stopcock. This connection is for convenience in filling the syringe, the stopcock, and the sharpening needle without the risk of introducing air bubbles into the system. We have used a syringe of narrow bore so that linear movement of the plunger of the syringe involves only a small volume change. The motor-driven syringe should be capable of movement in either direction, and the speed of the motor must be adjustable. In connecting the piston of the syringe 28 Thanks are due Drs. H. Hoch, G. Kegeles, T. E. Thompson, and J. L. Oncley for their suggestions regarding different procedures for boundary sharpening in diffusion experiments.

86

TECHI~IQUES FOR CHARACTERIZATION OF PROTEINS

[2]

to the arm driven by the motor, care must be taken that the coupling is flexible so that the plunger won't bind with the barrel of the syringe in case of slight misalignment. This flexible coupling must allow for movement in all directions except that in the direction of movement of the plunger. Here there must be firm, continuous tension at all times with no elasticity as with most rubber connections. Otherwise the movement of the plunger and the liquid will occur in spurts and the boundary sharpening will be difficult to control. Many workers prefer to use a siphon and allow the liquid flow to occur through the sharpening needle under the influence of a fixed pressure head rather than trust a motor-driven syringe to produce smooth action. With the sections of the cell completely aligned to form a U, the needle, the stopcock, and only a small part of the syringe filled with solvent, and the needle in position such that its tip is near the center of the boundary, sharpening is begun. First, the connection between the side arms of the extension tubes is closed by means of a hemostat, and then withdrawal of liquid is started. The motor is adjusted so that the flow is only about 0.1 ml./min. This is continued for about 4 minutes during which time the boundary is considerably sharpened owing to the formation near the tip of the needle of an interface between solvent and solution. The motor is then turned off for a few minutes, and some spreading of the boundary occurs because of diffusion. Again the motor is turned on and another 0.3 ml. of liquid is withdrawn at the same rate of 0.1 ml./min. The withdrawal of liquid is then stopped for a few minutes, and the boundary broadens a little due to diffusion. For the last time the motor-driven syringe is started and liquid is withdrawn for about 3 minutes at 0.1 ml./min. The boundary should be examined during this time to see that the fringes are straight, right up to the position where the boundary is being sharpened. The motor is turned off, the stopcock to the sharpening needle is turned 45 ° to isolate the needle, and the bottom section of the cell is moved to the right in order to break the connection between the left- and right-hand limbs. It is important that these operations be done almost simultaneously, with the aid of another individual if necessary. The time of stopping the flow and moving the bottom section is then recorded as the starting time of diffusion. The needle is withdrawn by careful operation of the rack and pinion. When this is completed, it is advisable to slide the center section to the left, dragging with it the bottom section which will now be in its normal position. The center section of the diffusion cell is now isolated from both upper and lower sections. Thus any dust which falls into the open reservoir tubes or moisture which condenses on the cold extension tubes cannot cause a disturbance of the boundary. As soon as the center

88

TECHI~IQUES FOR CHARACTERIZATION OF PROTEINS

[9.1

symmetrically, and the b o u n d a r y width when sharpening is discontinued should be between 0.3 and 0.4 mm. Calculations. Figure 11 shows a series of typical Rayleigh interference patterns obtained in a diffusion experiment with a peptic digest of ribonuclease. I t should be borne in mind t h a t the observed p a t t e r n is essentially a section of a plot of refractive index versus distance in the cell. Since the refractive index of protein solutions can be considered as varying in a linear fashion with the concentration of solute, the Rayleigh m e t h o d gives a plot of the concentration versus distance in the cell, and if we were

Fzo. 11. Representative Rayleigh interference patterns from a diffusion experiment on a peptic digest of ribonuclease. Pictures from top to bottom were taken at 9, 38, 99, 337 and 1183 rain., respectively, after formation of the boundary. to plot the fringe n u m b e r as a function of fringe position the curve obtained would be the integral of the curves obtained b y schlieren methods. For this reason, the Rayleigh m e t h o d is frequently called the integral fringe method. Thus the total n u m b e r of fringes, J, is a measure of the change in concentration across the boundary, and it is equal to a hn/)~, where a is the cell thickness (along the optical path), An is the difference in refractive index between solvent and solution, and X is the wavelength of light. T o compute the diffusion coefficient from Rayleigh patterns we first must evaluate the total n u m b e r of fringes, J . This is done most readily b y using different photographs. An early picture, before the fringes are completely resolved as in the 9-minute photograph, is used to determine the fractional part of J ; and a later photograph, after the fringes are distinctly separated as in the 337-minute photograph, is used to measure

90

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

will be straight except for small regions at the top and bottom of the channel where cementing of the end plates onto the optical glass windows causes some distortion. If the fringes are not straight throughout the entire length of the diffusion cell, it is advisable to choose the best region and to form the boundary at that level. There are a variety of methods of handling the data for the calculation of diffusion coefficients. For example, the equivalent of the wellknown height-area method can be used, since J is a measure of the area and we can readily determine the maximum height (of the gradient curve) by finding the minimum separation of fringes. This method has been used by Svensson ~9 and others, but the method used by Longsworth ~7 is recommended both because of its accuracy and because it uses much more of the experimental data and is therefore capable of detecting deviations from ideal diffusion behavior. Computations by the method of Longsworth involve the assumption that the concentration distribution in the boundary is Gaussian, and therefore we can use the Tables of Probability Functions (Federal Work Agency, Works Project Administration, Superintendent of Documents, Washington, D. C., 1941). These tables are very complete and the calculations with them can be made without the need of interpolation. If the concentration and cell thickness are such that there are about 20 fringes in the boundary, then the position and number of all the fringes are tabulated. The number of the fringe with which the cross hairs coincide outside the boundary is termed the zero'th fringe. If there are as many as 50 fringes in the boundary it is advisable, in order to reduce labor, to record the position and number of every other fringe. The method of calculation is illustrated by Table IV showing data obtained on a peptic digest of ribonuclease. In column 1, headed fl, are listed the number of the different fringes up to a value less than J/2. The second column lists the computed value, jk* = (2jk - J)/J, for each value of jk. As long as j~ is less than J/2, the sign of the computed value, j~*, in column 2 can be ignored. Corresponding to each value of jk there should be a value of jr, where j~ is greater than J/2. The values of jl are listed in column 3, and the computed values, jz* = (2jl - J)/J, are tabulated in column 4. It should be stressed that the pairing of fringes can be done in a variety of ways. Thus the pairing of fringes 2 and 10, 3 and 11, 4 and 12, etc., could be changed to 2 and 11, 3 and 12, 4 and 13, etc., or 1 and .10, 2 and 11, 3 and 12, etc., or to a variety of other combinations. Pairing of the fringes in different ways can furnish valuable information about the homogeneity of the diffusing substance and the dependence of diffusion coefficient on concentration. We usually omit the first and last 29 H. Svensson, Acta Chem. Scand. 5~ 72 (1951).

[9-]

ULTRACENTRIFUGkTIO~ DIFFUSION, AND VISCOMETRY

i <

Z ©

r~

~

×

N t~

N

r~ 0

u~ u~ ~

u~ u~ ue~ u~ U~

Z

u~

O

O

~t

91

92

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

fringes, since the accuracy in locating these fringes is low, owing to small curvature of these fringes at the ends of the boundary. The corresponding microcomparator readings for each jk and j~ are listed in columns 5 and 6 as Hk and H~, and the distance in centimeters between paired fringes, Hk -- Hz, is listed in column 7 as AH. The values of jk* and j~* correspond to

2 foe_a,d~ v7 listed in the probability tables in the right-hand column of each page. In the left-hand column are listed the values of x corresponding to each value of the integral. Thus the values of jk* and j~.*are found in the righthand column of the probability tables and the corresponding values of x determined. Since x is equal to h/(4Dt)~, these values are recorded for all values ofjk* and j~* in columns 8 and 9. The symbol h is the cell height and is equal to zero at the center of the boundary, where j = J/2. The values in columns 8 and 9 are added to give Ah/(4Dt) ~ shown in column 10. The values of column 7 are now divided by those in column 10 to give column 11, (4Dt) ~ AH/Ah. AH/Ah is the magnification factor of the camera lens relating distances in the diffusion cell to those on the photographic plate. This factor is determined by photographing an accurately ruled glass scale mounted in the appropriate place on the cell rack and comparing the distance between lines on the photograph and original scale. In our instrument AH/Ah is very close to unity, and the average of the values of column 11 is, therefore, (4Dr) ~, where D is the diffusion coefficient in square centimeters per second, and t is the time in seconds. The calculation of D follows immediately to give the value shown in the bottom of the table. This calculation is repeated for a number of pictures taken at different times. The values of D so obtained may show a slight trend downward with time because the initial boundary was, of course, not infinitely sharp. Correction for this effect can be made by determining the "zero time correction." This is most readily obtained from a plot of the apparent diffusion coefficients versus the reciprocal of time. The intercept of this linear plot at lit = 0, corresponding to infinite time, is the true diffusion coefficient, and the slope is D At, where At is the zero time correction. For small molecules like sucrose the zero time correction is of the order of 20 seconds, whereas for proteins it may be of the order of several minutes. The value of the diffusion coefficient obtained above corresponds to a certain solvent and a certain temperature. It is common practice to report diffusion coefficients, D~o.~, corresponding to a temperature of 20 ° in a solvent with the viscosity of water. Correction of the measured value to

94

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

microcomparator are recorded. It should be noted that the positions of the Gouy fringes correspond to the concentration gradients in the diffusion cell. Thus the zero'th fringe corresponds to the peak of the schlieren curve and the J / 2 fringe in the Rayleigh system. Also the top fringes in the Gouy system correspond to the ends of the boundary or the first and last Rayleigh fringes. The position of the undeviated slit image is also recorded. This l~osition can be determined from the fringe pattern resulting from the two horizontal slits in the Gouy mask. These slits are so oriented that light passing through them actually traverses the water bath and not the diffusion cell. In the resulting pattern, the center of the central bright fringe corresponds to the position of the center of the image of the light source. It is frequently found, however, that this bright central fringe is displaced if the same double-slit assembly is located in front of the diffusion cell filled with a homogeneous solution. Account must be taken of this deviation, which probably arises from optical imperfection in the ceil. The position of the undeviated slit image can also be obtained, apparently with sufficient precision, by careful location of the brightest fringe in the Gouy pattern. With the recorded values of the undeviated slit image and all the interference minima, a table of fringe number, j, and fringe distance, Y¢, in centimeters can be constructed. If the photograph is enlarged by the cylinder lens, then these distances on the plate must be divided by the magnification of the cylinder lens. For each value o f j the appropriate value of z~is obtained by adding ~ , i.e., zj = j + 3/~. A more accurate value of z¢ can be obtained by the use of Table 1 in the paper by Gosting and Morris. 3° With the values of z¢, the corresponding values of f(z) are computed for each fringe by the relationship (17) f(z) = jzj These values of f(z) are then used to compute values of e-~2 for each fringe. This is performed through the use of Table 1 in the paper of Kegeles and Gosting/' Since this table is rather abbreviated, considerable interpolation is necessary unless one computes a more extensive table from the equation defining f(z) in terms of e-~'~. With the values of e-*~' and Yj, the corresponding value of Ct can be calculated from the relation C,-

Y~

e-zi2

(18)

For an ideal diffusion curve, Ct should be independent of j. The average a0 L. J. Gosting and M. S. Morris, J. Am. Chem. Soc. 71, 1998 (1949). 31 G. Kegeles and L. J. Gosting, J. Am. Chem. Soc. 69, 2516 (1947).

[9.]

ULTRACEN'TRIFUGATION., DIFFUSION., &N'D VISCOMETRY

95

value of Ct is then used to calculate an apparent diffusion coefficient, D,pp., (Jkb)~ Dap~. - 4~.Ct2t

(19)

In this equation, b is the optical lever arm or the distance between the schlieren lens and the schlieren diaphragm (this value is supplied by the manufacturer), k is the wavelength of the light used, and t is the time in seconds. As in the Rayleigh method, photographs at different times are measured and the apparent diffusion coefficients plotted against 1/t to give the time diffusion coefficient and also the zero time correction.

IV. Viscometry Introduction. Although our theoretical understanding of the viscosity of pure liquids is rather limited at present, much is known about the viscosity of solutions of macromolecules. In conjunction with other physical-chemical data, studies of the viscosity of solutions of large molecules can yield valuable information about the molecular size and shape of the dissolved molecules. The coefficient of viscosity of a liquid is generally considered as a measure of its resistance to flow when the liquid is subjected to a shearing force. It can be shown very readily that the coefficient of viscosity is a direct measure of the amount of energy needed to maintain a given rate of flow in a liquid. If particles or large macromolecules such as a protein are added to the liquid, the normal flow patterns in the liquid are disturbed by these particles and more energy is dissipated in the maintenance of flow. Einstein first showed that the increase in viscosity due to the presence of large, rigid, uncharged, spherical particles was proportional to the volume fraction occupied by the particles. For particles known to be spherical, viscosity measurements provide information about the volume concentration of the dissolved macromolecules. Since the dry weight concentration of the solute is generally known, the viscosity data may, for the particular case of spherical particles, be interpreted in terms of the amount of solvent associated in solution with a gram of dry material. In general, of course, the shape of protein molecules is not known, and it is likely that many of them cannot be approximated by rigid, impermeable spherical particles. Anisometric particles such as rodlike or platelike objects are subjected to Brownian motion in solution, and as a result of the rotation of the particles the effective volume occupied by the particles is much larger than their true geometric volume. As a consequence, anisometric particles which are randomly oriented in solution cause a greater increase in viscosity than do spherical particles. Reliable theories are now available relating the viscosity of solutions of

96

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

anisometric particles to both the shape and effective volume of the large particles. Unless some simplifying assumptions are made regarding the macromolecules, viscosity data must be combined with other data to determine both the size and shape of the macromolecules. Experimental Procedure. The determination of the absolute viscosity of a liquid is a difficult experimental problem, and recourse is usually made to comparative studies of different liquids and calibration of instruments through the use of liquids of known viscosity. The most popular viscometer is the so-called capillary viscometer in which measurements are made of the volume rate of flow of the liquid through a capillary of known, length and diameter under the influence of a known pressure head. Poiseuille showed m empirically the interrelationships among the various experimental factors, and theories have now been developed to explain the observed results. If a volume of liquid, v, in milliliters, flows through a capillary of length 1 and radius r, in centimeters, under a pressure head, h, in centimeters, the viscosity can be calculated from

hgpr4t~ -

81v

(20)

where g is 980 cm./sec. ~, p is the density in grams per milliliter and ~ is the viscosity in poises. Since we are interested in comparative results for different liquids, a Fro. 13. second liquid can be introduced into the instrument and C a p ill a r y measurements of the volume rate of flow or outflow time viscometer. can again be made. In general, the instrument is used in a reproducible manner, and the quantities h, g, r, l, and v will be the same for the two liquids. We can write, therefore, .

~/~0 = (t/t0)(p/p0)

(21)

where T0, to, and p0 are the viscosity, outflow time, and density for the reference liquid. Figure 13 shows a capillary viscometer, commonly termed an Ostwald viscometer. A given volume of liquid is placed in the reservoir bulb by means of a pipet. With gentle suction applied at the right-hand limb of the viscometer, the liquid is forced through the capillary into the drainage bulb. Suction is continued until the liquid level is above the line scratched in the glass tube above the drainage bulb. There should be sufficient liquid in the viscometer to fill completely the drainage bulb, the capillary tubing, and part of the reservoir bulb. In the same way as the line is engraved above the drainage bulb so there is also engraved a line below the bulb at a distance about 2 cm. from the

[2]

ULTRACENTRIFUGATION, DIFFUSION, AND VISCOMETRY

97

bulb. After the liquid level is raised above the upper engraved line, the negative pressure is released so that there is atmospheric pressure operating on both sides of the viscometer. The liquid flows due to the hydrostatic head, and the time required for the meniscus to pass between the two engraved lines is the outflow time for the volume of liquid contained in the drainage bulb. With this outflow time, the density of the liquid, and measurements of the physical characteristics of the viscometer--i.e., length and radius of the capillary, volume of liquid contained between the two engraved lines, and the mean hydrostatic head between the drainage and reservoir bulbs--the coefficient of viscosity of the liquid can be calculated according to equation 20. As indicated above, however, it is a better procedure to measure the outflow time and density of the unknown liquid, such as a solution of enzyme of known concentration, and compare these results with those of a known liquid, such as water. In the latter case the relative viscosity is calculated from the relative outflow times and the relative densities according to equation 21. Since the viscosity of water at different temperatures is readily available i~ handbooks, the viscosity of the unknown solution can be calculated. There are many precautions to be exercised in the design, construction, and use of a viscometer of the type shown in Fig. 13. With regard to the use of the viscometer for precise work the obvious requirements are accuracy in measuring the outflow times and the densities of the liquids. To facilitate accurate measurements of the outflow time, we have used fine slits made by scratching through a thin film of black cement which coats the glass, in place of the customary engraved lines above and below the drainage bulb of the viscometer. The passage of the meniscus across the slits causes a bright light flash to be visible, due to internal reflections ill the tube, and thus the time at which the liquid passes a given point can be obtained through reflex responses rather than through the more subiective action of judging when the meniscus reaches an engraved line. With the use of a mechanical stopwatch which makes one revolution every 10 seconds, five successive readings of the outflow time for a given liquid are found to lie within about 0.05 second for total outflow times of the order of 60 seconds. As will be seen later, the use of viscosity data for studies of enzymes requires measurements on dilute solutions for which the outflow time is only slightly larger than that of the solvent. Therefore, extreme accuracy is required for the results to have any validity. In such studies, measurements with an electric timer, such as Model S-1 of the Standard Electric Time Co., which makes one revolution per second are recommended. No~ infrequently, five successive readings of the outflow time for the same sample will show an average deviation of only a few hundredths of a second. Densities of the liquids can be measured

98

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

with satisfactory precision by using standard pycnometric procedures. With a pycnometer of 5-ml. capacity the density of a liquid can be measured readily to _+0.0001 g./ml. The density term, p/po, of equation 3 is seldom as great as 1.010, and generally it is in the neighborhood of 1.002. If we measure the viscosity of a solvent such as 0.2 M sodium chloride, then P/POrepresents the density of 0.2 M sodium chloride relative to that of water, and the value of p/po is close to 1.01. When the measurements involve a protein solution, the reference liquid will contain the same inorganic salts and the difference in density between the solution and solvent is attributable only to the protein. In the absence of experimental measurements of the density term, due, for example, to inadequate supply of protein for pycnometric studies, the density term can be calculated with satisfactory precision by assuming additivity of the weights and volumes of the protein and the solvent. Thus a solution of protein in water containing 1 g. per 100 ml. of solution can be considered as being composed of 1/1.35 ml. of solid protein (the density of mosrTj 18.24 0 T6l TD 1 1 1

[9.]

ULTRACENTRIFUGATION~ DIFFUSION~ AND VISCOMETRY

99

flowing back and forth in the viscometer. This can be reduced somewhat if care is exercised so that the liquid flows slowly through the capillary when suction is applied above the drainage bulb. In order to ensure that the hydrostatic head of liquid, h, is the same ~t all times, the viscometer should be placed in a metal frame which ~llows for alignment of the viscometer in a vertical position. Once the viscometer is placed in the frame it should not be removed, even for cleaning, unless restandardization is performed with a known liquid. Care should be exercised so that the frame can be mounted on a support in a reproducible manner and so that vibrations due to the stirring of the water in the bath do not affect the viscometer. Capillary viscometers are subject to several limitations which can be eliminated or at least minimized by careful design. Since the liquid is forced both to enter the capillary tube from the drainage bulb and then to leave the capillary into the reservoir bulb, there will be energy losses in establishing the flow pattern characteristic of the capillary. This effect has been termed the kinetic energy effect, and corrections sometimes need be made. If the design is such that the volume rate of flow of liquid is small, the kinetic energy correction can be reduced sufficiently so as to be negligible. This is most readily accomplished not by reducing the hydrostatic head of liquid which introduces other complications or by reducing the radius of the capillary which makes more troublesome the problem of dust particles, but rather by increasing the length of the capillary. If the reservoir bulb is rather large in cross section, slight errors in pipetting the solutions in the viscometer will cause only a small variation in the mean hydrostatic head. Other corrections such as the drainage correction and the surface tension correction are generally almost negligible, since comparisons are made between dilute protein solutions and the solvent. In this laboratory, the capillary tubing used for viscometers has a diameter of about 0.6 mm. and a length between 50 and 100 cm. The diameter is generally measured by weighing a snlall piece of capillary tubing with and without a small column of mercury in the capillary. From measurement of the length of the mercury column and the weight of the mercury whose density is known, the diameter of the capillary is readily determined. Uniformity of the capillary with respect to diameter is tested by using the whole length of capillary and sliding a small amount of mercury along the inside so that the length of the mercury column can be measured in different positions of the capillary. If the length of this mercury column is independent of position in the capillary, the uniformity of the bore of the capillary is demonstrated. The drainage bulb is generally made of tubing with a diameter about 2 ram. by careful glass

100

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[2]

blowing to produce a symmetrical bulb of approximately the volume desired. Since many proteins are in scarce supply, we use a bulb of about 0.7-ml. capacity, the volume of which is determined roughly with mercury. The bulb with several centimeters of 2-mm. tubing on either side of it is then fused to one end of the capillary tubing which is then bent carefully about every 20 cm. so that the over-all size of the instrument is not cumbersome. Care is taken in making the turns in the capillary so as to avoid constricting the capillary. The reservoir bulb with a 25-cm.long glass tube (15-mm. inside diameter) is then fused to the other end of the capillary tubing so that the vertical distance between the drainage bulb and the reservoir bulb is about 15 cm. At. the top of the drainage bulb a small piece of glass tubing of 4-ram. diameter is fused onto the 2-mm. tubing, and then cross braces of glass rods are added at various positions to strengthen the viscometer and reduce the risk of breakage. With an instrument of the type described, the outflow time is about 80 seconds and the kinetic energy correction, pv/8~rlt, which should be subtracted from equation 2 is less than 0.1% of the term h g p r 4 t r / 8 1 v . Once the instrument is constructed, measurements with water are made and the viscosity is calculated according to equation 20. This is done not to measure the absolute viscosity of water at the temperature of the water bath, but rather to show that no unforeseen complications have arisen, such as constriction of the tubing, during the construction of the instrument. If the viscosity calculated in this way is within 10% of the known value, then the instrument can be considered satisfactory for use. C a l c u l a t i o n s . It is the increment in viscosity caused by the addition of a known volume fraction of the solute which is of interest to the biochemist, since this is related to the physical properties of the macromolecule. As Einstein pointed out, the theories require that measurements be made at sufficiently low concentrations in order to minimize hydrodynamic interactions among the macromolecules. Therefore, experiments should be conducted at several different concentrations and the data plotted in a manner which permits extrapolation to infinite dilution. If the viscosity of the solvent is n0, the viscosity of a solution, n, of concentration c can be expressed in the form of a power series, 7/70 = l $

Ac $

Bc ~-~

• • •

(22)

where n / n o is generally written 7,e~., the relative viscosity. The term specific viscosity, 7sp., is used quite frequently and is equal to nr~,. - 1. If the specific viscosity is divided by c, we obtain n,p./c = A + B c +

• • •

(23)

[2]

ULTR~_CEN'TRIFUG~-TION~ DIFFUSION, AND VISCOMETRY

101

where ~,p/c is frequently termed the reduced viscosity. At infinite dilution the second and following terms in the power series become negligible, and we obtain equation 24, where [7] is defined lim (~,,./c)c--,o = A = [~]

(24)

as the intrinsic viscosity. Note t h a t it has the units of reciprocal concentration. If, as is rarely the case, the concentration, c, is in volume units, VISCOSITY OF RIBONUCLEASE

at pH 2.3

16.0

"<)"-0"----~

0

O--

"qsp/C cc/gm 8.0

I

i

I

L

I

heated o heated with HSCH2CH20H

1.160

k080 ql/~lo ~

~

t~.."

1.000 2

6 t0 CONC. IN mg/cc. FIO. 14. Plot of n,p./c and n/n0 versus concentration for ribonuclease treated in different ways. i.e., milliliters of solute per milliliter of solution, then [7] would be dimensionless. If the concentration is in grams per milliliter or grams per 100 ml. the intrinsic viscosity will have the units milliliters per gram or deciliters per gram, respectively. Since it is the intrinsic viscosity which is needed, the q u a n t i t y n,p./c should be plotted against c to give curves such as t h a t shown in Fig. 14. Unfortunately such plots usually show wide scattering of the points, especially at low concentrations, and it is sometimes difficult to extrapolate to a reliable value for the intercept, which is the intrinsic viscosity, [7]. I t is advisable in such cases to plot the d a t a also as m,. versus c, which yields a curve such as t h a t shown in Fig. 14.* To evaluate the intrinsic viscosity from such a curve we must * The plot would be identical to that shown in the lower part of the figure but the ordinates would be decreased by 1.000.

[2]

ULTRACENTRIFUGATION, DIFFUSION, AND VISCOMETRY

103

solution is represented as an impermeable rigid ellipsoid of revolution whose hydrodynamic behavior in solution would be equivalent to that of the protein. This is not to say, of course, that the protein molecule in solution is actually an ellipsoid of revolution, and in fact the size and shape of the actual molecule are likely not to be the same as those calculated for the equivalent particle. It should be stressed that the value of f3 defined by Scheraga and Mandelkern is extremely sensitive to slight experimental errors. Oncley, at a much earlier time, 9 proposed another method for handling the dilemma of evaluating the independent contributions of hydration and shape to the hydrodynamic behavior of protein molecules in solution. In effect, he assumed that the volume of a protein molecule in solution is only slightly greater than that calculated from the partial specific volume, with the slight increase being due to hydration. With an assumed value for the hydration, it is then a straightforward matter to calculate the shape of the kinetic unit. The validity of such an assumption for the volume of a protein molecule in solution is a matter of some controversy; though it may be correct for some native proteins, it is almost certain that the more general approach of Scheraga and Mandelkern is more meaningful for denatured proteins. It is beyond the scope of this article to discuss in detail this difficult problem, and the reader is referred to the above papers and the discussion by EdsalP ~ for the different points of view. For detailed calculations the reader will find useful tables in Cohn and Edsall 2~ which relate the frictional ratio and the intrinsic viscosity to the shape of the hydrodynamic unit. Also the contour plots presented by Oncley for these relationships will be of considerable use. It should be pointed out that molecular weights can be calculated from sedimentation and viscosity data or from diffusion and viscosity data if it is either known or assumed that the molecules in solution do not depart significantly from rigid, impermeable spheres. For these calculations the following equations should be used: M = 4690(s2°'w)~['/]~

(1 M-

-

(26)

?p)~

6.58 X 10-'6 (D20.w)3in]

(27)

In equations 26 and 27 a value of ~ equal to 2.16 X 10s has been employed and will provide reliable results even for materials with axial ratio about 5 to I. The sedimentation coefficientsare in Svedbergs, and the intrinsic viscosityin reciprocal concentrationunits, i.e., (g./I00 ml.)-'. ~ J. T. Edsall, in "The Proteins" (Neurath and Bailey, eds.), Chapter 7. Academic Press, New York, 1953.

[1A]

PAPER ELECTROPHORESIS

21

[IA] Paper Electrophoresis B y A. HELGE F. LAURELL

Introduction T h e basic elements of p a p e r electrophoresis m a y be said to h a v e had forerunners in experiments in which filter p a p e r was soaked in different salt solutions a n d dipped into beakers connected through p l a t i n u m electrodes to the poles of V o l t a ' s pile (see the collected v o l u m e s of F a r a d a y and of Berzelius for references), b u t it was p r o b a b l y not until the end of the nineteenth c e n t u r y t h a t the use of electromigration as a potential separation process was conceived. T h e applicability of the m e t h o d of p a p e r electrophoresis seems to h a v e been realized in 1937 b y KSnig in Brazil, who used it for separating v e n o m toxins. I t was later independently reintroduced in 1948 b y H a u g a a r d and K r o n e r and b y Wieland and Fischer and through the publications in 1950 of D u r r u m , Biserte, G r a s s m a n n and Hannig, T u r b a a a d Enenkel, Cremer and Tiselius, and M c D o n a l d et al.; the m e t h o d is now widely used. No a t t e m p t will be m a d e to give a complete bibliography, as the technique during the last few years has been the subject of several monographs, ~-~ reviews, 6-1~ a n d symposia, ~e-15 to which the reader is referred. T h e applicability 1 C. Wunderly, "Die Papierelektrophorese." Verlag H. R. Sauerli~nder, Frankfurtam-Main, 1954. 2 R. J. Block, E. L. Durrum, and G. Zweig, "A Manual of Paper Chromatography and Paper Eleetrophoresis." Academic Press, New York, 1955. 3 H. J. McDonald, "Ionography. Electrophoresis in Stabilized Media." Year Book Publishers, Chicago, 1955. 4 M. Lederer, "Introduction to Paper Electrophoresis and Related Methods." Elsevier Publishing Co., Amsterdam, 1955. hA. Dittmer, "Papierelektrophorese. Grundlagen-Methodik-Klinische Betrachtungen." Gustav Fischer Verlag, Jena, 1956. 6 W. Grassmann, Naturwissenscha]ten 38, 200 (1951). A. Tiselius and P. Flodin, Advances in Protein Chem. 8, 461 (1953). 8 H. G. Kunkel, "Methods of Biochemical Analysis," Vol. 1, p. 141. Interscience Publishers, New York, 1954. 9F. Turba, "Chromatographische Methoden in der Protein-Chemie," p. 324. Springer-Verlag, Berlin, 1954. 10R. L. M. Synge, "Modern Methods of Plant Analysis," Vol. 1, p. 55. SpringerVerlag, Berlin, 1956. ~1H. J. McDonald, Federation Proc. 14, 733 (1955). 12Symposium Utrecht. Chem. Weekblad 49, 229 (1953). 13 let Colloque du Laboratoire de l'H5pital Saint-Jean a Bruges, Union Chimique Belge S. A., 1953. ~42~. CoUoque du Laboratoire de l'HSpital Saint-Jean ~ Bruges, Editions Arscia, Bruxelles, 1954. 15 Ciba Syrup. on Paper Electrophoresis, London (1956).

22

TECHI~IQUES FOR CHARACTERIZATION OF PROTEINS

[1A]

of the method is in most cases obvious and will only be discussed briefly. Applicability Paper electrophoresis may be used for following the distribution of activity among the different protein fractions in each successive step in the course of fractionations by conventional methods such as extraction, salting out, or adsorption. If, in a certain step of a purification procedure, one intends as an alternative to apply zone electrophoresis in columns, paper electrophoresis may be used to find a suitable pH and buffer in which to perform the separation. It is an advantage that only about 0.5 mg. of protein has to be used in each of the preliminary experiments. In many cases, preparative separations can be carried out on a fairly large scale on thick filter papers (see Schultze and Bie118), but zone electrophoresis in columns is probably to be preferred in most cases because of its greater capacity (up to at least 10 to 20 g. of protein) and because of the possibility of using supporting media which give negligible adsorption in a given case. Relative mobility measurements are made by running, on the same paper, the test sample together with a sample of known mobility. If a homogeneous reference substance is found which is not adsorbed to the paper and which migrates an equal distance as the test sample, the mobility is probably of the same magnitude, especially if this equality in migration should hold over different concentrations of hydrogen ion. The method is not suitable for absolute mobility measurements, however, even though an approximate value for the isoelectric point may be found from data collected at different pH's. Approximate mobility values may be arrived at in certain types of apparatus where the evaporation is prevented by placing the paper between two parallel glass plates, as done by Kunkel and Tiselius.17 It is, however, technically difficult to get good separation patterns in this type of apparatus. If enough material is available, mobility measurements will best be "performed with the moving boundary technique. Wallenfels and Pechmann TM were among the first to apply paper electrophoresis in enzyme studies. These authors detected fractions with amylase, protease, lipase, and phosphatase activities in a commercial enzyme preparation (takadiastase). M a n y different enzymes from various origins have since been studied, such as proteinase,i~9,2°pepsin, 21.52 le H. E. Schultze and H. Biel, Behringwerkmitteilungen 30, 72 (1955). 1TH. G. Kunkel and A. Tiselius, J. Gen. Physiol. 35, 89 (1951). 18K. Wallenfelsand E. Pechmann, Angew. Chem. 63, 44 (1951). lg j. M. Gillespie, M. A. Jermyn, and E. F. Woods, Nature 169, 487 (1952).

[IA]

PAPER ELECTROPHORESIS

23

t r y p s i n , 23 c a t h e p s i n , 2' f i b r i n o k i n a s e , 2° a m i n o p e p t i d a s e , :°,~4 r i b o n u c l e a s e 25 a n d its r e a c t i o n p r o d u c t s , 26 m a l t a s e 27,2s i n v e r t a s e , 27 d e x t r i n a s e , 27 cellulase, 29 sucrase, ~9 amylase,'9,:~, 30 glucuronidase,~9, a~ h y a l u r o n i d a s e , a2 lactase, ~7 l y s o z y m e , 33 aldolase, 34 enolase, 35 p h o s p h o r y l a s e , 38 A T P a s e , 37 p h o s p h o m o n o e s t e r a s e , ~s phosphatases,20,~0.3'. 39-43 cholinesterase,30.44, 4~ lipase,S° peroxidase46,47 dehydrogenases,34.4S~,b a n d c y t o c h r o m e c. 49 T h e a b o v e - m e n t i o n e d references give some e x a m p l e s of d i f f e r e n t t e c h n i q u e s for d e t e c t i n g e n z y m a t i c a c t i v i t y on t h e paper. T h e p a p e r strip n m y be d i v i d e d i n t o t w o e q u i v a l e n t strips, one of which is used for s t a i n ing the p r o t e i n zones a n d t h e o t h e r for d e t e c t i o n of e n z y m a t i c a c t i v i t y . T h i s can be d o n e b y c u t t i n g t h e p a p e r i n t o s e g m e n t s each of which is s u b s e q u e n t l y e l u t e d a n d t e s t e d for a c t i v i t y b y c o n v e n t i o n a l m e t h o d s . I n m a n y cases it is possible to s p r a y t h e s u b s t r a t e d i r e c t l y o n t o the p a p e r - p r e f e r a b l y before t h e p r o t e i n f r a c t i o n s h a v e dried on t h e paper. A f t e r 20 F. Lundquist, T. Thorsteinsson, and O. Buus, Biochem. J. 59, 69 (1955). 2~ W. Grassmann, K. Hannig, R. Merten, and G. Schramm, Z. physiol. Chem. 289, 173 (1952). 22 W. D. Heinrich, Biochem. Z. 323, 469 (1953). 23 E. Nikkilh, K. Ekholm, and H. Silvok, Acta Chem. Scan& 6, 617 (1952L 2~ D. H. Spackman, E. L. Smith, and D. M. Brown, J. Biol. Chem. 212, 255 (1955). 25 A. M. Crestfield and F. W. Allen, J. Biol. Chem. 211, 363 (1954). 26 F. F. Davis and F. W. Alien, J. Biol. Chem. 217, 13 (1955). 27L. R. Wetter and J. J. Corrigal, Nature 174, 695 (1954). 2s L. R. Wetter, Biochim. et Biophys. Acta 18, 321 (1955). 29 M. A. Jermyn, Australian J. Sci. Research B5, 433 (1952). 30 A. Delcourt and R. Delcourt, Compt. rend. soc. biol. 147, 1104 (1953). 3~ G. T. Mills and E. E. B. Smith, Biochem. J. 49, vi (1951). ~2 A. Caputo, Nature 17-% 358 (1954). 33 p. Caselli and H. Schumacher, Z. ges. exptl. Med. 124~ 65 (1954). 34 G. Toschi, Boll. soc. ital. biol. sper. 30, 565 (1954). 35 B. MalmstrSm, Arch. Biochem. and Biophys. 46, 345 (1953). ~6G. Toschi, Boll. soc. ital. biol. sper. 30, 563 (1954). a7 G. Toschi, Boll. soc. ital. biol. sper. 30~ 567 (1954). 38 K. M. MOller, Biochim. et Biophys. Acta 16, 162 (1955). 39 j. Roche and S. Bouchilloux, Bull. soc. chim. biol. 35, 567 (1953). 4o G. Eisfeld and E. Koch, Z. ges. inn. Med. u. ihre Grenzgebiete 9, 519 (1954). 4~R. S. Levy and D. Mazia, Arch. Biochem. and Biophys. 44, 280 (1953). ~ G. Nylander, Scand. J. Clin. Lab. Invest. 7, 254 (1955). ~a R. W. Baker and C. Pellegrino, Scand. J. Clin. Lab. Invest. 6, 94 (1954). 44 p. G. Togni and O. Meier, Experientia 9, 106 (1953). 45 C. A. Zittle, E. S. DellaMonica, J. H. Custer, and R. Krikorian, Arch. Biochem. and Biophys. [i6, 469 (1955). 4~ M. A. Jermyn, Nature 169, 488 (1952). 4~ M. A. Jermyn and R. Thomas, Biochem. J. 56, 631 (1954). ts~ E. Mitidieri, L. P. Ribeiro, O. R. Affonso, and G. G. Villela, Biochim. el Biophys. Acta 17, 587 (1955). 4sb T. Wieland and G. Pfleiderer, Angew. Chem. 69, 199 (1957). 49S. Paleus, Acta Chem. Scand. 6, 969 (1952).

24

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[].A]

an incubation period the zones of a c t i v i t y are located. This can be done b y using ultraviolet absorption or a suitable color reaction for either the s u b s t r a t e or the product. I n c o r p o r a t i o n of radioactive isotopes in enzymes or substrates which are studied b y p a p e r electrophoresis greatly facilitates their localization on the paper. This procedure has, for instance, been used for studying the p u r i t y of P32-1abeled hexose phosphates. 50 The fate of intramuscularly injected labeled D F P (diisopropylfluorophosphonate), with special regard to its distribution a m o n g the serum esterases, has also been studied b y this method. 61 The radioactive zones are m a d e visible as r a d i o a u t o g r a p h s or are located b y direct m e a s u r e m e n t or b y a recording a u t o m a t i c scanning device. Techniques for combining p a p e r electrophoresis and p a p e r c h r o m a t o g r a p h y , 1-4,5~ either in one or in two separate operations, 53 are useful in cases of low molecular weight substances. T h e introduction of volatile buffers 54-5s for the first electrophoretic separation has facilitated the subsequent chromatographic procedure 59-6~ which otherwise is a p t to be disturbed b y salt effects. A p p a r a t u s for twodimensional p a p e r electrophoresis is likewise reviewed in the m o n o g r a p h s already mentioned. 1-4 A new construction has recently been described b y Mead. ~ After electromigration in one direction, the p H of the buffer in the p a p e r is changed b y exposure to a certain constant v a p o r pressure of ammonia. Electromigration perpendicular to the previous direction is then initiated at the new pH. F o r special demonstrations p H gradients perpendicular 63 or parallel 64 to the electric field h a v e been introduced; the latter alternative has been used b y H o c h 65 for electrophoretic separations of v e r y dilute protein solutions without prior concentration. 50K. T. Schild and L. Bottenbruch, Z. physiol. Chem. 292, 1 (1953). 51R. Goutier, Biochim. et Biophys. Acta 19, 524 (1956). 59B. KickhOfen and O. Westphal, Z. Naturforsch. 7b, 659 (1952). 53 W. Grassmann, H. Hannig, and M. P15ckl, Z. physiol. Chem. 299, 258 (1955). 54R. Consden, A. It. Gordon, and A. J. P. Martin, Biochem. J. 40, 39 (1946). 55H. Mich], Monatsh. Chem. 82, 490 (1951). 56C. H. W. Hirs, S. Moore, and W. H. Stein, J. Biol. Chem. 195, 669 (1952). 57G. G. F. Newton and E. P. Abraham, Biochem. J. 58, 103 (1954). 5s j. Porath, Nature 175, 478 (1955). 59E. L. Durrum, J. Colloid Sci. 6, 274 (1951). 80G. B. Marini-Bettolo, Gazz chim. ital. 84, 1155 (1954). 61R. J. Block, E. L. Durrum, and G. Zweig, " A Manual of Paper Chromatography and Paper Electrophoresis," p. 379. Academic Press, New York, 1955. 82T. It. Mead, Biochem. J. 59, 534 (1955). 8aH. Michl, Monatsh. Chem. 83, 218 (1952). 84H. J. McDonald and M. B. Williamson, Naturwissenschaften 42, 461 (1955). 85H. Hoch, Science 122, 243 (1955).

[1A]

PAPER ELECTROPHORESIS

25

Different paper electrophoretic techniques have been used for studying interactions among various substances in solution. Grassmann and Hiibner 66 showed the possibilities of the method by demonstrating the formation of weak complexes between dyes by means of their twodimensional method. McDonald et al. 67,6s used a different approach for studying interactions between protein and dyes, and Zilversmit and Hood ~9 developed still another technique for detecting interactions of serum proteins with Y~' and Ca 4~. Maurer and Miiller 7° have clearly pointed out the applicability of labeled molecules and used a most interesting technique for studying different forms of interactions, chiefly among serum proteins. In certain cases, the interaction between, for instance, enzymes and substrates or metal activators may be studied by specially adapted techniques of paper electrophoresis. Differences among a number of competitive inhibitors for a certain enzyme may thus be demonstrated if each of the inhibitors in turn is incorporated in the buffer solution in which the filter paper is soaked and a narrow zone of the purified enzyme is allowed to migrate. The protein zone should be retarded if the inhibitorenzyme complex has a net charge which is less than that of the free enzyme. If the equilibrium between free and bound inhibitor is reached instantaneously, the zone should move as a narrow band; if the equilibrium between the two is reached only slowly, the zone should show a considerable tailing. Among the many different forms of apparatus for paper electrophoresis which have been constructed and which are dealt with in detail in the monographs mentioned, three principal types may be distinguished : for low-voltage gradients, for high-voltage gradients, and for continuous separations. Most of these, at least the recent constructions, are capable of giving satisfactory separations, as judged from published patterns; details of construction are given in the original publications. A discussion of physicochemical aspects in relationship to the design of the apparatus has been given by Svensson. 71 It is felt that a discussion of the factors which favor a good separation might be of more value than a detailed technical manual for the handling of a special apparatus. 66 W. G r a s s m a n n and L. Htibner, Naturwissenschaften 40, 279 (1953). 67 R. H. Spitzer a n d H. J. McDonald, Federation Proc. 14, 285 (1955). 6s H. J. McDonald, E. W. Bermes, a n d R. H. Spitzer, Federation Proc. 14, 733

(1955). 69 D. B. Zilversmit and'S. L. Hood, Proc. Soc. Exptl. Biol. Med. 84, 573 (1953). 70 W. Maurer a n d E. R. Miiller, Biochera. Z. 324, 255 (1953). 7, H. Svensson, Ciba Syrup. on Paper Electrophoresis, London p. 86 (1956).

26

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[1A]

Low-Voltage Apparatus It is generally accepted that good separations are easily obtained in appratus of the moist-chamber type with the paper either horizontally 72,73 stretched or more or less vertically TM supported. Most factors to be discussed are relevant to both types of apparatus, and in all types gradients of conductivity and pH are established after a certain time, which are due to evaporation. Each type of apparatus has its own parameters as to time, voltage, ionic strength, etc., for obtaining optimal resolution in a given case.

FIG. 1. Schematic drawing of a paper electrophoresis apparatus.

Series of duplicate analyses of protein solutions, such as serum, give very similar results with both types of apparatus, however. 75-77 With certain filter papers it is difficult to avoid a considerable depression in the paper when it is horizontally stretched in the moist chamber, and in these cases it may be advantageous to support the paper on rows of plastic pins. ~,Ts Figure 1 is a schematic drawing of an apparatus incorporating some features which have been found advantageous when working with the two types of apparatus quoted. ~7,7s The even placing of a moist and fragile sheet of filter paper (A) in the electrophoresis chamber (B) is facilitated by temporarily removing the two outer vertical walls (C). The two electrode vessels (D) are filled and leveled by means of a siphon well above the wall which divides each electrode vessel into two compartments, one of which contains a horizontal electrode (E). To keep the 72 W. Grassmann and K. Hannig, Z. physiol. Chem. 290, 1 (1952). ,a T. Wieland and K. Dose, Biochem. Z. 325, 439 (1954). 74 F. G. Williams, E. G. Pickels, and E. L. Durrum, Science 121, 829 (1955). 76 W. P. Jencks, M. A. Jetton, and E. L. Durrum, Biochem. J. 60, 205 (1955). ,6 W. Grassmann and K. Hannig, Klin. Wochschr. 32, 838 (1954). 7~ C. B. Laurell, S. Laurell, and N. Skoog, Clin. Chem. 2, 99 (1956). ~8 E. Valmet and tt. Svensson, Science Tools 1, 3 (1954).

[1A]

PAPER ELECTROPHORESIS

27

supporting plane of the electrophoresis chamber even, a strong metal plate (F) is permanently screwed the chamber (B). The moist chamber is closed by a lid (G) which fits tightly b y means of a v a c u u m seal (H). I t is i m p o r t a n t to have the supporting plane horizontally adjusted and to avoid draft and direct sunshine on the electrophoretic chamber. The structure of the paper, the a m o u n t of buffer solution and other non-conducting solutes held in the paper, the temperature and the electric current, and any liquid flow through the paper are all factors which in an ideal paper electrophoretic separation should remain constant during the experiment. 71,n These conditions are v e r y seldom fulfilled, but knowledge of how the factors influence the separations is essential for understanding why different types of apparatus m a y produce different separation patterns and also for understanding why different sites of application of the zone m a y produce patterns with unequal resolution even if all other factors remain constant. Structure of the Paper. W h a t m a n No. 1, n W h a t m a n 3MM, 75 Schleicher and Schuell 2043aM, n Schleicher and Schuell 2043a and b, 76 and Macherey and Nagel No. 819 76 have all been recommended for paper electrophoresis. For preparative purposes, thick filter papers, such as Munktell No. 20 79,80 or Schleicher and Schuell 2230 sl are used. T h e latter paper seems to adsorb al-lipoprotein far less than does W h a t m a n No. 1. Recently, Gedin 82 has found t h a t W h a t m a n No. 54 adsorbs less lipoprotein from serum than W h a t m a n No. 1. Differences which are not easily predieted are thus found among different papers, and it is advisable to test the q u a n t i t y of substance adsorbed with different papers prior to electrophoresis. This can be done in a simple chromatographic experiment. In m a n y cases the small n u m b e r of carboxyl groups in the paper m a y account for the adsorption; proteins are generally adsorbed less when they are negatively charged. 8~ In m a n y types of apparatus the filter paper is used in form of strips, but in other constructions there are facilities for applying the paper in form of sheets. T h e latter arrangement makes it easier to compare directly two substances migrating in the same paper under identical conditions. Buffer Solution. A sheet of filter paper is dipped into the buffer solution, and the excess moisture is allowed to drain. I t is better to have the paper a little too d r y rather than too wet when it is placed for equilibra79H. G. Kunkel and R. J. Slater, J. Clin. Invest. 31, 677 (1952). 80A.-B. Laurell, Acta Pathol. Microbiol. Scand. Suppl. 103, 18 (1955). 8xH. E. Schultze and H. Biel, Behringwerkmi~leilungen 30, 74 (1955). 83H. Gedin, unpublished observation. sa A. Tiselius and P. Flodin, Advances in Protein Chem. 8, 471 (1953).

28

TECHNIQUES FOR CHAR~kCTERIZATION OF PROTEINS

[1A]

tion in the m o i s t chamber. I n certain t y p e s of a p p a r a t u s it has been found an a d v a n t a g e to leave the a p p a r a t u s with the current on for some hours before the sample is introduced, b u t as an a l t e r n a t i v e the filter p a p e r can be dipped in a buffer with a b o u t 4 0 % higher ionic strength t h a n the buffer in the electrode vessels. ~7 Buffers with ionic strength less t h a n 0.1 are m o s t l y used. T h e i m p o r t a n c e of choosing n o t only a suitable p H b u t also appropriate buffer salts in a t t e m p t s to separate, for instance, an e n z y m e f r o m impurities is d e m o n s t r a t e d in Fig. 2. T h e figure shows the stained protein

FIa. 2. Paper electrophoresis of crude and purified enolase. A crystalline preparation and a crude fraction, both with enolase activity, were subjected to electrophoresis on filter paper (Whatman No. 1) in two different buffers at pH 8.4, ionic strength = 0.05, 130 volts, 2 ma, for 15 hours. To left, the stained pattern in phosphateborate buffer; right, in barbital buffer. Both cells were allowed to run for 3 hours before the samples were applied. The same amount of the two preparations were used in both eases. The two samples were kindly supplied by Dr. B. MalmstrSm. See text for explanation. p a t t e r n s f r o m two parallel experiments with p h o s p h a t e - b o r a t e and barbital buffer of p H 8.4 and ~ = 0.05. A crystalline p r e p a r a t i o n with enolase activity, shown in the ultracentrifuge to consist of only one b o u n d a r y , except for a 5 % impurity, was used in b o t h experiments. T h e stained p a t t e r n to the left represents an earlier stage in the purification of the enzyme. 84 M a r k e d discrepancies are shown between the resolution of the p a t t e r n s in the two buffer solutions. T h e crystalline enolase preparation in b o t h buffers separates into two components, b o t h of which h a v e enolase a c t i v i t y according to Dr. M a l m s t r S m ' s findings. T h e i m p u r i t y which, according to the centrifuge diagrams, should a m o u n t to 84 Both preparations were kindly supplied by Dr. B. MalmstrSm.

[1A]

rAPZR ELECTROPHORESIS

29

5 % is clearly shown in the original stained patterns. At least six components can be detected in the crude enolase preparation, judging from the stained diagram which was separated in phosphate-borate buffer. Such a separation is not achieved with the barbital buffer. The importance of choosing the appropriate buffer is demonstrated if an a t t e m p t at isolating any of the fast-moving components is to be carried out with zone electrophoresis in columns. For minimizing influences due to oxidation during the migration in the paper of a protein zone, the moist chamber has been filled with an inert gas 8~ such as helium which at the same time increases the heat conduction about five times. Oxidation of SH- groups in cysteine m a y occur during the conditions prevailing in paper electrophoresis and might to some extent be prevented b y adding NaHSO~ to the buffer. 8~ Higher yields of alkaline phosphatase activity is obtained, however, if 0.004% H~O2 is added 86 to the buffer solution. Studies of redox systems involved in inactivation of enzymes m a y thus be i m p o r t a n t for retaining activity during the separation. Sucrose has been added to buffers of low ionic strength for preventing globulins from precipitating, s~ Evaporation. In all moist-chamber apparatus, uncontrolled evaporation more or less limits the strength of the electric field which m a y be used in the paper. The evaporation causes a flow of buffer solution from both electrode vessels into the paper. As a consequence of the evaporation, the buffer solution in the paper becomes more concentrated, which in turn leads to an inhomogenous electric field distribution in the paper. 88 Macheboeuf et al. 8~ have used a special apparatus which permits excessive evaporation for demonstrating a phenomenon of dynamic equilibrium called " e l e c t r o r h e o p h o r e s e " which has been used, for example, for studying dilute solutions. E v a p o r a t i o n m a y be diminished or suppressed if an appropriate a m o u n t of relatively inert substances 78,9°,91 such as glycerol or ethylene glycol is added to the buffer. In the apparatus shown in Fig. 1, strips of filter paper soaked in distilled water are placed on B, without contact with A. 85H. J. McDonald, in "Ionography, Electrophoresis in Stabilized 'Media," p. 52. Year Book Publishers, Chicago, 1955. s6 H. E. Schultze and H. Biel, Behringwerkmitteilungen 30, 92 (1955). s7 H. Laurell, Ciba Syrup. on Paper Electrophoresis, London, p. 123 (1956). ss Z. Pucar, Arhiv Kern 25, 205 (1953). s9 M. Macheboeuf, P. Rebeyrotte, J-M. Dubert, and M. Brunerie, Bull. soc. chim. biol. 35, 334 (1953). 90E. L. Durrum, J. Am. Chem. Soc. 73, 4875 (1951). ,t H. J. McDonald, J. Chem. Educ. 29, 428 (1952).

30

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[1A]

Application of the Sample. To obtain good resolutions even of minor c o m p o n e n t s it is advisable to a p p l y 8 to 10 ~l. of a clear b u t concentrated (2 to 4 % ) solution as a thin line onto the paper. This can be done b y m e a n s of special applicators, or b y drawing a pipet sev--7 eral times across the application site, as it is emptied slowly through a v e r y fine orifice. M e t h o d s for sharpening zones in columns 92,~3 m a y be applied to p a p e r electrophoresis. A zone of lower electrolyte concentration m a y be applied behind the initial line b y means of a d r y and a moist thick filter paper. For concentrating dilute protein solutions, m e m b r a n e s according to Mies 94 are recommended. A m e m b r a n e in the f o r m of a tube is suspended in a buffer solution, and a partial v a c u u m of a b o u t 300 m m . H g is applied (~ee Buffer Fig. 3). Depending on several factors, such as the voltage gradient and the e v a p o r a t i o n occurring, a suitable starting line has to be found empirically for each a p p a r a t u s to obtain m a x i m a l resolution. Fixation and Staining. M a n y dyes h a v e been shown useful for staining proteins, b u t for reproducible results it is essential t h a t the stain be a well-defined chemical entity. As is well known, m o s t commercially available stains are mixtures of different components, and one b a t c h usually does not h a v e the same composition as another. A few stains are obtainable in a "chromatographically" pure form, however and a m o n g t h e m bromophenol blue, n a p h t h a l e n e black 12B200 (Amido Black 10B, TM Leverkusen, G e r m a n y ) , and lissamine green 95 h a v e been widely used. A staining m e t h o d which is applicable to amino acids and polypeptides, as well as proteins, is described b y Reindel and H o p p e 2 e As to the staining procedure, a choice m a y be m a d e between certain modifications with due regard to the fact t h a t the cost of the rinse solutions m a y v a r y within wide limits. A detailed investigation of staining with b r o m o p h e n o l blue has been carried out b y D u r r u m . 75 A new modification of G r a s s m a n n ' s Amido Black 10B procedure has

FIG. 3. App a r a t u s for c o n centrating dilute protein solutions. The dilute protein solution is p l a c e d in a tube-formed collodion sack (supplied from Membranengesellschaft, GSttingen) and a pressure of 300 m m . t t g is applied above the buffer solution in which the collodion sack is placed.

99 H. Haglund and A. Tiselius, Acta Chem. Scan& 4, 959 (1950). 98j. Porath, Biochim. et Biophys. Acta 22, 160 (1956). 94H. J. Mies, KEn. Wochschr. $1, 159 (1953). g5 E . M . Abdel-Wahab and D. J. R. Laurence, Biochem J. 60~ xxxv (1955). 96F. Reindel and W. Hoppe, Chem. Ber. 87~ 1103 (1954).

[1A]

PAPER ELECTROPHORESIS

31

recently been described. 73 Although many modifications may be equally satisfactory, the following procedure 77 has been found to give reproducible results and can also be used for semi-quantitative estimations of different fractions. 1. The moist paper sheet is dried at 110 ° for 15 minutes. 2. The dried paper is sewed on a special bent glass rod and suspended for 10 minutes in the staining solution (1% bromophenol blue, 20% w / v HgC12 in methanol). 3. The sheet is transferred and rinsed three times for 10 minutes in 0.5% (v/v) acetic acid (about 6 1. of fresh rinsing solution to each of the rectangular jars). The staining capacity of the bromophenol blue solution is slightly impaired after some months, as its composition is altered by evaporation and the addition of salts. It is thus recommended prior to fixation to apply to the moist paper, with a constriction pipet, 10 ~l. of a 2 % albumin solution as an internal standard. For semiquantitative determinations the paper is cut into pieces corresponding to the fractions seen in the pattern. A piece of paper which has not been in contact with protein fractions is used as a blank. All the dry paper pieces are weighed and transferred to equal volumes of 0.1 N Na2CO3 for elution for 3 hours. The absorbance is read at 575 rap, and due corrections are made for the absorbance per milligram of dry paper found in the blank. Equally satisfactory results may be obtained by direct scanning of the filter paper with the dyed zones, if the apparatus has been properly calibrated.

High-Voltage Apparatus and Apparatus for Continuous Separations Paper electrophoresis apparatus with high-voltage gradients have chiefly been used in connection with separations of low molecular weight substances, and striking results have been obtained. For details the reader is referred to Werner and Westphal2 7,* As to paper electrophoresis apparatus aiming at continuous separations, several modifications 98 have appeared since the pioneer work of Svensson et a l 2 ~ and Grassmann and Hannig. 1°° Both Grassmann's apparatus (Bender & Hobein, Munich) and a construction by Durrum (Spinco Co., Belmont, California) are commercially available. Besides many obvious applications they are most useful in cases where adsorption tends to blur the obtained separations. 97 G. Werner a n d O. Westphal, Angew. Chem. 67, 251 (1955). 9s E. S. Holdsworth, Biochem. J. 59, 340 (1955). 99 H. Svensson and I. B r a t t s t e n , Arkiv Kemi 1, 401 (1949). 100 W. G r a s s m a n n and K. Hannig, Z. physiol. Chem. 292, 32 (1953). * See also 48b.

104

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[3]

[3] Infrared Spectrophotometry By D. L. Wood

Introduction The energy of a molecule, according to quantum theory, is not allowed to have a continuum of values. Rather it is required that the total energy at any instant have one of a set of discrete characteristic values. The molecule may change its energy by the absorption or emission of a photon or by collision, but it may only change from certain allowed energy levels to certain others according to well-established selection rules. The absorption or emission of radiant energy which accompanies a molecular energy change is possible only if the radiated or absorbed photon has an energy which exactly fits the difference between two energy levels of the molecule. Thus in absorption or emission a discrete spectrum is observed, made up of radiant energies fitting the allowed energy changes for the particular molecule. Most molecular spectrophotometric studies are concerned with absorption of radiant energy, and consideration will not be given here to infrared emission spectra. Therefore, the term spectrum in this discussion means a plot of the discrete energy absorbed by a sample as a function of the frequency or wavelength. The energy of a molecule is usually considered to be a sum of three terms: one from the energy of motion of the electron cloud, one from the vibrational motion of the constituent atoms, and the third from the energy of pure rotation of the whole molecule. Although some electronic transitions are observed in the infrared, most of the spectra under discussion arise from transitions between vibrational and rotational energy levels. Furthermore, in liquids and solids, where molecular rotation is hindered or impossible, only the vibrational part of the energy remains quantized. Thus, except in the case of gases, the infrared spectrum arises solely from changes in the energy of vibration of molecules. The vibrations of a molecule and the energies associated therewith are very sensitive to the strength of chemical bonding and the particular configuration of atoms comprising the molecule. This strong dependence of the spectrum on even the smallest structural detail makes infrared spectrophotometry a useful tool. The following applications illustrate how the sensitivity of the spectrum to composition, structure, configuration, and environment gives the method widespread utility. 1. It is obvious that if the infrared spectrum is highly characteristic of a particular molecule it may be used in the identification of that molecule in a mixture in much the same way that a fingerprint may be used to

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identify an individual in a group of people. For example, even stereoisomers like trans- and gauche-l,2-dibromoethane ~ have infrared spectra sufficiently different to enable one to distinguish between the two forms. Thus infrared spectra are widely used in characterizing unknown pure compounds and in identifying the components of mixtures or solutions. 2. This is also true of individual groups or chemical bonds in a molecule so that the presence of these groups or bonds in a molecule of unknown structure may be detected by their characteristic absorption bands, and thus considerable information about the molecular structure can be obtained. 3. These aspects of qualitative analysis can also be made quantitative, giving a convenient method for quantitative analysis which is extremely useful, since the sample is not destroyed and only a very small quantity of sample is required. 4. A knowledge of the energy levels of a molecule allows the computation of various thermodynamic data 2 so that the infrared spectrum can be useful in the theoretical side of chemistry. 5. The molecular differences in members of homologous series, the effect of environment on molecular structure (e.g. solvent effects), and the progress of chemical reactions may all be followed by this technique. It is safe to say, therefore, that any change in a molecule, however slight, can usually be studied by means of the accompanying change in the infrared spectrum. Procedures Infrared Absorption Measurements. The recording of spectra for molecular structure studies or analysis involves two phases: the sample preparation, and the measurement of energy absorbed as a function of frequency or wavelength. With regard to the energy measurements the operating procedures usually are described in the manufacturer's instruction book, and these instructions make clear the proper choice of recording time, slit width, amplifier gain, and detecting system response time to give the best possible spectrum for the particular sample. Operating procedures, therefore, will not be discussed as far as they pertain to the recording of data. Useful information supplementing that of manufacturers' handbooks will be found in the articles by Clark 2a and Miller. 2b

J. K. Brown and N. Sheppard, Discussions Faraday Soc. 9, 144 (1950). G. Herzberg, "Infrared and Raman Spectra." Van Nostrand, New York, 19~5. 2~C. Clark, in "Physical Techniques in Biological Research" (Oster and Pollistcr, eds.), Vol. 1, pp. 206-316. Academic Press, New York, 1955. 2bF. A. Miller, in "Organic Chemistry" (H. Gilman, ed.), Vol. 4, pp. 122-158. Wiley, New York, 1953.

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The spectrophotometers in common use fall into two classes, either single beam or double beam. In the single-beam type, the energy passing through the sample is simply recorded as a function of distance on a paper chart. In a double-beam instrument, the paper chart record presents the ratio of energy passing through the sample to the energy passing through an identical optical path with no sample, as a function of frequency or wavelength. The two principal advantages of the double-beam over the single-beam system are that the recording of energy ratios directly in the double-beam system eliminates much tedious processing of data, and that the double-beam system eliminates atmospheric absorption bands. One application of the direct recording of ratios by the double-beam type of spectrophotometer is in the measurement of "differential" or, more properly, "ratio" spectra. 3 Suppose two samples have a common major component but a minor component not in common, and it is desired to record the spectrum of the minor component alone. If the two samples are put into cells of appropriate thickness and one introduced in each beam of the double-beam instrument, the major-component absorption bands will be compensated out, leaving those of the minor component. An obvious example is that of a sample in solution where the solvent absorption may be balanced out, leaving just the solute spectrum. There are difficulties in this method, however, the most important of which is that very strong absorption by the component to be balanced out may render the instrument inoperative over the region of strong absorption. That is, the spectrophotometer requires a certain minimum energy to drive the pen across the chart paper. If this amount of energy is not transmitted by the two samples, the detecting system will have no signals to balance and thus no energy to drive the recording pen. Much information can thus be lost in the very regions in which the method might be expected to be most useful. By opening the monochromator slits or advancing the gain of the detecting system in these regions of strong absorption this effect can be minimized, although the best technique is to use only samples in which the absorptions to be balanced out are not over 80 to 90 % of the incident energy. A special case of "ratio spectra" which has been useful in protein structure work is the recording of dichroism by this method. A sample in which the molecules have been suitably oriented may absorb more strongly when the electric vector of the incident infrared radiation is polarized in some particular direction. This is dichroism, and it gives information about the orientation of the specific molecular groupings or chemical bonds involved in a particular absorption band. If two identical oriented samples are located in the two spectrophotometer beams and 3 D. Z. Robinson, Anal. Chem. 24, 619 (1952).

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the radiation in one beam is polarized at 90° to that of the other, a "ratio spectrum" related to the dichroism of the sample will be recorded. This is particularly useful where the dichroism is weak. The polarization of infrared radiation may be accomplished by the use of reflection4 or transmission polarizers usually made of selenium ~ or silver chloride 6 sheets. The silver chloride transmission polarizers are commercially available. In the choice of ordinate and abscissa for presenting the spectrum, present practice 7 tends toward frequency in cm. -1 (kaiser) as abscissa versus absorbance as ordinate, although wavelength in microns (abscissa) versus % transmission (ordinate) is also widely used. The former is TABLE I PRISM MATERIALS Material Glass *Quartz LiF *CaF2 *NaCI KBr *CsBr KRS-5 (TIBr-TII) CsI

Useful range, era. -~ 8000-12,500 4000-12,500 1600- 4000 1100- 4000 650- 1500 370700 300700 270700 195400

preferable, since line widths are not distorted, although the linear wavelength abscissa gives a more linear representation of the actual resolving power throughout the range of a single prism. Ideally, however, more than one prism is used to cover the whole spectrum, and in this case a more uniform resolving power may be obtained on the linear frequency scale. There is a wide choice of prism materials now available, the most common of which are listed in Table I along with their most useful spectral ranges. The four noted with asterisks make a convenient set giving maximum resolving power throughout the spectrum. In actual practice most spectra are recorded with the NaC1 prism, and the others are used in supplementary experiments. It is possible to use any prism at frequencies higher than the limits indicated in the table, although there will usually be another material available which gives higher resolving power in that region. 4 A. H. Pfund, J. Opt. "Soc. Amer. 37, 558 (1947). A. Elliott, E. J. Ambrose, a n d R. B. Temple, J. Opt. Soc. Amer. 38, 212 (1948). 6 R. N e w m a n a n d R. S. Halford, Rev. Sci. Instr. 19, 270 (1948). 7 W. R. Brode, J. Opt. Soc. Amer. 39, 1022 (1949).

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For very high resolving power an echelette diffraction grating can be used as dispersing agent, and recent developments in instrumentation 7a have brought this type of equipment within the means of most infrared laboratories. The limit of resolution of a grating spectrophotometer is well below 0.5 cm. -1, whereas the limit for a prism spectrophotometer in the most favorable regions of the spectrum is about 2-3 cm. -1. One of the time-consuming operations in single-beam spectrophotometry is the reduction of the records of background, sample, and zero energies versus arbitrary frequency calibration units to graphs of absorbance versus frequency. There are several ways of doing this, some of which involve modification of the recording system. 8-I~ A common practice is to use a mechanical device to compute the ordinate ratios for various frequencies located by means of a paper or plastic strip abscissa calibration.1~ Such mechanical devices are usually based on the fact that one leg of a right triangle can be divided into a number of equal parts by drawing lines parallel to the other leg through equidistant points laid out on the hypotenuse. From the same hypotenuse, any number of shorter lengths can be so divided by constructing the appropriate right triangle and drawing in the lines parallel to the base. 12 S a m p l e Preparation. The proper preparation of infrared samples is of in~portance because the spectrum may be different for different procedures and therefore may affect interpretations. The preparative procedures involve the confining of the sample to a layer with two parallel fiat faces through which the radiation may pass, and for many samples this may be done without complication. Gases are usually confined in cylindrical glass or metal tubes with infrared transmitting windows sealed on the ends with a suitable gasket, wax, or cement. Appropriate outlet and inlet tubes usually are of glass with stopcocks and either glass taper joints or glass ball and socket joints for connecting with the gas handling system. The usual length for pressures of 5 to 76 cm. Hg is 10 cm., though shorter (5 em., 2.5 cm.) and longer (up to 10 meters) 13are sometimes useful. The gas pressure and cell length may be adjusted to suit the sample, if it is remembered that higher pressures broaden the infrared bands. It is sometimes desirable to raise the temperature of the cell to raise the vapor pressure of the sample, and v~R. C. Lord and T. K. McCubbin, Jr., J. Opt. Soc. Amer. 46, 441 (1955). 8 C. E. Zerwekh, Rev. Sci. Instr. 20, 371 (1949). 9 R . M . Fuoss and D. J. 5Mead, Rev. Sci. Instr. 16, 223 (1945). 10R. A. G. Carrington and J. G. Reynolds, J. Sci. Instr. 29, 197 (1952). 11R. W. Foreman and W. Jackson, Jr., Instruments 22, 497 (1949). 12H. A. Willis and A. R. Philpotts, Trans. Faraday Soc. 419 187 (1945). 13j. U. White, J. Opt. Soc. Amer. 32, 285 (1942).

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this m a y easily be done electrically. M a n y low-temperature cells have been described, and these m a y sometimes be used to good advantage. TM There is a wide choice of window materials available for infrared cells. T h e choice is usually made on the basis of the transmission limit and chemical or physical stability. The properties of several materials are listed in Table II. The most commonly used materials are: For the highfrequency region, fused quartz. For the region from 2800 to 400 cm. -1, TABLE II WINDOW MATERIALS

Material Glass SiO2 LiF CaF2 BaF2 As~S~ Se

Useful frequency Water range, cm. -1 stability Down to to to to to to to

3000 2800 1600 1000 900 800 600

NaCl KBr AgCl

to 600 to 400 to 400

CsBr KRS-5(T1Br-TII) CsI Polythene

to to to to

250 250 190 100

Remarks

Excellent Excellent Good Good Excellent Good. Excellent

1.0 mm. thick Strong, durable Cleaves, brittle Cleaves, brittle Cleaves, brittle A deep-red glass, soft Amorphous form only; opaque in visible Poor Brittle, fogs easily Poor Brittle, soft, fogs easily Good Soft, plastic, photosensitive, chemically unstable Poor Plastic, fogs easily Good Slightly plastic, deep red, poisonous Poor Fogs easily Excellent Sheets, absorption bands present

NaC1 or K B r for samples without water, and AgC1 for samples containing water. For the low-frequency region CsBr is good where water is not present, but KRS-5 is b e t t e r where water is present. The other window materials included in the table are useful in special cases. Although windows of these transmitting materials may be obtained commercially, it is often desirable to make or refinish windows, especially those of the water-soluble crystals like NaC1, KBr, CsBr, and CsI. The techniques involved are not difficult, and detailed procedures are given in ref. 14. The AgC1 must be protected from photodegradation, particularly by fluorescent lights. Incandescent lighting requires considerable time (days) to cause objectionable decomposition. More serious than this is the problem of keeping all metals below Ag in the electromotive series away L4R. C. Lord, R. S. McDonald, and F. A. Miller, J. Opt. Soc. Amer. 42, 149 (1952).

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from contact with the AgC1. The decomposition by replacement of the Ag by the metal is very rapid. Silver chloride windows may be polished or flattened by pressing between glass plates in a hydraulic press, or rolling between highly polished rolls. They can also be worked into special shapes by pressing or extrusion and welded together with an incandescent electrically heated platinum wire loop. Cells for holding liquid samples are usually much thinner than those for gases (of the order of 0.1 mm.). Their construction is the same in principle as those for gaseous samples but with shorter spacers. Appropriate thicknesses can be accomplished with shims of lead amalgam or other suitable gasket materials, or by a more elaborate construction involving a screw adjustment for variable thicknesses. 1~-17 The filling and emptying of the cell is accomplished through holes drilled in the windows or shims and connecting with hypodermic syringe adapters. The handling of liquid samples in syringes is very convenient. The thickness of an infrared liquid cell can be measured in any one of several ways. Probably the best method requires measuring the chanael spectrum ~s of the empty cell. Because there is interference between rays passing through the cell with rays reflected twice at the two interfaces of the cell windows, there will be a series of maxima and minima in the spectrum of the empty cell. The separation of two successive maxima in terms of wavelength will give the cell thickness, t, according to the formula t = ~ k ~ / 2 ( ~ 1 - ~ ) where ~ and ks are the wavelengths of the two successive transmission maxima. Other methods involve direct measurement with a micrometer, weighing full and empty, the use of known absorption coefficients19 (e.g. benzene in the ultraviolet), and the use of Talbot's fringes, 19 or of visible interference fringes. A great deal of work with liquid samples is done with solutions of compounds. It is important, of course, that the solvent have no absorption bands in the regions where the sample absorbs. The best solvent, therefore, is the one with fewest absorption bands. Carbon tetrachloride and carbon disulfide are the two most commonly used, and if the sample will go into solution in these solvents they make a useful pair, since each is clear of absorption where the other has bands. For convenience in choosing solvents a chart prepared by the Spectroscopy Laboratory at the Massachusetts Institute of Technology is shown in Fig 1. One other 15j. U. White, Rev. Sci. Instr. 211 629 (1950). 19A. V. Stuart, J. Opt. Soc.,Amer. 43, 212 (1953). 17V. J. Coates, Rev. Sci. Instr. 22, 853 (1951). 18D. C. Smith and E. C. Miller, J. Opt. Soc. Amer. 34~ 130 (1944). 19W. C. Price, Trans. Faraday Soc. 41, 189 (1945).

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useful solvent not included on the chart and whose properties make it desirable for solid sample preparation is hexachlorobutadiene. 2° Besides considering the absorption of a solvent one also should consider the effect it may have on the molecule and therefore the spectrum of the solute. The effects of solvents on the spectrum are discussed later in the chapter, and it is well to bear in mind that such effects may be observed. The preparation of gaseous and liquid samples is relatively straightforward compared to that of solid samples. For convenience the preparative techniques for solids may be divided into those applicable to particulate samples and those applicable to continuuus samples. Particulate samples will not in general yield good spectra if the powder alone is placed in the radiation path of the spectrophotometer. The scattering of the radiation by the particles in the sample render it essentially opaque in much the same way as with visible light. The scattering is avoided, however, if the powder is mixed to a paste or slurry with some liquid having nearly the same refractive index as the sample grains. It is convenient if the liquid used in making the paste or mull has enough viscosity to prevent settling of the grains in the cell. It is also desirable, of course, that the mulling liquid have as few absorption bands as possible. It is common practice to choose complementary liquids, the one being free of absorption where the other absorbs. The most commonly used pair is mineral oil (Nujol) and hexachlorobutadiene, although Nujol and Fluorolube or Fluorokerosene are also a successful combination. Since powder samples are not continuous, the radiation may pass unabsorbed between the grains of the mull if the particles are not small compared to the thickness of the layer. For this reason it is desirable that the particle size be as small as possible. A specific procedure for powder sample preparation consists of several steps. First the sample is ground to as fine a particle size as possible in a mortar, ball mill, or air jet mill. Second, a specific amount is weighed out into a small mulling mortar and ground briefly with a drop or two of the liquid mulling agent. Third, the paste is transferred to a suitable infrared window and squeezed into a uniformly thin layer with a second window. The sandwich of two windows with the mull between is clamped in a cell holder and is ready for the spectrophotometer. The matter of proportions of powder and mulling liquid will be dictated by the results, it being necessary to adjust the flow properties and thickness of the mull to suit the circumstances. The order of magnitude of sample size required is 5 to 10 mg./cm. 2 of window area, depending to some extent on the efficiency of transfer of sample from the mulling mortar to the window. 20 j . S. Ard, Anal. Chem. 25~ 1743 (1953).

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A convenient mulling mortar may be made in the form of a hemispherical cup of Pyrex glass about 6 ram. in diameter with a closely fitting spherical Pyrex pestle ground in with No. 240 Carborundum. A small nickel spatula is convenient for transferring the mull from mortar to window. Another recently introduced method of preparing powder samples is a useful supplement to the mulling method. This is the pressed KBr pellet technique first proposed by Sister M. M. Stimson ~1 and independently by Scheidt. ~ In this method the sample powder is mixed with finely divided KBr powder and pressed in an evacuated die under rather high mechanical pressure until a homogeneous transparent plate is produced. Since pure KBr has no absorption at frequencies higher than 400 cm. -1, the spectrum of the plate will be that of the powder sample. It is obviously a great advantage to reduce scattering without introducing absorption bands due to the suspension medium. Dies appropriate to the method are commonly 0.5 inch in diameter and cylindrical with highly polished ends on the plungers. An exhaust port is provided to remove the air from the powder before compression, and "O"-rings are used to seal the plungers. The hydraulic press may be of modest capacity, 10 tons total force on a 0.5-inch-diameter die being sufficient for pressing times of the order of minutes. Both dies and press are commercially available, or they can be made in a well-equipped machine shop. The specific procedure for making a KBr pellet sample 12 mm. in diameter involves the following steps: The KBr powder which has been previously dried at 120 ° for 4 hours, ground, and sieved through a No. 200 mesh screen is weighed out with the sample to make a mixture of 0.1 to 0.5 % of sample in the KBr. The powder mixture is ground in a mortar with acetone to dryness, with care taken to avoid water condensation due to the cooling of the evaporating acetone. About 200 mg. of mixture is put into the die and evacuated. After the 10 tons of force has been applied to the plunger for 1 to 5 minutes, the pellet is removed from the die and is ready for the spectrophotometer. The pellet should be transparent to visible light and without cracks. Opaque spots may be due to moisture contamination, low pressures, or poor evacuation of the die. It is essential that the sample be finely divided for the same reason that mull powders should be finely divided, namely, that radiation will pass between particles unabsorbed if the particle size is not small compared to the thickness of the plate. It has been reported 23 that, the smaller 21 M. M. Stimson, J. Am. Chem. Soc. 74, 1805 (1952). 22 U. Scheidt and H. Reinwein, Z. Naturforsch. 76, 270 (1952); U. Scheidt, ibid. 78, 66 (1953). 2~Perkin-Elmer Instrument News 4, No. 3 (1953).

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the particle size, the better will be the sharpness and separation of absorption bands in pressed KBr samples. Several other methods besides grinding in a mortar have been suggested for rendering the KBr and sample mixture more homogeneous. The ball mill and sonic vibrators may be useful, but freeze-drying of KBr-sample solutions in a suitable solvent is probably more effective, though more elaborate. Satisfactory dispersions have also been made by spraying a solution of the sample over KBr powder and drying the mixture. Whatever the method, however, it should be recognized that the final particle size of the powder to be pressed can affect the quality of the spectrum greatly. Other techniques have been developed for measuring spectra of powder samples. In one method 24 ammonium stearate is used as a solubilizing or dispersing agent for producing stable suspensions of powders in CS2 or CC14. The ammonium stearate has but one more band than mineral oil and has wide regions of no absorption. Another method 2~ reduces scattering by reducing the particle size to considerably less than one wavelength of the absorbed radiation. The powder is ground to as fine a particle size as possible and suspended in a tall cylinder of alcohol. After settling for a suitable length of time, the top fraction containing the smallest particles is decanted, centrifuged, and decanted again, leaving a concentrated suspension of the finest particles. This suspension is spread on a window and the alcohol evaporated to dryness. The sample is then ready for the spectrophotometer. A third method 26 involves recording spectra not of the material itself but of its pyrolysis products. This has been useful for completely intractible solids, but the interpretation of such spectra may be somewhat involved. Fortunately many solids can be prepared in the form of suitably thin transparent films or plates so that scattering of the radiation is not a problem. Thin films can be made by casting from solution or melt, or by cutting up bulk material. Vacuum evaporation 27 has also proved a useful technique. The two factors of paramount importance are that the film be clear and not scatter the radiation and that it be thin enough for good spectra. The proper thickness may be as small as 0.1 ~ for strong absorption bands. It is likely that many water-soluble proteins as well as polymers and even crystalline compounds will form uniform clear coatings on silver chloride plates if a drop of solution is spread and allowed to dry on the plate. There will be, perhaps, some difficulty in making quantitative 24 M. Dolinsky, J. Assoc. OJ~c. Agr. Chemists 34~ 748 (1951). 26 j . M. Hunt, M. P. Wisherd, and L. C. Bonham, Anal. Chem. 22, 1478 (1950). ~6 D. L. Harms, Anal. Chem. 25, 1140 (1953). 2~ j. E. Tyler and S. A. Ehrhardt, Anal. Chem. 25, 390 (1953).

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measurements on such films unless great care is taken to make the thickness uniform and to determine its magnitude. In some cases the thickness can be followed if the films are first cast on the surface of water or mercury where interference fringes in visible light can be observed. For films thicker t h a n 1 or 2 ~, interference fringes in the infrared m a y be used to determine the thickness b y the same method as for e m p t y liquid cells. Solid films of known thickness can also be prepared b y filling a fixed liquid cell with the melt and allowing it to cool below the melting point. Great care is necessary, however, in heating optical crystal windows because thermal stresses from uneven heating will easily crack such materials. For cutting bulk material or large crystals to make infrared specimens two methods m a y be employed if the material has no convenient cleaYage like mica. Some materials will slice in a microtome sufficiently well to be prepared this way. T h e material is waxed to a wooden block with paraffin or a hard beeswax and rosin mixture and sliced in the usual manner. For hard materials such as fibrous proteins or nylon a knife with a 90 ° cutting angle 2s is v e r y helpful, although the standard knife works better on soft materials. The slice usually rolls up into a tight coil during the cutting, but it can be unrolled easily on the surface of or under water. The water can then be removed in an alcohol b a t h and a n y wax removed by a subsequent bath in xylene. Such a procedure will produce excellent selfsupporting films, 3 ~ thick, of such intractable materials as keratins. The second method for cutting bulk material which the author has found useful requires t h a t a conveniently thin slice be cut with a saw of some kind and the slice cemented or waxed to a glass plate. Optical pitch is a good adhesive. T h e free surface is then worked down with various grades of abrasive paper, either wet or dry, on a wheel or by hand, until the surface is fiat, smooth, and polished. T h e slice is then removed from the glass, turned over, and waxed down again so t h a t the polished face is parallel with that of the glass plate. T h e n the second surface can be worked down until an appropriate thickness is obtained. E v e n the most intractable materials such as the keratins or mineral crystals can be handled this way, although some skill is required for preparing the thinner samples (1 to 30 ~ thick). In m a n y cases where the compound can be easily crystallized in the form of thin plates either from solution or from the melt a great deal more information about molecular structure can be discovered from the spectra, since the molecules in a crystal have certain particular orientations. The growing of large single crystal sections (larger than 5 X 10 ram.) is a very specialized art, but if micro-sampling techniques are available it is often the case t h a t crystals selected from a bulk crystalline sample will be 28 E. J. Ambrose and A. Elliott, Proc. Roy. Soc. A206, 206 (1951).

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ble to the source itself where the dimensions of the radiation beam are smallest. Actually it is the entrance slit of the monochromator or its image which determines the "microcell" dimensions, since the two may be equal for the largest slit width used. This reduces the actual sample size required to the order of 1 X 12 mm., or 24 X 10-8 ml. for a thickness of 20 t~. Such cells are available for most spectrophotometers and are quite convenient. A second reduction may be accomplished by masking the slit height to reduce the necessary sample length. 32~ This can best be done at the entrance slit of the monochromator, since in double-beam spectrophotometers individual masking of sample and reference beams in the sample space usually cannot be done precisely enough to preserve the compensation of one beam with the other. As a result of even a small inequality of masking the atmospheric absorption bands will appear and spoil the record. Masking at the entrance slit avoids the difficulty, since the two beams are superimposed at that point. This procedure reduces the energy passing through the monochromator and results in a loss of resolving power or an increase in recording time. For a reduction by a factor of 4 to 1 × 3 ram. in sample size, however, this objection is not serious for modern instruments, since for liquids and solids the bands are broad enough so that no detail is lost. A similar reduction in sample size may be made in spectrophotometers which are so constructed that a pair of lenses of infrared transmitting material (e.g., AgC1) located one focal length each side of the sample will produce a reduced image of the source (or more important the slit) so that very small samples may be used2 ~ This arrangement makes possible spectrophotometric measurements of samples about 1 X 3 ram. in size without loss in resolving power. The ultimate in small sampling is accomplished by means of a "microspectrometer" attachment, one model of which has recently been made available commercially. 3~ One arrangement of such an attachment is in effect a microscope with totally reflecting condenser and obiective so arranged that the enlarged image of the sample, illuminated by an infrared source, falls on the entrance slit of the monochromator of the spectrophotometer. The radiation from the sample image on the monochromator slit has passed first through the very tiny sample and thus the spectrum is recorded. With such an attachment, spectra have been recorded from fibers about 17 t~ in diameter, 34 though the normal sample size is about 650 × 220 u with a sacrifice in resolving power of considerably ~" D. L. Wood, Rev. Sci. Instr. 26, 787 (1955). 33 D, H Anderson and O. F. Miller, J. Opt. Soc. Amer. 4-% 777 (1953). 3~ V. J. Conies, A. Offner, and E. H. Siegler, J. Opt. Soc. Amer. 43, 984 (1953).

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less than a factor of 2. If a sample could be confined to the small area of the normal sample size it would have a volume of about 3 × 10-7 ml. for a 20-~ thickness. The advantages of such small sampling volumes are obvious.

Interpretations Band Assignments, Qualitative Analysis, and Structure Determination. The infrared region from 10,000 to 250 cm. -1 which embraces the present convenient limits of prism spectrophotometry is divided into two parts according to the origin of the absorption bands found in each part. The low-frequency region (250 to 3700 era. -1) contains primarily the bands d u e to fundamental vibrations, whereas the high-frequency region (3700 to 10,000 cm.-0 contains bands due to overtones or combinations of vibrations whose fundamentals are in the low-frequency region. Although the high-frequency region shows promise of being very useful, 84a,b,° most of the data available on infrared assignments is concerned with the low-frequency fundamentals. The fundamental vibrations giving rise to infrared absorption may be divided into four classes: stretching, or valence vibrations; deformation, or bending vibrations; group vibrations where only the atoms in a particular chemical group are involved in the vibration; and the vibrations of the molecule as a whole. Not all the vibrations of a molecule produce infrared absorption, since the absorption requires a dipole moment change, and the dipole moment may remain constant in certain cases even though the molecule is vibrating. The rules governing this matter are called selection rules, and they can be derived from consideration of the symmetry of the molecule. ~ The stretching, deformation, and group vibrations and their characteristic infrared absorption frequencies are really special cases of vibrations of the molecule as a whole. Only in special cases is it possible to separate and assign a normal vibration to a particular part of the molecule. Indeed, this is never completely true, since the motion of one atom in a molecule will surely have some effect on the motion of every other atom in the molecule. It happens in these special cases, however, that the vibrational motion of one group has only a small effect on those of other atoms in the molecule, and in this sense belongs to that particular group. In other words, although all oscillations belong to the molecule as a whole, in some special cases the motion or more properly the energy of the vibration is not distributed uniformly over the molecule but is 34a W. Kaye, Spectrochim. Acta 6, 257 (1954). 34b W. Kaye, Spectrochim. Acta 7~ 181 (1955). 84~K. T. Hecht and D. L. Wood, Proc. Roy. Soc. A285, 174 (1956).

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concentrated in a particular group. T h e separable frequency is a useful concept, b u t it m u s t be applied with care, since e v e r y frequency involves the whole molecule to some extent. T h e b a n d s in an infrared s p e c t r u m m a y serve to identify certain groups b y the presence or absence of b a n d s assigned to actual vibrations of the group. T h e same kind of identification can be m a d e with bands whose actual vibrations are u n k n o w n b u t which have been found empirically to be associated with certain groups in pure compounds. T h e b a n d s whose exact assignments are known are m u c h more helpful t h a n the empirically correlated b a n d s in structural studies. T h e empirically TABLE I I I CARBON-HYDROGEN VALENCE FREQUENCIES

Type of vibration CH3 CH.CH (tertiary) CH (aromatic)

Frequency 2962 cm. -1 (symmetric) 2926 cm. -~ (symmetric) 2890 cm. -1 3030 cm. -~

2872 cm. -~ (asymmetric) 2853 cm. -1 (asymmetric)

correlated bands are of assistance, however, for analysis and characterization of molecules. I t is possible in the limited space available here to review only rather briefly the large b o d y of information now available 35-3'~ on infrared b a n d correlations and assignments. Consequently only the r u d i m e n t s of b a n d assignment procedure will be discussed and further details m a y be gleaned f r o m the specialized references cited. Of particular interest are the books b y B e l l a m y ~5 and b y Randall e! al. 36 and the chapter b y 5/[iller 37 in G i l m a n ' s text on organic chemistry. Stretching or valence vibrations involving hydrogen are rather high in frequency because of the small mass of the proton. T h e y are essentially separate from other bands and lie in the region near 3000 cm. -1. T h e carbon-hydrogen valence frequencies, some of which are listed in T a b l e I I I , are found in m o s t organic compounds. T h e y are usually close together so t h a t it requires the high resolving power of LiF or CaF2 prisms to record spectra in which these b a n d s can be assigned with confidence. T h e C H b a n d s are not often selected as principal identifying features in a~L. J. Bellamy, "Infrared Spectra of Complex Molecules." Methuen, London, 1953. a6 H. M. Randall, N. Fuson, R. G. Fowler, and J. R. Dangl, "Infrared Determination of Organic Structures." Van Nostrand, New York, 1949. 3~F. A. Miller, in "Organic Chemistry" (H. Gilman, ed.), Vol. 4, pp. 122-158. Wiley, New York, 1953. as N. B. Colthup, J. Opt. Soc. Amer. 40, 397 (1950). a9 R. B. Barnes, R. C. Gore, U. Liddell, and V. Z. Williams, "Infrared Spectroscopy." Reinhold, New York, 1944.

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and nitrogen or oxygen produce absorption bands which are highly characteristic of the bond type because the force constant is higher than that of a single bond. Bands due to double-bond oscillations occur in the region from about 1650 cm. -1 to about 1850 cm. -1. The various environments in which the double bond is found affect its frequency. Thus the acid anhydride carbonyl has a band near 1820 cm. -1, whereas the ketone carbonyl is lower near 1715 cm. -1, and the C ~ N link usually exhibits a band in the region from 1640 to 1690 cm. -1. Carbon-carbon double bonds such as those found in the alkenes also fall in this region, giving bands from 1600 to 1680 cm. -I. It is possible in some cases to differentiate conjugated (near .1600 cm. -1) from unconjugated (1620 to 1680 cm. -1) C ~ C bonds. Triple bonds are equally characteristic in the frequency of their absorption bands. Cyanides, nitriles, and acetylenes all have bands in the region between 2000 and 2400 cm. -~ characteristic of their triple bonds. Few other absorption bands occur in this region, so that these groupings are very easily identified. There are many bands in the spectrum of nearly every compound which do not arise from hydrogenic, double-bond, triple-bond, or group vibrations. These are the vibrations of the molecule as a whole, and because of their origin they are very useful in following subtle changes in molecules. For qualitative analysis or structure determination a procedure may be set up so that in principle any unknown compound or mixture may be identified: 36 First a check of the separable frequencies (hydrogenic, double- and triple-bond, or group frequencies) reveals something about the molecule. Perhaps the fact that many frequencies are not there is most important. Second, the "fingerprint region" is checked with spectra of known compounds until one or a superposition of several will match the unknown spectrum. Again, the consideration of bands missing from the unknown may be just as valuable as consideration of bands present. For both of these steps a correlation chart ~ 9 is very helpful, but in the end it will be desirable to compare the unknown spectrum with that of a pure compound or of a known mixture of pure compounds. It is essential, therefore, to have access to a file of pure compound spectra for qualitative analysis and structure determination. In the past it has been necessary for each laboratory to compile its own file, but this information is beginning to become available from various organizations (e.g., American Petroleum Institute, National Research Council, Samuel P. Sadler & Co.). There are great advantages in the use of punched card sorting for this kind of analysis, ~2 and the commercially available data are tending toward this method of presentation. 42 A. W. Baker, N. Wright, and A. Opler, Anal. Chem. 25, 1457 (1953).

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Since all proteins have similar spectra it is likely that the infrared method will not be useful in qualitative analysis of enzymes, enzyme mixtures, or large peptides. It will, however, be valuable in analysis of mixtures of the smaller molecules such as amino acids 43 commonly employed in experiments with enzymes. Another drawback of the infrared method is its lack of sensitivity in many cases. Although some examples exist where extremely small concentrations can be detected, it is a general rule that the quantitative determination of components present in concentrations of less than 1 to 10% is unlikely, depending on the strength of the absorption bands in question. Auxiliary fractionation or concentration techniques such as distillation or chromatography will, of course, greatly reduce this threshold. 43~ It should be pointed out that in addition to band positions as an index of molecular constitution, recent work has shown that band intensities 44 may also be used for qualitative analysis. This technique has been applied to analysis of carbonyl types in steroids 45 and may also be helpful elsewhere. Two other techniques may contribute to a knowledge of molecular constitution and structure. These involve the use of isotope substitution and dichroie spectra. When an isotope of different mass but identical chemical properties is substituted for an atom in a molecule, the frequencies in which this particular atom participates will change. A common example is substitution of deuterium for hydrogen. Since the mass changes by a factor of 2, there may be a drastic shift of about V ~ in the hydrogen frequencies to the corresponding deuterium frequencies. If the origin of the hydrogenic frequencies is known, then the knowledge of which hydrogens are affected by deuterium substitution may give information about molecular structure. Conversely, deuterium substitution can be used to assign infrared bands to hydrogenic oscillations by noting which bands shift with the substitution. Other isotopes such as N '5 are useful, although the mass effect is much smaller. Dichroic spectra give information about the relative orientations of various parts of a molecule or of the various molecules in a crystal. This comes about because there can be absorption only for the component of the electric vector of the incident radiation which is parallel to the 4aS. E. Darmon, G. B. B. M. Sutherland, and G. R. Tristram, Biochem. J. 42, 508 (1948). 4~ H. M. Randall and D. M. Smith, J. Opt. Soc. Amer. 43, 1086 (1953). ~4D. A. Ramsay, J. Am. Chem. Soc. 74, 72 (1952). 45R. N. Jones, D. A. Ramsay, D. S. Kier, and K. Dobriner, J. Am. Chem. Soc. 74, 80 (1952).

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direction of the dipole moment change characteristic of the particular vibration. If the molecules of a sample are oriented so that corresponding dipole moment changes are aligned in some direction, strong absorption will occur when the electric vector of the incident radiation is parallel with this direction, and no absorption occurs when it is perpendicular. If the direction of the dipole moment change is known for an oscillation of a particular group in the molecule, then the orientation of the group with respect to the rest of the molecule can be determined. This has been applied with success to the peptide NH and carbonyl stretching frequencies in proteins, 46 where those proteins with folded chains can be distinguished from those with extended chains by means of the dichroism of these two bands. It is not always true, however, that the dipole moment change of a given oscillation takes place along a chemical bond, and for this reason care is necessary in interpreting dichroie spectraW With these powerful methods it is possible to extend analytical work beyond structure determination in the chemical sense to the configurational level, as in distinguishing between stereoisomers, in protein structure, or in the location and orientation of molecules in crystals. Quantitative Analysis. The use of infrared spectra for quantitative analysis is based on the validity of the Beer-Lambert law. This law states that if, at some frequency v, I0~ is the incident radiation intensity and I , the transmitted radiation intensity, their ratio will be exponentially related to path length (0, concentration (c), and the extinction coefficient, or power of the sample to absorb at that frequency (av). Thus Iv _ e_.oc,

I0,

or

l o g -I0~ ~ = D~ = a~ct

The quantity D~ is called optical density, or absorbance. If several components are absorbing at a given frequency the total absorbance is the sum of the absorbances of the individual components:

i

i

If, now, the total absorbance of a mixture of n components is measured at n frequencies where the n individual extinction coefficients are known, a set of n equations in n unknowns may be set up and solved for the individual concentrations. For more than two or three components the method is cumbersome in the solution of so many equations, although machine computation may often be available. For rough quantitative 46 E. J. Ambrose, A. Elliott, and R. B. Temple, N a t u r e 163, 859 (1949). 47 R. B. D. Fraser and W. C. Price, N a t u r e 170, 490 (1952).

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incorrect absorbances and thus cause errors in composition determined this way. A second difficulty arises in the determination of the effective thickness of the sample. This can be avoided by including an internal standard in the form of a powder of known extinction coefficient to which all absorbances may be referred. A convenient material will have bands in a region normally free of absorption so that standardization may be unambiguous. An obvious choice on this basis might be a compound containing a triple bond, such as a cyanide or thioeyanate. The exact choice depends more on the handling properties of the standard than its actual composition. A third difficulty in quantitative work with solids, and for that matter in qualitative work also, is the fact that many solids may have several different forms which are common enough so that calibration work may be done with one form and the analysis performed on another. If the spectra of the two forms are slightly different, as indeed in many cases they are, the analyses may be rendered incorrect. Some Additional Effects. It has already been mentioned that solvents may affect an infrared spectrum by shifting or changing intensities of bands. The most common solvent effect occurs with hydrogen-bonding compounds. When molecules associated through intermolecular hydrogen bonding are put into dilute solutions, the bonding is broken and the free molecules predominate. Any band whose frequency and intensity have been affected by the bonding will return to the unbonded values. Such shifts are found, for example, in secondary amides where the bonding formed between NH and CO of neighboring molecules shifts the NH stretching frequency by 160 cm. -~ and the CO stretching frequency by 50 cm. -~. Many other systems show hydrogen bonding and its effects in their infrared spectra. 49 Most solvents act through the change in electrostatic forces between interacting molecules due to the dielectric properties of the solvent. Any interaction effect of an electrostatic nature, therefore, is likely to be affected by solvents and thereby produce an effect on the infrared spectrum. The effects of pH on the infrared spectrum of polyelectrolytes5° and proteins ~,~'~ have been noted in the literature, and the effect of ionization on the spectra of amino acids (zwitterion form) has been reviewed by Sutherland. 4° In the measurement of solid-state samples in the form of powders 49 G. 50 G. 51 H. ~2 H.

B. B. hl. Sutherland, Ann. Rev. Phys. Che~n. 4, 189 (1953). Ehrlich and G. B. B. M. Sutherland, Nature 172, 671 (1953). L e n o r m a n t a n d M. Herisson, Compt. rend. 232, 815 (1951). L e n o r n m n t and E. R. Blout, Nature 172, 770 (1953).

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one often observes an anomalous scattering of the radiation near an absorption band (Christiansen effect). 53 This is due to the fact t h a t scattering power depends on the difference in refractive index between the suspension medium and the particles. Because the refractive index changes rapidly with wavelength near an absorption band (anomalous dispersion), the scattering power does also, giving a sharp inflection on the high-frequency side of the band and a broad tailing off on the lowfrequency side. T h e effect, which m a y obscure structure on the sides of absorption bands, m a y be minimized by improving the match between the particle refractive index and t h a t of the suspension medium. When spectra are measured from aggregates of small crystals as in the case of films cast from a melt, one often observes anomalous intensity ratios t h r o u g h o u t the spectrum. This m a y be due to the fact t h a t some absorption bands are produced by highly oriented dipole moment oscillations, whereas others are due to disoriented dipoles. If in the simplest case the film consists of crystallites one layer deep, then for the oriented dipoles only the component of the incident radiation whose electric vector is parallel to the dipoles can be absorbed, but the component perpendicular passes through unchanged. For the unoriented dipoles the whole radiation can be absorbed. Thus the intensity of bands going with oriented dipoles relative to t h a t of bands going with unoriented dipoles will be less in the crystal aggregate than in fine powders or the melt where effectively all dipoles are unoriented. Another similar case occurs when the crystallites grow in the film with one axis perpendicular to the surface of the film b u t with random orientation around t h a t axis. Any oscillation whose dipole m o m e n t change is along the axis will not absorb, since for all incident radiation the electric vector is perpendicular to the m o m e n t change. This also gives anomalous relative intensities.

Acknowledgments I am much indebted to Dr. G. B. B. M. Sutherland for many helpful suggestions and discussions and for reading the manuscript. I am also greatly indebted to Dr. Norman Wright and the staff of the Infrared Spectrophotometric Laboratory at the Dow Chemical Company for helpful suggestions and discussions, particularly in connection with analytical procedures. Thanks are due to Dr. R. C. Lord of M. I. T. for permission to use the solvent chart of Fig. 1. 5a R. B. Barnes and L. G. Bonnet, Phys. Rev. 49~ 732 (1936).

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[4] X-Ray Diffraction of Protein Crystals B y F. H. C. CRICK

Introduction The X-ray study of protein crystals is a difficult and highly specialized field. Eventually, when the structure of a few proteins has been unraveled, the results will be of vital interest to ensymologists, since they should give information about the spacial arrangement of the "active site" of the enzyme. But meanwhile X-ray methods will have only a secondary interest for enzymologists. The equipment required for X-ray work is expensive and will normally not be available in an enzymological laboratory. This chapter has therefore been written on the assumption that the X-ray work will be carried out in collaboration with a professional crystallographer. It aims to show both the enzymologist and the X-ray crystallographer--who may not be familiar with the X-ray work on proteinsl.2--the kind of information that can usefully be obtained in a fairly short time. A knowledge of normal crystallographic terms and techniques ~ is therefore assumed, but all the special techniques required are described in detail. The most useful information that can be obtained from an X-ray examination of protein crystals is a rather good value for the molecular weight. If crystals of a reasonable size are available, a figure correct to a few per cent can be obtained in a few days. This method has been neglected in the past, and enzymologists might well find it helpful. Occasionally it can be shown that two proteins from different sources have very similar structures, and more detailed studies may, in favorable cases, yield some information on the shape of the molecule. Identification It might be thought that the very detailed X-ray picture of a protein crystal would be like a set of fingerprints and that it would provide a good method of identifying a protein. This is not so, for two reasons. First, the same protein may form, under slightly different conditions, quite different crystals, having radically different unit cells. Polymorphism is 1B. W. Low, in "The Proteins" (Neurath and Bailey, eds.), Vol. 1, Part A, p. 235. Academic Press, New York, 1953. 2j. C. Kendrew, in "Progress in Biophysics" (Butler and Randall, eds.), Vol. 4, p. 244. Pergamon Press, London, 1954. a See, for example, C. W. Bunn, "Chemical Crystallography." Oxford, New York, 1945.

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the rule rather than the exception in protein crystals. With present techniques there is no method of deciding from the X-ray pattern that such different crystals contain the same protein. Second, proteins which are known to be similar, but are in fact different from the point of view of the protein chemist, can form identical crystals and give substantially identical X-ray pictures. 4 This is because the X-ray picture depends mainly on the broad architecture of the protein molecule, and small changes, such as the substitution of one amino acid for another, make such very slight changes to the X-ray intensities that in practice they can hardly be detected. It should be realized, however, that if two "different" proteins (e.g., from different sources) are found to give identical crystals, having very similar X-ray patterns, it is certain that the broad features of the structure of the two proteins are very similar. Such a case occurs, for example, among the myoglobins, in which it is found that crystals obtained from porpoise myoglobin and from three different species of whale give essentially the same X-ray diagrams. 2 Thus in special circumstances protein crystals can give limited information about the identity of proteins. Crystallization • Nothing will be said about the search for the conditions under which crystals can be obtained from amorphous protein, as the protein chemist is familiar with this problem, and there appear to be no general rules to act as guides. It will be assumed that crystals of some sort have already been obtained. Protein crystals must be a certain size, however, if they are to be studied conveniently by X-rays. The optimum is about 0.3 to 0.5 ram. in all directions, though preliminary work can sometimes be done on crystals as small as 0.1 ram., or even smaller. The protein chemist is usually satisfied if his crystals are large enough to be seen in the microscope, so it will often be necessary as a first step to grow larger crystals, especially as crystals as large as 1 ram. are required for very accurate molecular weight determination. There are many ways of doing this, but in essence they all consist in growing as few crystals as possible, and in growing these few rather slowly, so that fresh crystal nuclei cannot easily form. Proteins vary considerably: some may form quite large crystals without any special precautions, whereas others produce such small crystals that considerable effort must be expended to get them large enough. An example of the first is chick lysozyme; with this enzyme from an amorphous precipitate large crystals will often form overnight. Trypsin inhibitor (from bovine pancreas), on the other hand, requires some weeks 4 M. F. Perutz, A. M. Liquori, and F. Eirich, Nature 167, 929 (1951).

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to form adequate crystals. The difference may depend on whether or not there is any marked difference in the solubility of the amorphous and the crystalline material. If fairly large quantities of the protein are available, it is simplest to make up a series of test tubes in which the protein concentration or the solvent composition is varied in very small steps from the conditions under which small crystals are formed. Those tubes, which initially show no crystals, may produce quite large crystals if left for a few days. If little protein is available a better method is to adjust the solvent so that from a concentrated protein solution a very slight precipitate is obtained. This should be carefully filtered to give a saturated solution free from crystal nuclei, and then very slowly concentrated further by evaporation. This can conveniently be done by placing a shallow layer of the solution, say a few millimeters thick, in the bottom of a fiat-bottomed tube. The tube is then sealed with a rubber bung containing a length of capillary (about 2 cm. long and 1 ram. internal diameter) to restrict the rate of evaporation, placed in a constant temperature desiccator, and left for some days or even weeks. If the crystals produced are too small the procedure should be repeated with a finer capillary tube or with a slightly less concentrated solution. Some workers favor the use of seed crystals. Large crystals of ribonuclease have been successfully grown by adding to the lyophilized protein powder a minute amount of crushed dried crystals to act as seeds and then adding solvent. The smaller the amount of seeds, the larger are the resulting crystals. Other methods are: the slow concentration of the protein solution by forcing it through a semipermeable membrane; a controlled and gradual change of temperature (if the solubility varies with temperature) ; or slow salting out by diffusing more salt into the solution through a semipermeable membrane (if alcohol forms part of the solvent, it may be allowed to diffuse in through the vapor phase). It may even be possible to dispense with a membrane and merely place two layers of liquid (one of which contains the protein and the other alcohol or salts) one above the other, the lighter on top of the denser. Crystals may form on the walls of the test tube near the interface. Since these processes are all slow, trouble may be encountered from bacterial growth or from molds, unless the solvent is one which resists such contamination, such as strong salt solutions or alcohol-water mixtures. It may be necessary to pass the protein solution through a bacterial filter and v~ork under sterile conditions. It sometimes happens that, although crystals grow readily, they do so in an unfavorable habit, such as long thin needles or, even worse, as

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hedgehogs of needles. A protein crystal can always be cut, so extreme length is no handicap as long as the smallest dimension is not too small, which is unfortunately often the case when needles form. Whereas the problem of increasing the size of the crystal can be tackled in a rational manner, that of altering the habit cannot be, and the only method is to try empirical alterations in the conditions of crystallization, by varying the pH, by altering the salt used, or the type of alcohol if from alcoholwater mixtures, or by adding small amounts of heavy metals. These may, of course, not only alter the habit but produce a new type of crystal. It is for this reason that the conditions under which the crystallization is carried out should always be carefully reported.

The Nature of Protein Crystals Protein crystals differ from almost all other crystals in containing within themselves a large amount of the solvent from which they were grown, much of which is still in the liquid state. Typically about half the volume of the crystal is solvent. If such crystals are exposed to the air, liquid evaporates and the crystals shrink. This usually causes considerable deterioration of the optical properties of the crystal and the quality of the X-ray pattern. The crystals are often deformed and may split. Such crystals, although still containing some water, are referred to as "dry," and the original crystals, as grown, as "wet." If the humidity is changed in a controlled manner it is sometimes found ~,6 that between the wet and the dry stages one or more "hydration stages" or "shrinkage stages" exist, each of which, like the wet stage, has well-defined cell dimensions and a typical X-ray pattern. The dimensions of the dry stage, which is usually rather disordered, are less precisely defined than the wet stage and may vary a little, depending on the exact method of drying. Because of the liquid state of the solvent molecules inside the crystal it is often possible for small molecules to diffuse into the crystal without disturbing the arrangement of the protein molecules to any appreciable extent. The salt concentration of the crystal may be changed in this way, or, if the crystal is grown from alcohol-water mixtures, the concentration of the alcohol may be altered, or a different alcohol allowed to penetrate the crystal. Even comparatively large molecules, like dyes, can sometimes diffuse into protein crystals, and it would not be surprising if an enzyme inhibitor could be made to combine with the enzyme while the latter was in the form of a crystal. Naturally it may happen that such a combination will change the unit cell somewhat, but it is quite possible that the unit cell dimensions will hardly alter at all. M. F. Perutz, Trans. Faraday Soc. 42B, 187 (1946). 6 H. E. Huxley and J. C. Kendrew, Acta Cryst. 6, 76 (1953).

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Optical Examination The optical examination must be done on the wet crystals immersed in the mother liquor, as the optical quality of the dry crystals is usually very poor. This restriction makes some of the usual optical measurements very awkward, and in practice the only properties measured are those which can be studied with reasonable convenience. It is often difficult, for example, to view a rodlike crystal end on. The infrared and ultraviolet dichroism of protein crystals have been measured in a few cases, but these techniques are outside the scope of this discussion. If the protein is conjugated with a molecule which absorbs in the visible region, the crystal should be examined for dichroismJ

Mounting Crystals To obtain X-ray pictures of a wet protein crystal it is obviously necessary to mount the crystal in an enclosed space. This is usually done by placing it in a thin-walled capillary, made of low-absorption glass, which is then sealed at both ends. Such capillaries, typically 11/~ mm. in diameter and several centimeters long, can be made by heating a suitable glass tube in a flame and then pulling it out very rapidly. For accurate work they should have very thin walls (say 20 ~), but for a preliminary examination thicker walls can be tolerated. Suitable capillaries can be obtained commercially from Paul Raebiger, Berlin-Spandau (British Sector) Franzstrasse 43, Germany. Some workers find it an advantage to coat the inside of the capillary with a hydrophobie film, as this makes for a cleaner mount. It also reduces any effects due to the alkalinity of the glass. A technique for doing this has been worked out by King. 8 To commercial Desicote add 5% watersaturated ether, and dip the capillary into this mixture. This step produces partial polymerization on the glass surface. Then bake in an oven (about 150°) to remove the solvents, and complete the polymerization by immersing the capillary in hot water (100°) for a few minutes. The advantage of this technique is that it removes all traces of HC1, which otherwise may produce a low pH. The crystal is mounted with as little mother liquor in actual contact with it as possible, but a few drops of mother liquor are included elsewhere in the capillary to maintain the correct humidity. The crystal will usually cling to the wall of the capillary of its own accord. The mounting of such crystals is tricky, but the technique can be mastered in a day. The usual method is to insert the crystal, inside a drop of mother liquor, into the capillary, and then manoeuvre the drop until the crystal is in thc 7 M. F. Perutz, Acta Crgst. 6, 689 (1953). 8 M. V King, Acta Cryst. 7, 601 (1954).

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desired region. The excess liquid is then withdrawn (for example, with a special fine pipet), and the crystal is gently pushed with a fine glass fiber into the required orientation on the capillary wall. Finally a further drop of mother liquor is introduced at a distance from the crystal, and the tube is sealed at both ends, care being taken to avoid heating the crystal or the drop of mother liquor in the process. Some workers use black wax (picien) for this purpose. This is unsuitable if there is alcohol in the mother liquor, and in such cases the capillary may have to be sealed with a flame. It is sometimes easier to seal off one end of the capillary before starting to mount the crystal. Whatever method is used, the seal must be perfect or the crystal will dry out. Each experimenter has his own favorite variation on this basic method. 8,9 As protein crystals are often rather soft (though this varies from protein to protein), considerable care must be taken in handling them, but, although the initial efforts are apt to be discouraging, a little experience soon overcomes this. Dry crystals can usually be mounted in the more customary manner by sticking them gently to a fine glass rod. They are often less fragile than wet crystals. Alternatively a crystal may be mounted wet in a capillary, which is left unsealed so that the crystal dries in position, though there is the danger that such crystals sometimes drop off the capillary walls after drying. It is often an advantage to study the same crystal in the wet and the dry state, as this may help to decide which axes of the dry cell correspond to those of the wet. After the studies on the wet crystal have been completed the capillary can be gently cracked, the crystal realigned if necessary, and X-ray pictures taken at intervals until the crystal has become dry and shrinks no further. Typically this will take some hours, or even a day or so.

X-Ray Technique Beyond the problem of mounting, the X-ray techniques employed are fairly standard. As the unit cell is large, much longer exposures are necessary than for smaller organic molecules. Reflections rarely extend to spacings shorter than 2 A., whereas cell dimensions greater than 100 A. are quite common. The region of interest of the reciprocal lattice is therefore nearer the origin than is customary. For this reason a precession camera is a very favorable tool for studying protein crystals. The experimenter should use a fine collimator, ideally little bigger than the size of the crystal, and as small a backstop as possible. A very good arrangement 9 j. Boyes-Watson, E. Davidson, and M. F. Perutz, Proc. Roy. Boc. Algl, 83 (1947).

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consists of a hanging backstop, with some method of adjusting its position, placed 2 cm. in front of the film, as this reduces air scattering near the backstop. Certainly an effort should be made to observe the lowest orders of the diffraction pattern, as important information may otherwise be missed. If only very small crystals are available it may pay, for preliminary work, to reduce the film distance from, say, 6 cm. to as little as 2 cm., as this will decrease the exposure time considerably. It will then usually be necessary to use a very fine collimator, or alternatively a lead pinhole, of about the same diameter as the crystal, mounted immediately in front of the crystal. If this pinhole is tapered (so that it acts both as a pinhole and a guard pinhole combined), diffraction from the lead itself can be avoided. Such pinholes can easily be produced with a little care. The backstop must be correspondingly reduced in size. Naturally the small film distance reduces the accuracy with which the cell dimensions can be measured, but the space group and the approximate dimensions can be obtained without prohibitively long exposure times. Protein crystals, especially when wet, deteriorate with long exposure to X-rays. This deterioration takes the form of a fading away of the X-ray intensities. Typically it occurs after exposure times of about 100 or 200 hours. In obtaining the molecular weight from a dry crystal the accuracy of the method is usually limited by the precision with which the X-ray spacings can be measured, and the experimenter should adjust his technique to try to obtain the greatest possible accuracy. Owing to the disorder in such crystals this cannot be very high, but ___~% on cell dimensions is not impossible in favorable cases, especially if several crystals are measured.

The X-Ray Picture There are two features of protein X-ray pictures, arising out of the small reciprocal spacings, which are worth mentioning although their interpretation is straightforward. A "still," taken with the X-ray beam parallel to an axis of the unit cell, will show, on a flat film, concentric circles of reflections. These are clearly due to the intersection of successive planes of the reciprocal lattice with the sphere of reflection, and from the diameters of such circles the length of that particular axis of the unit cell can be computed. The circles show up because of the dense packing of these planes with reciprocal lattice points. If the axis is mis-set a few degrees the circles become eccentric, and the degree of this eccentricity can be used to estimate the degree of mis-setting. This is the standard

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method for aligning protein crystals, after a preliminary optical alignment. The final adjustment may be confirmed by an oscillation or precession photo of 2 ° or 3 ° amplitude. The second feature is that a nonprimitive lattice can often be recognized at a glance from inspection of the X-ray photograph, say a 5 ° oscillation, because the reciprocal cell being so small, its arrangement can be visualized, and it is often unnecessary to index the reflections and use the well-known rules for systematic absences. In their place the following equivalent rules can be followed: A body-centered reciprocal cell corresponds to an all-face-centered real cell. An all-face-centered reciprocal cell corresponds to a body-centered real cell. A side-centered reciprocal cell corresponds to a side-centered real cell.

Space Group Determination The space group of a protein can usually be determined with a minimum of ambiguity, since the polypeptide chain-the backbone of the protein structure--contains many asymmetric carbon atoms, all of which are believed to occur only in the L-configuration. Because of this, mirror planes, glide planes, and centers of symmetry are impossible. It is therefore well worth while to look for the systematic absences which distinguish screw axes from rotation axes, and the preliminary study of a protein crystal should not be regarded as complete until this has been done and the space group determined. It may not be possible to do this so convincingly for the dry crystal, but in all cases investigated so far the space group of the dry cell appears to be the same as that of the wet. At least one case of pseudosymmetry1° of the wet crystal has been found (in which the real symmetry was P21 but the pseudosymmetry P22~2), and this possibility should be watched for. For this reason it is useful to take precession pictures of the principal zones of the wet crystal, since any pseudosymmetry or other striking features of the X-ray pattern can often be seen at a glance. From the space group and the unit cell volume the volume of the asymmetric unit can be obtained. If the approximate molecular weight 11 (M) of the protein is known (say, to within ___30 %) from other methods, the number of molecules in the asymmetric unit can be calculated. If the molecular weight of the asymmetric protein unit is denoted by A, the volume of the dry unit cell by V (units A.3), and the number of asymmet10 j . C. Kendrew a n d I. F. Trotter, Acta Cryst. 7, 347 (1954). ~ T h e term molecular weight is used t h r o u g h o u t in the loose sense commonly employed.

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ric units in the unit cell by n, we may write a

~

1 V k n

--

.

- -

It is found empirically that k is never far from 1.5, the observed values lying between 1.35 and 1.75. This rule is possible because most proteins have about the same density, and about the same degree of hydration in the dry cell. With k = 1.5, therefore, the number of molecules in the asymmetric unit is the nearest integer, or reciprocal integer, to A / M . The term reciprocal integer is included to cover the case where the asymmetric unit is, say, one-half or one-third the "molecular weight." For example, in horse hemoglobin the molecular weight normally observed in solution is about 67,000, but the asymmetric unit in the usual crystals (space group C2) is half this, 9 showing clearly that the "molecule" consists of two essentially identical halves related by a diad axis. Note that such an internal symmetry of the molecule is not necessarily shown by the space group. Another type of horse hemoglobin crystaP shows an asymmetric unit of 67,000, and there are numerous cases where the asymmetric unit of a protein crystal consists of two or more molecules. The value of k = 1.5 will not, of course, apply to a protein containing a substantial quantity of a nonprotein component, such as ferritin. In such cases more precise methods must be employed, and the density and nonprotein content of the crystals must be measured.

The Implications of Protein Axes In considering the relationship between the molecule in solution and the molecule in the crystal the difference in the implication of screw axes and rotation axes should be realized. In a space group without rotation axes and with one molecule in the asymmetric unit there is no particular association of the molecules of the crystal into small groups. One would not expect, therefore, to find the molecules in the mother liquor forming dimers, for example. If the space group contains a rotation axis, however, say a twofold axis, there is a very real sense in which the molecules of the structure are related in pairs, and it would not be suprising if dimers were found in the corresponding mother liquor. Insulin provides a very pretty example of this. 1~,1~ The usual crystals have the space group R3, which has a threefold axis, and under rather similar pH conditions the molecule is found to have a molecular weight not far from three times the molecular weight of the asymmetric unit of 12,000. At low pH different forms of the crystal are found, one of which has the space group P212121 which 1~ H. Gutfreund, Biochem J. 50, 564 (1952). 13 For a full discussion and references, see J. T. Edsall, in " T h e Proteins" (Neurath and Bailey, eds.), Vol. 1, Part B, p. 717. Academic Press, New York, 1953.

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[4]

contains only screw axes, and it is therefore not surprising that at low pH the molecular weight in solution is found to be 12,000. Thus the space group of the crystal may give a very strong hint as to which condition in solution will show an unaggregated molecule. Notice here, as mentioned earlier, that the fact that the asymmetric unit is 12,000 gives no evidence as to whether the " t r u e " molecular weight of insulin is half this.

Molecular Weight Determination I t will be realized from what has been already said that the molecular weight as determined from the crystal alone may be a multiple of the " t r u e " molecular weight and a multiple or a submultiple of the molecular weight found in solution. This fact will not be referred to again, and in what follows the term "molecular weight" should be read as "crystal molecular weight." A rough value of the molecular weight can be obtained from the volume of the dry unit cell by the empirical rule described earlier and with k = 1.5. Note that this rule seems to apply irrespective of the amount of salt or alcohol in the wet crystal. This rule should be accurate to - 1 0 to + 2 0 % and will often be within - 5 to +10%. There are, in fact, few other methods of molecular weight determination which will give a value as accurate as this from a single simple experiment. To obtain a better value it is necessary to measure the density of the crystal when it is in some well-defined state. This will allow the mass of the unit cell to be obtained from the observed volume. From this figure for the mass must be subtracted the mass of the nonprotein components of the unit cell--namely, the volatile material, such as water or alcohol, and the nonvolatile nonprotein material (such as salt) remaining after drying. The former is found by measuring the percentage loss of weight in taking the crystal from the original state (in which the density can be measured) to the vacuum-dried condition. The latter must then be found by chemical methods. What remains after subtracting these corrections is considered to be the molecular weight of the protein. There are three obvious choices for the "well-defined" state of the crystal: the wet crystal, the air-dried crystal, and the vacuum-dried crystal. Experience has shown 14 that the last of these is not suitable. The effect of vacuum drying is to remove the solvent from the spaces between the protein molecules and to leave holes. If the density of such a crystal is determined (by immersion in a fluid), the fluid may enter these holes, and the observed density may not correspond to the density of the unit cell but be more nearly that of the protein molecule itself. One must therefore start from either the wet crystal or the air-dried crystal. ~4 B. W. Low and F. M. Richards, J. Am. Chem. Soc. 76, 2511 (1954).

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137

At the moment of writing it is not clear which of these methods is the better for the enzymologist. In theory the wet crystal should yield a more accurate figure, but in practice the technique is more difficult, and this may perhaps lead to errors. On the other hand, the air-dried method can be carried out with rather small crystals, whereas the wet-crystal method requires large crystals. Both methods will therefore be described. They are followed by detailed accounts of the techniques of density determination and composition measurements.

Air-Dried Crystals Air-dried crystals are not precisely defined for two reasons. First, the dimensions of the unit cell and the degree of disorder usually depend somewhat on the method of drying. Slow drying usually produces a more ordered crystal. Second, the cell dimensions and the water content vary with the humidity, though not very greatly--certainly much less than they do for wet crystals. Reasonable care should therefore be taken to keep the humidity the same for the different sets of measurements--on the cell dimensions, on the density, and on the nonprotein content. It is convenient to measure the cell dimensions first and to carry out the other measurements at the humidity of the atmosphere at the time of the X-ray measurements. It should be not far from 40 %. The rate of drying of a crystal can be reduced by obvious methods, such as restricting the entry of the dry air. A less obvious way is to immerse the wet crystals in xylene2 The water of the crystal is only slightly miscible with xylene, and because of this it diffuses out of the crystal at a very reduced rate. The perfection of an air-dried crystal depends partly on the nature of the crystal. A given protein may have one unit cell which, when dried, still gives X-ray reflections at spacings of 4 A., whereas a second type of unit cell of the same protein may under similar conditions give no reflections beyond 7 A. (the latter amount of disorder is the more customary for dry crystals). Thus, if several forms of crystal exist, it is worth while trying them all. High concentrations of salt are usually a disadvantage as, on drying, the salt in the crystal tends to become saturated and crystallize out separately. This not only disrupts the crystal somewhat but introduces complications in the other determinations. Thus the protein should be crystallized with as small a quantity of salt as possible. Alternatively the crystals may be grown under one set of conditions, but measured under another. For example, Low and Richards TM in their studies on dimeralbumin and f~-lactoglobulin crystals grew their crystals from threecomponent systems containing protein, water, and either salt or alcohol.

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TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[4]

After the crystals had grown they washed them thoroughly with a saturated aqueous solution of the protein before any measurements were made. Analysis showed t h a t less than 0.5% salt or alcohol was left in the crystal.

Wet Crystals The unit cell dimensions of a wet crystal can be measured with considerable accuracy, as can their density. The main difficulty comes in measuring the a m o u n t of solvent in the crystal. On the one hand, the crystal must be weighed without excess m o t h e r liquor clinging to it; on the other hand, it must not be allowed to dry. The wet crystal is exceedingly sensitive to h u m i d i t y changes, and some solvent is lost v e r y easily. I t is therefore almost impossible to measure the nonprotein content of the wet cell by using a mass of small crystals. The measurement must be made on large individual crystals. The details are described in a later section. In certain cases it m a y be possible to calculate the protein content of the unit cell. This happens when the solvent has only one component, water. If one assumes t h a t the apparent specific volume of the protein in the crystal is equal to the partial specific volume of the protein in dilute solution (which can be measured in the usual way) and t h a t the water in the crystal has the same partial specific volume as free water, namely unity, then one can easily calculate the value of w, the grams of water in the crystal per gram of dry protein, from the formula of Adair and Adair: ~ W

(Dp - D) (D - D1)

D1 Dp

where D -- density of crystal. D~ = density of water. Dp -- reciprocal of the partial specific volume of the protein. This version of the formula, the derivation of which is straightforward, is usually, but not always, correct for a wet crystal. 14 I t does not give correct results for an air-dried crystal. 14 I t should in any case be used with discretion. The formula cannot be followed when the solvent has more than one c o m p o n e n t - - f o r example, when it is a strong salt solution or an alcoholwater mixture. Measurements have shown ~ t h a t the concentration of salt solution inside a protein crystal is always less than t h a t of the m o t h e r liquor, as if some of the water of the crystal was bound to the protein and not available to the salt. 15 G. S. Adair and M. E. Adair, Proc. Roy. Soc. B120~ 422 (1936).

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X-RAY DIFFRACTION OF PROTEIN CRYSTALS

139

Density Measurements There are three methods of density measurement: flotation, gradient column, and microbalance. The microbalance method, 16,17 in which the apparent mass of the crystal is measured while it is immersed in its mother liquor, and the volume of the crystal computed from photographs, demands an instrument which, though not too difficult to make, will not normally be available to an enzymologist. It will therefore not be described further. In the other two methods--flotation and gradient column--the protein crystal is immersed in a liquid which is immiscible with the liquid in the crystal, and whose density is the same as that of the crystal. In the flotation method the density of the liquid is adjusted by trial and error until the crystals neither float nor sink decisively. In the gradient column, which consists of a long vertical liquid column, the density of which increases steadily from top to bottom, the crystals fall to a level where the density of the liquid is the same as their own. In both these methods centrifugation may be used to make the method more sensitive or more rapid. The flotation method requires little special comment. A series of tubes may be prepared containing mixtures of slightly different density in a regular sequence. Crystals are added to each, and the density is considered to be midway between those of the two adjacent tubes in which the crystals just sink and just float. Care should be taken to prevent evaporation of the liquids if they are volatile, and to avoid excessive temperature gradients which may set up convection currents. Alternatively the density of a single tube containing the crystals may be adjusted in steps until the experimenter judges that the crystals arc very close to equilibrium. For wet crystals the precautions mentioned in the description of the gradient column should be followed--large crystals should be used, they should be wiped as quickly as possible, and the measurements taken within a few minutes. The gradient column is based on that originally devised by Linderstr0m-Lang, is and modified for rapid though less accurate work (0.1%) by Jacobson and Linderstr0m-Lang.19 As is well known, such columns are prepared by placing two liquids of different density in a tong vertical column, the heavier below the lighter. A linear gradient of density develops near the interface. Manipulation of a plunger-type stirrer in a vertical tube can extend the gradient over the greater part of the column. ~(~B. 17 F. is K. ~9 C.

W. Low a n d F. M. Richards, Nature 170, 412 (1952). M. Riehards, Rev. Sc~. Instr. 24, 1029 (1953). Linderstr¢m-Lang, Nature 139, 713 (1937). F. Jacobson a n d K. Linderstr0m-Lang, Acta Physiol. Scan& 2~ 149 (1940).

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TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[4]

Since extreme accuracy is not required, a reasonably high temperature gradient is acceptable, and therefore no special precautions need be taken concerning accurate temperature control and vibration-free mounting as would be required for a more sensitive column. A column formed in this way is surprisingly stable and will maintain its gradient virtually unchanged for many months. A suitable column for general use has been described by Low and Richards. 2° The column was 20 cm. high, made of bromobenzene and xylene, and covered a density range from 1.15 to 1.35. The position of the crystal could be observed to within 0.5 mm. from a millimeter rule supported alongside the column. The gradient was calibrated by means of drops of about 0.01 ml. and of known specific gravity, of potassium phosphate solutions, with density increments of 0.002. Low and Richards 2° also used gradient columns set up in 10-ml. centrifuge tubes and spun for 1 or 2 minutes at 2500 r.p.m. During this time the heating was negligible. The useful range of length, developed by stirring, was about 5 cm. They did not assume that the density gradient was linear but bracketed the crystal position with two calibration drops. As the amount of material was small, the entire column was discarded after each measurement. For proteins Low and Richards 2° used mainly 10-cm. columns of bromobenzene-kerosene or bromobenzene-xylene at 24 °. Centrifugation proved unnecessary for the larger protein crystals. For wet crystals the column used had a density range of 1.10 to 1.20. The density of the crystals was measured after a standard time of 5 minutes. The large crystals reached an equilibrium position in about 3 minutes, but smaller crystals took up to 5 minutes. It was found 14" to make no observable difference during the first 10 minutes whether the column was made up with dry components or water-saturated components. After that time, however, crystals in both types of column showed a perceptible increase of density due to loss of water to column components. Within 24 hours t~-lactoglobulin crystals in the dry column had attained densities of about 1.26, whereas those in the water-saturated ones gave values of about 1.16 to 1.17. To prepare the crystal for the column the excess mother liquor was removed by the method described in the next section, and the time taken from the moment the crystal left its mother liquor to the moment that it was dropped in the column was recorded. For ~-lactoglobulin it was found 2° that the lowest density was obtained with the largest crystal and with drying times of approximately 25 seconds. They ~°state: "Comparing crystals of different sizes (e.g. weighing 2.0 to 0.2 mg.) it was evident that ~0 B. W. Low a n d F. M. Richards, J. Am. Chem. Soc. 74~ 1660 (1952).

142

TECHNIQUES FOR CHARACTERIZATION" OF PROTEINS

[4]

why a mass of small crystals should not be used, provided little amorphous material is present. The correction for nonvolatile nonprotein material most obviously depends on its chemical nature. It will therefore not be discussed here. As already stated the salt concentration of the crystals should be reduced to as low a value as possible. There seems to be no case recorded in the literature in which an attempt has been made to obtain an accurate molecular weight from a protein crystal containing a large amount of salt. Air-dried lysozyme chloride, which contains only a small amount, was found 21 to have 0.5% Na and 3.15% C1, and a correction was applied to the molecular weight for the bound chloride, and for the effect of the NaC1, which formed small crystals on the surface of the protein crystal, on the observed density. These corrections were small, but in the case of a protein crystallized from, say, 2 M (NH4)~S04 the correction might be considerable. There would seem to be no reason, however, why the wet crystal method should not be reasonably accurate, provided there is a good micromethod for estimating the nonvolatile nonprotein component. The accuracy of the molecular weight found with the air-dried crystals might be considerably reduced, however, owing to the type of effect described above for lysozyme chloride.

Summary of Molecular Weight Methods We can summarize the methods for determining molecular weight by classifying them according to the accuracy the experimenter wishes to obtain.

Accuracy +_15% Obtain the space group of the wet unit cell. Obtain the dimensions of the air-dried unit cell. Use the empirical rule to calculate the molecular weight.

Accuracy +_5% Obtain the space group of the wet unit cell. Obtain the dimensions of the air-dried unit cell as accurately as possible. Measure the density of the air-dried crystals (small crystals may be used). Measure the percentage loss of weight on vacuum drying (a mass of small crystals may be used). Measure the percentage mass of the nonvolatile nonprotein component (a mass of small crystals m a y be used). ~z K. J. Palmer, M, Ballantyne, and J. A. Galvin, J~ Am. Chem. Soc. 70t 906 (1948).

[4]

X-RAY DIFFRACTION

OF PROTEIN CRYSTALS

143

The accuracy will depend mainly on the precision with which the cell dimensions can be measured, unless the crystal contains much salt, which may reduce the accuracy further. Accuracy as High as Possible

Obtain the space group and cell dimensions of the wet unit cell. Measure the density of single, large, wet crystals. Measure the percentage loss of weight on drying of single, large, wet crystals. Measure the percentage mass of the nonvolatile nonprotein component. The accuracy will depend mainly on the accuracy of the last two measurements. If the solvent is water it is sometimes possible to perform only the first two operations and find the water content from the formula of Adair and Adair, but this should not be done if the highest accuracy is required. Further Work

Further work may be undertaken for two reasons: first, to find the arrangement of the molecules in the crystal and the general shape of the molecule; second, to discover whether there are any features of the crystal which suggest that it should be studied more intensively. It is not possible here to discuss the information, often rather limited, which may be obtained from a study of Patterson projections. Fortunately an excellent review, in nontechnical language, has been published by Kendrew, ~ and this should be read as a preliminary to any further studies on the protein crystal, as should the very comprehensive review by Low. 1

The Shape of the Molecule X-Rays will " s e e " the shape of the molecule only if there is a good contrast between the average electron density of the protein and that of the solvent. The average electron density of protein is usually about 0.43 electrons/A2, whereas that of hydrated protein is rather lower, say 0.41 or 0.40. The electron density of water is 0.335 electrons/A. 3, whereas that of 4 M (saturated) ammonium sulfate is about 0.41. Alcohol-water mixtures are less dense than water. Thus if the solvent is water, an alcohol-water mixture, or a salt solution of low concentration, its electron density will be well below that of the hydrated protein, and the very low-order X-ray reflections may give information about the shape of the

144

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[4]

protein. If the solvent is a strong salt solution, its electron density may be rather close to that of the hydrated protein, and the very low orders are likely" to be weak or absent. It may happen that for very strong salt solutions, or for salts of high molecular weight, the electron density of the solvent may exceed that of the hydrated protein by so much that the very low orders are strong because of the contrast the other way round. Such cases are not common, however. Even if the salt has reduced the contrast between the salt solution and the protein it is possible to obtain valuable information by measuring how the low-order intensities alter with changes of salt concentration. This method has been utilized for hemoglobin .by Bragg and Perutz. ~ Thus, if the electron density of the solvent is low, it is worth while to measure all the very low orders (spacings ~ 20 A.) in the reciprocal lattice of the wet crystal--there will not usually be very many of them. If it is found that only two or three of the reflections are outstandingly strong it may be reasonable to calculate a very low resolution Fourier from these terms alone. Such a Fourier is likely to give information on the arrangement of the molecules in the cell, and also possibly something about the shape of the molecule. Thus the very great strength of the 001 and the 110 reflections in normal wet salt-free horse methemoglobin (space group C2) immediately gives an idea of the general arrangement of the molecules. ~2 It is advisable in such cases to measure the absolute intensities of the strong reflections, as this makes the interpretation more certain. More detailed studies on hemoglobin, ~ with data from various shrinkage stages, have made it possible to obtain the general shape of the hemoglobin molecule at low resolution. In this work on hemoglobin the packing arrangements in various other crystals of hemoglobin were considered, 28 and helped to re-enforce the argument for the shape. The possibility of using packing arguments should therefore be kept in mind. For example, if there are sufficient polymorphic forms of the crystal it is not improbable that the smallest diameter of the molecule is about the size of the smallest (primitive) cell dimension found in any of the unit cells, but such arguments should be considered with caution. Points to Look For

The following features are ones which might make it worth while to study the crystals further. 1. A very small unit cell, or one with one dimension very small ( < 20 A.). A small unit cell will arise only with a protein of low molecular 22 W. L. Bragg and M. F. Perutz, Acta Cryst. 5, 277 (1952). 2a W. L. Bragg and M. F. Perutz, Acta Cryst. 5, 323 (1952).

146

TECHNIQUES FOR CHARACTERIZATION" OF PROTEINS

[4]

confuse the situation. A number of lighter atoms (say bromine) will not be acceptable instead of a heavy atom except possibly in the case where they are bound together in a group of a definite shape. Finally, the unit cells of the protein with and without the heavy atom should have identical dimensions. It can be seen that the chemical requirement of attaching one heavy atom, or at the most two or three, with high efficiency onto a few specific sites is a formidable one. Any protein to which it can be done becomes a very favorable object for X-ray studies. It may be possible, for example, to find the general shape of such a protein molecule rather quickly. I t should not be forgotten that the active site of an enzyme offers great possibilities as a unique site of attachment, and that an enzyme inhibitor containing a heavy atom might rather easily fulfill the above requirements. If such a case comes to the knowledge of an enzymologist he should certainly bring it to the attention of a protein crystallographer. Program of Work In this section is set out a typical program of a preliminary examination as it might be made by a protein crystallographer. It is not suggested that the whole of this program should always be carried out, but it may serve as a guide to general practice. 1. Optical examination of the wet crystals. 2. Cell dimensions and space group of the wet cell and the air-dried cell. 3. Density measurements (as discussed). 4. Composition measurements (as discussed). 5. Precession pictures of three projections of the wet cell: Spacings out to 7 A. or perhaps to 3 A. 6. Calculation of the Pattersons of these projections. 7. Preliminary exploration of shrinkage stages. 8. Preliminary examination of lhe intensities of the dry cell. 9. Measurement of the three-dim~nsional low orders of the wet cell (in cases where the solvent has a low electron density). Acknowledgments I should like to thank Drs. Barbara W. Low and F. M. Richards for allowing me to read their manuscript prior to publication, and my colleagues at the Protein

Structure Project for helpful criticism.

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LIGHT-SCATTERING MEASUREMENTS

147

[5] Light-Scattering M e a s u r e m e n t s By M. BIER Introduction When a colloidal solution is illuminated by a parallel beam of white light, a faint bluish light can be laterally observed. This phenomenon, caused by the scattering of light, is commonly known as the Tyndall effect. 1 The scattering of light is, however, not limited to colloidal solutions, where it is the most pronounced, but is observable in all transparent media, be it a gas, a pure liquid, a solution, or a crystal. Lord Rayleigh 2 formulated in 1871 the fundamental laws of the scattering of light by calculating the polarizability of individual gaseous molecules placed in the oscillating electromagnetic field of a light beam. It is the polarized molecules that then act as sources of secondary radiation, re-emitting the energy of excitation, thus giving rise to the scattered light. Various authors have further contributed to the theories of light scattering, and several reviews adequately cover the field2 -s The intensity of the scattered light depends on a number of measurable quantities and can be expressed as a function of the number of centers of scattering (i.e., molecules) per unit volume. Its quantitative measurement can thus be used for the determination of Avogadro's number] or it can be applied to the determination of molecular weights, if a value for Avogadro's number is adopted. The first applications of light scattering to the determination of particle weights in colloidal systems seem to be due to Smirnov and Bazenov 8 and Putzeys and Brosteaux. 9 Despite this long history it is only since the simplification of the theories by Debye 1° that the light-scattering method has become a practical tool for the study of macromoleeular systems. For the full characterization of the scattering of a nonabsorbing solution we have to know the relative intensity of the scattered light with re, J. Tyndall, Proc. Roy. Soc. 17, 223 (1869). 2 j. W. Strutt (Lord Rayleigh), Phil. Mag. [4] 41, 107, 274, 447 (1871). a S. Bhagavantam, "Scattering of Light and the Raman Effect." Chemical Publishing Co., Brooklyn, 1942. 4 H. Mark, in "Frontiers in C h e m i s t r y " (Burk and Grummitt, eds.), Vol. 5: Chemical Architecture, p. 121. Interscienee Publishers, New York, 1948. 5 G. Oster, Chem. Revs. 43, 319 (1948). J. T. F,dsall and W. B. Dandliker, Fortschr. chem. Forsch. 2, 1 (1951). 7 j . Cabannes, Ann. phys. [9] 15, 5 (1921). 8 L. V. Smirnov and N. M. Bazenov, Colloid J. (U.S.S.R.) 1, 89 (1935). 9 p. Putzeys and J. Brosteaux, Trans. Faraday Soc. 31, 1314 (1935). t0 l'. Debye, J. Phys. & Colloid Chem. 51, 18 (1947).

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[5]

spect to the incident beam, its angular distribution, and its depolarization. Furthermore, the optical relationship between solvent and solute has to be determined and is usually expressed in terms of the specific refractive index increment, ( n - no)/C. From the proper combination of these values we can derive not only the molecular weight but also data on the size and shape of the dissolved macromolecules as well as information on the thermodynamic properties of the system. Owing to the great rapidity with which optical measurements can be carried out, light scattering offers also unique possibilities of following the kinetics of macromolecular reactions involving a change in the size or shape of the dissolved particles. ~1-14 Theory A detailed discussion of the theories of light scattering is far beyond the scope of this article, which shall be limited only to the presentation of the equations of immediate use in the evaluation of light-scattering data. In the consideration of the equations attention should be given to the limitations imposed on the systems to which they apply. The turbidity, T, of a system is defined by the equation I = Ioe -t~

(1)

and expresses the exponential loss of the intensity of light, I, on passage through any nonabsorbing medium of path length I. In appearance it is analogous to Beer's law, defining the loss of light due to specific absorption of light by " c o l o r e d " media. In practice, however, it is distinguished by two important facts. First, the absolute value of r is for most systems considerably lower than the corresponding extinction coefficient of Beer's law for usual "colored" solutions. Second, the energy lost by the transmitted beam is not transformed into heat as in colored solutions, but is immediately re-emitted, with the same wavelength as the incident light, in all directions, the scattering molecules acting as secondary sources of radiation. Whereas the above equation gives the total loss of light intensity in the transmitted beam, the Rayleigh ratio Ro - i°r2

(2)

I0 11 M. Bier and F. F. Nord, Proc. Natl. Acad. Sci. U.S. $§~ 17 (1949). 1~ G. Oster, J. Colloid Sc/. 2, 291 (1947). 13 j . D. Ferry, S. Shulman, K. Gutfreund, and S. Katz, J. Am. Chem. Soc. 74, 5709 (1952). 14 S. Katz, S. Shulman, I. Tinoeo, Jr., I. H. Billick, K. Gutfreund, and J. D. Ferry, Arch. Biochem. and Biophys. 47, 165 (1953).

[5]

LIGHT-SCATTERING MEASUREMENTS

149

defines the intensity, i0, of the scattered light measured at the angle 0 and distance r as a function of the intensity, I0, of the incident light. It is the Rayleigh ratio, sometimes also referred to as the reduced intensity of the scattered light, that is usually measured. The relation between T and R~ is given by 7 = s/./~R0 = 16/t~Rg0 (3) In macromolecular solutions, the solvent is assumed to be continuous, and only the additional scattering, due to the solute molecules, is considered in the above equations. The molecular weight, M, of the solute is then calculated from either the turbidity or the Rayleigh ratio by means of the following simple equations:

Hc

l

-

211

(4)

and

Kc (1 R~

+ cos" 0)

=~

1

(5)

or

Kc Rg0

1 -

M

(6)

These equations apply only to infinitely dilute solutions of isotropic and dielectric molecules of relatively small size as compared to the wavelength of light (<)~/20). The terms H and K group together all the pertinent optical constants for the solute-solvent system at a particular wavelength of light: H=

3NoXo 4

2rr'no' (n c n--~°)2 K=

(s)

NoXo~

Equations 4 to 6 presuppose that the incident light is unpolarized, which then can be considered as the superposition of two polarized beams of light, oscillating at right angles to each other and incoherent in phase. The intensity of the scattered light, due to the beam of light having an electrical vector oscillating in a plane perpendicular to the plane defined by the incident beam and the direction of observation, will be the same at all angles of observation, whereas the angular distribution of the scattered light produced by the horizontally polarized light will be a

150

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[5]

function of cos 2 0. The addition of the two, results in the angular distribution of the scattered light, (1 + cos 2 0), indicated in equation 5. As a consequence, the scattered light at 90 ° should be fully polarized in the vertical direction, and the intensity of scattering in the forward direction (0 - 90 °) is symmetrical to that in the backward direction (90 - 180°). The similarity of equations 4 and 6 to van't Hoff's limiting equation for osmotic pressure, P, P c RT - M (9) is not purely fortuitous, as was shown by Einstein 15 in his analysis of the scattering in more concentrated systems. In condensed systems, the total observed scattering is less than would be obtained by the summation of the scattering from each individual molecule, owing to the destructive interference of the scattered rays. As an extreme case, in an ideal crystal, in which all the molecules would be arranged in a perfect lattice without thermal vibration, any of the volume elements of the crystal would contain exactly the same number of scattering centers, each polarized exactly with the same amplitude and in the same phase, when exposed to incident radiation. As a consequence, for each scattering molecule another one could be found, placed at such a distance as to cause the mutual interference of the secondary radiation. Such a crystal would scatter no light. The observable scattering in real systems is therefore due to the local random fluctuation in the density of the system. Thus, the same number of molecules will scatter much more light in gaseous state than in liquid state, and the liquid will scatter much more than a crystal. The excess scattering of a solution, in which we are interested here, is, by analogy, due to the local fluctuation ill the concentration of the solute molecules, i.e., in the osmotic pressure. This finds its expression in the following equation: Hc _ O(P/RT) (10) 7

Oc

Van't Hoff's equation (9) expresses the osmotic behavior of highly dilute solutions of low molecular weight compounds. For most colloidal solutions, however, it fails at very low concentrations, and the following empirical equation expresses the dependence of the pressure on concentration: P c R - T -- -M -~ Bc~ (11) 1~ A. Einstein, Ann. Physik 83, 1275 (1910).

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LIGHT-SCATTERING MEASUREMENTS

151

Inserting equations 10 and 11 in equations 4 and 6, we obtain Kc

Hc

1

Rg0

T

M

+ 2Be

(12)

These expressions are the ones usually employed in the determination of molecular weights. It can be seen that by plotting either Kc/R~o or H c / r versus concentration a straight line will be obtained, the intercept with the ordinate corresponding to the reciprocal of the molecular weight, while the half-slope, B, is identical with the slope of the osmotic pressure equation. Its meaning will be discussed later on. Although there is thus a great similarity between light-scattering and osmotic pressure measurements, and data obtained by the two methods are frequently compared, there arises an important difference in the results, when the colloidal solutions are not monodisperse, but contain molecules of different weights. In such a case, osmotic pressure measurements give a so-called number average molecular weight, M,~-

Zm,M~

Zm~

(13)

where mi is the molar concentration of the macromolecular component, i, and Mi its molecular weight. By light-scattering measurements, however, a different average is obtained, namely the weight-average molecular weight, Z m i M i2 M w - Y.miM~ (14) The difference is due to the fact that osmotic pressure depends only on the number of particles, whereas the light scattering depends also on the weight of the particles. As a consequence, in polydispersed solutions lightscattering measurements will always yield a higher average molecular weight, and this method is particularly sensitive to dust particles, or other giant-sized impurities. Before going into the question of further data derivable from lightscattering measurements, we have first to extend the theory so as to be able to determine the molecular weight in two of the cases so far excluded by our restrictions, relating to the isotropy and size of the molecules. In the above equations it was assumed that the molecules are optically perfectly symmetrical, i.e., that their polarization is perfectly parallel to that of the exciting beam of light. For most molecules, however, a more appropriate model is that of an ellipsoid of polarization, i.e., they have their own preferential directions of polarization. The plane of polarization of the induced secondary radiation will therefore be slightly inclined with

152

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[5]

respect to that of the primary radiation, and the light scattered at 90 ° will have a small horizontally polarized component besides the normal vertically polarized light. The scattering of such a system is therefore higher than that which would be expected from only the fluctuation in concentration, as it also contains the scattering due to fluctuation in orientation of the anisotropic molecules. For the calculation of molecular weights the latter factor has to be subtracted from the total measured light scattering. The correction factor for Rg0 is (6 - 7p)/(6 ~ 6p), and for r it is ( 6 - 7p)/(6-]-3p), whereby the depolarization ratio, p, is defined as the ratio of the horizontal to the vertical component of the scattered light at 90 ° with unpolarized incident light. 1~ In most macromolecular systems this correction is rather small, p amounting to about 0.004 to 0.04. This is true also in all proteins TM so far studied, although they may be quite asymmetric as in the case of the tobacco mosaic virus. Although this simplifies the use of the method for the determination of molecular weights, it is possibly unfortunate that it precludes fuller use of measurements of depolarization to the study of the shape of the molecules. More information on the actual size and shape of the molecules can be gained from the study of the angular distribution of scattered light from molecules of size comparable to the wavelength of light. Particles which are small compared to the wavelength of light can be treated as point sources of radiation. Particles approaching the dimensions of the wavelength of light (in the particular solvent, i.e., X.ol~o~t= X...... :n,olvont) will, however, have many scattering elements. Thus, interference will occur with a resulting decrease in the observed intensity of the scattering. It was repeatedly shown that the interference will be much greater in the backward direction than in the forward direction, causing a dissymmetry of the angular distribution of the scattering. Equation 5 has therefore to be multiplied by factor P(0) to give the correct angular distribution of the intensity of scattered light: K_c (1 + cos 20)P(O) Re

=

1

(15)

In Table I are listed the values of the functions P(0) for the three usual macromolecular models, namely, for spherical molecules, for rodlike particles, and for randomly coiled linear polymers, in terms of a parameter, x, for the first two and as a function of ~v/X for the coils. The le Cabannes, J., "La Diffusion mol6culaire de la Lumi~re." Presses Universitaires de France, Paris, 1929. 16~ E. P. Geiduschek, Or. Polymer Sci. 13, 408 (1954).

[5]

LIGHT-SCATTERING MEASUREMENTS

153

TABLE I PARTICLE SCATTERING FUNCTIONS FOR THE THREE MACROMOLECULAR MODELS P(O) x"

Spheres

Coils

Rods

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2,2 2,3 2.4 2.5 2.6 2.7 2.8 2.9 3.0 3.2 3.4 3.6

0.998 0,992 0.982 0.968 0.951 0. 930 0.906 0. 879 0.849 0.816 0.782 0.745 0.707 0.668 0.628 0. 587 0. 547 0. 506 0. 466 0.427 0. 388 0. 351 0.316 0. 282 0.249 0. 219 0. 191 0. 165 0.141 0. 119

1.000 0. 986 0.971 0.949 0.922 0. 890 0. 855 0. 817 0.777 0.736 0. 694 0.653 0.612 0.573 0.535 0. 500 0. 466 0. 434 0. 405 0. 377

0. 999 0. 996 0.990 0. 983 0.973 0. 961 0. 948 0. 932 0.916 0.897

0. 329

0. 627

0. 287

0. 583

0. 252

0.54'3

0. 223

0. 506

0. 198

0.473 0.443 0.417 0.395

0. 857 0.813 0. 767 0.719 0. 672

'~ For random coils = x/a:. p ~ r ~ m e t e r , x, is For spheres

D x = 2v~sin

(16)

For rods

x = 2~ L s i n 0

(17)

F o r coils

R2 0 8 r~ ~ sin2 2_ x = .~

(18)

154

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

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The critical dimensions of the models are the diameter, D, of the sphere, the length, L, of the rod, and the root mean square, R, of the distance between the two ends of the random coil. The parameter R is also the largest distance within the coil in its most probable configuration and can be approximated by R2 = Na 2 1 - p l+p

(19)

where N is the number of segments in the chain, a their distance, and p the cosine of the angle between two successive segments. ~7 The above equation assumes a free rotation of the various segments. Experimental results indicate, however, that the size of the coils is usually larger and can be better expressed by R2 = N a ~1 - p 1 ~ c o s ¢

(20)

1 ~ p 1 - cos where the rotation of each segment is assumed to be restricted to an angle ~.~8 With Table I the values of P(O) can be found for any scattering angle or particle size. It is a fortunate characteristic of this function, P(0), that it is sufficient to determine the intensity of the scattering at any two angles for the calculation of the particle size. The two symmetrical angles of 45 ° and 135 ° are usually chosen, and the ratio of scattering intensities at the two angles is referred to as the dissymmetry of scattering, z. In Table II are listed the values of this dissymmetry in function of the critical particle size. Also, in the same table are given the corresponding values of the reciprocal of P(90), which is the correction factor by which the calculated molecular weight has to be multiplied to correct for the interference. The above tables apply only to systems at infinite dilution. The particle size and correction factor 1/P(90), is therefore calculated from the so-called intrinsic dissymmetry, i.e., the dissymmetry extrapolated to zero concentration. Although not strictly valid, the same correction factor is applied also to the slope, 2B, of the conventional light-scattering plots. The shortcoming of the above method is that it depends on the proper selection of the macromolecular model, although the dissymmetry for most systems is rather low, where there is no great difference between the 17W. Kuhn, KoUoid-Z. 68, 2 (1934). is p. j. Flory, "Principles of Polymer Chemistry." Cornell University Press, Ithaca, N.Y., 1953.

[5]

LIGHT-SCATTERING MEASUREMENTS

155

TABLE II DISSYMMETRY AND CORRECTION FACTORS AS A FUNCTION OF PARTICLE SIZE Spheres

D/x 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 0.26 0.28 0.30 0.32 0.34 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 0.52 0.54 0.56 0.58 0.60

l)issym- Correction metry factor (z) [1/P(90)] 1.002 1.01o 1.02t 1.037 1.05s 1.08~ 1.11~ 1.15s 1.202 1.257 1.32o 1.394 1.48, 1.582 1.699 1.837 2.000 2.192 2.41, 2.691 3.01~ 3.417 3.90~ 4.51~ 5.29~

1.002 1.01o 1.013 1.026 1.04o 1.059 1.08, 1.107 1.13s 1.173 1.214 1.26o 1.31a 1.373 1.441 1.517 1.604 1.702 1.81~ 1.94, 2.08~ 2.25, 2.44o 2.650 2.91~

Coils

Rods

1)issym- Correction merry factor (z) [1/P(90)]

Dissym- Correction merry factor (z) [1/P(90)]

1.00 1.012 1.025 1.04o 1.062 1.09o 1.12a 1.162 1.205 1.255 1.31o 1.37o 1.436 1.50s 1.582 1.663 1.74s 1.83s 1.93o 2.02o 2.12~ 2.22~ 2.328 2.42s 2.53s 2.64o 2.74~ 2.84s 2.95, 3.05~

1.00 1.005 1.017 1.02s 1.04s 1.064 1.08s 1.115 1.14s 1.183 1.223 1.26s 1.317 1.37, 1.43o 1.49~ 1.563 1.63~ 1.72o 1.80~ 1.89s 1.99~ 2.090 2.20~ 2.32~ 2.44: 2.57~ 2.70~ 2.84: 2.99~

1.00 1.005 1.011 1.02o 1.03, 1.045 1.061 1.08, 1.102 1.127 1.154 1.183 1.21o 1.25, 1.28s 1.32T 1.37o 1.414 1.46o 1.50~ 1.55s 1.60~ 1.65~ 1.70s 1.75~ 1.80~ 1.84o 1.89~ 1.93a 1.97~

1.00 1.004 1.00s 1.014 1.022 1.032 1.043 1.057 1.072 1.09o 1.10~ 1.131 1.154 1.18o 1.20~ 1.237 1.27o 1.304 1.34~ 1.38o 1.42~ 1.46~ 1.51o 1.55s 1.6(b 1.659 1.71~ 1.76~ 1.827 1.88s

three models. For larger particles the method fails to give any indication of the actual shape of the particles. This can be obtained by a more complete determination of the angular distribution of the scattered light.. In this mei hod 19 the observed angular i~ltensities of the scattering are simultaneously graphically extrapolated to zero angle and zero concentration, whereby the effects of interference disappear, as P(0) is always l For 1~B. |1. Zimm, J. Chem. Phys. 16, 1093, 1099 (1948).

156

TECHI~IQUESFOR CHARACTERIZATION OF PROTEINS

[5]

still larger particles the more complex theory of Mie 2° has to be applied. ~1 Light-scattering measurements can also yield valuable data on the thermodynamics of solvent-solute interaction. I t is sufficient to recall equation 10 and the fact that, according to classical thermodynamics, PIT"l = - A F 1

(21)

where 171 is the partial molal volume of the solvent, and AP~ is its partial molal free energy. The latter thus contains an e n t r o p y term and a heat content term, both referring to the mixing of the solvent and solute. These terms are to be found in the slope, 2B, of equation 12, and the heat content term can be determined by measuring the slope at different temperatures. T h e interpretation of the results in solutions of randomly coiled polymers has received considerable attention from various authors ~s,~2-24 but is beyond the scope of this article. I t will suffice to mention the following qualitative conclusions. In a good solvent, i.e., where there is a pronounced energetic interaction between the solute and solvent, the slope 2B will be large. In a poor solvent, however, i.e., in systems where the solvent and solute have but small affinity, the slope will be small, zero, or even slightly negative. Negative slopes are indicative of approaching phase separation, i.e., solute precipitation, the occurrence of which prevents strongly negative slopes. ~,25,26 Light scattering results in mixed solvents or solvent-precipitant mixtures employed in polymer fractionation should, however, be interpreted with caution, owing to the possible preferential absorption of one of the solvents. 27 In solutions of charged macromolecules and proteins in particular, the large effects on light scattering of the electrostatic forces surrounding them have also to be considered. Scatchard's theoretical t r e a t m e n t of such systems ~s was applied b y him and co-workers to a detailed s t u d y of the osmotic pressure of bovine serum albumin 29 and can be adapted *oG. Mie, Ann. Physik 25, 377 (1908). ~ A. S. Kenyon and V. K. LaMer, J. Colloid Sci. 4, 163 (1949). ~ M. L. Huggins, J. Am. Chem. Soc. 64, 1712 (1942); Ann. N.Y. Acad. Sci. 45, 1 (1942). 2ap. j. Flory, J. Chem. Phys. 10, 51 (1942); 13, 453 (1943); 17, 1347 (1949). 24A. R. Miller, "The Theory of Solutions of High Polymers." Oxford University Press, New York, 1948. 36F. F. Nord, M. Bier, and S. N. Timasheff, J. Am. Chem. Soc. 73, 289 (1951). 26S. N. Timasheff, M. Bier, and F. F. Nord, J. Phys. & Colloid Chem. 55, 1134 (1949). 27R. H. Ewart, C. P. Roe, P. Debye, and J. R. McCartney, J. Chem. Phys. 14, 687 (1946). 38G. Scatchard, J. Am. Chem. Soc. 68, 2315 (1946). 32G. Seatehard, A. C. Batchelder, and A. Brown, J. Am. Chem. Soc. 68, 2320 (1946).

158

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[~]

logarithm of the activity coefficient, % of the protein with the change in protein concentration: ~22 = 0 In ~protoin/Omp~ote~n (23) The term has a meaning similar to that of the slope 2B in solutions of uncharged macromolecules, as it represents the deviation of the system from the behavior of ideal solutions (having an activity coefficient -y = 1). The third term results from the three-component nature of such protein solutions, namely, the presence of solvent, protein, and salt. It represents the effect of the concentration of salt on the activity coefficient of the protein. It is usually of minor influence in comparison to the first two. Instruments The direct determination of the turbidity by measuring the attenuation of the intensity of light on passage through a cell of known length is rarely applicable, as the turbidity of most colloidal solutions is of the order of magnitude of r = 10-3 cm. -1. Excessively long cells would have to be used in order to measure it with sufficient accuracy. In practice, however, it is possible to determine with much greater accuracy the intensity of the light scattered laterally in appropriately constructed instruments. The instruments should provide for the determination of: 1. The intensity of the light scattered at 90 ° as compared to the intensity of the incident light (Rg0). 2. The angular distribution of the intensity of scattered light, or at least the dissymmetry, z, i.e., the ratio of intensities at 45 and 135°. 3. The depolarization of the scattered light, p. A number of instruments have been constructed for this purpose. Some of the early instruments were based on the visual comparison of the intensities of the incident and scattered light. 4 With the development of phototubes and particularly photomultip]ier tubes they became outdated. Two commercial instruments are available. The Phoenix ~3 instrument has been described in detail with particular attention given to its absolute calibration. 34 The Aminco 3~instrument utilizes a more compact design and has a particularly efficient amplification of the current emitted by the photomultiplier tube. Both instruments utilize a rectangular cell for the determination of the light scattered at 90 ° and a semioctagonal cell for the dissymmetry measurements. They measure the intensity 33 Phoenix Precision I n s t r u m e n t Co., Philadelphia, Pa. 34 B. A. Brice, M. Halwer, and R. Speiser, J. Opt. Soc. Amer. 40, 768 (1950). 3~ American I n s t r u m e n t Co., Silver Springs, Md.

[5]

LIGHT-SCATTERING MEASUREMENTS

159

either of the scattered light or of the incident light, but they do not measure directly the ratio iO/I. For the determination of the entire lightscattering envelope an instrument has been designed which utilizes a potentiometric bridge circuit, directly giving the above ratio. TMThe solution is contained in a small thin-walled conical glass bulb, and to minimize the reflections of light from the glass itself the bulb is immersed in a liquid having an index of refraction similar to that of the inside of the cell. A light-scattering instrument of very simple construction and application, presented in Fig. 1, has been utilized with satisfaction26 The light from a mercury arc lamp (General Electric AH-4) is focused by a simple optical system on the ceflter of the cell retaining the solution to be h

@ T

D

FIG. 1. Light-scattering photometer, a6 A, light source and housing; B, optical tube; C, semioctagonal light-scattering cell and housing; D, photomultiplier search unit; E, self-generating photocell; F, galvanometer; G, d.c. amplifier and measuring unit.

examined. The optical system is placed in an optical tube, which contains light filters to isolate the desired band of the mercury arc spectrum (Wratten filters No. 2A and C5 for X = 4358 A.). The housing for the cells is cylindrical, the cells being centered by a system of semicircular double recesses. It is fitted with a double bottom for temperature regulation by water circulation. The heavy wall of the housing possesses radial apertures at angles of 45 ° , 90 ° , 135 ° , and 180° to the incident beam of light. Into these circular apertures slides a short metal tube connected to the window of the search unit, which it supports. The search unit is part of an electronic photomultiplier photometer (Photovolt Corp., New York, New York). Thus, either the intensity of the transmitted light or the scattering intensity at the three symmetrical angles can be measured. The window of the search unit is equipped with a photographic shutter and the above-mentioned metal tube. This can be provided with a polarizer and contains a reversed collimating system, holding a lens (f = 5 cm.) and a screen with a central pin-hole, placed in 3~ M. Bier and F. F. Nord, Rev. Sci. Instr. 20, 752 (1949).

160

TECHNIQUESFOR CHARACTERIZATIONOF PROTEINS

[15]

the focal plane of the lens. This system is useful for more accurate delimitation of the angle of scattering. The intensity of the primary beam of light is continually checked by a self-generating photoelement. The line voltage to the mercury arc lamp is regulated by a constant-voltage transformer. The lamp housing is water-cooled. A rotating plate with circular apertures of varying diameter, is interposed between the light source and the optical tube, permitting an easy adjustment of the intensity of the primary beam. Semioctagonal

j "

:vi-

,I,I

3v

@~

~600V 2_

Fro. 2. Potentiometric circuit) 7A, spot galvanometer; GM Laboratories, 570-301; B, C, tubes IC5-GT; D, Amperite 2 HT-I ballast tube; E, potentiometer; F, phototube 929; G, photomultiplier tube IP21. cells are used for measurements of dissymmetry. To avoid reflections from the back of the cell with consequent increase in measured dissymmetry, a coating of a dull black glass cement, fused on the outer surface of the back wall, was found to be indispensable2 6 Although the above apparatus permits a continuous check on the intensity of the primary light source, it does not directly compare its intensity with that of the scattered light. This was accomplished in a simple modification of the same apparatus by introducing a mirror deflecting part of the light incident to the cell and the following potentiometric bridge circuit (Fig. 2). The circuit 37 permits accurate measurements of the scattered light over a wide range of intensities. The current ~7Suggested by Mr. S. E. Krewer of Photovolt Corp., New York.

[~]

LIGHT-SCATTERING MEASUREMENTS

161

from the photomultiplier tube, exposed to the scattered light, is balanced by that from the phototube, on which part of the incident beam of light is focused. Comparatively low voltages across the dynodes of the photomultiplier tube provide for good reproducibility of readings and absence of fatigue. The balanced d.c. amplifier is matched for drift-free performance and operates at low current loads. The null detector galvanometer has a sensitivity of 0.02 t~amp./div, and fast response. The power is supplied by dry cell batteries which are well insulated and shielded. One of the most important problems with all instruments is that of their absolute calibration. Debye L° has standardized his instrument with a solution of polystyrene in toluene, the absolute turbidity of which was determined by photographic means, comparing its scattering intensity to the intensity of the primary beam, attenuated by a known factor through multiple reflection on glass plates. This sample of polystyrene was made available in dry form to several laboratories, which utilized it as their primary standard, although its turbidity was consequently re-evaluated 37a as being r = 3.50 X 10-3 cm. -~ at 0.5% concentration in toluene. 34 Other authors have preferred to utilize pure liquids of known turbidity for calibration, for example, benzene '9,3' or carbon disulfide. 4° The calibration of the apparatus with such primary standards is not unambiguous, however, as it does not take into account the varying amount of stray light present in every instrument. The best defined molecular weight of a standard sample of polystyrene is probably that. reported by Frank and Mark. 4°~ The sample was the object of a collaborative study by a number of laboratories, employing osmotic pressure, light scattering, sedimentation and viscosity measurements. In calibrating the apparatus there are also other factors to be considered.~9.34 Thus, the divergent beam of scattered light, coming from the scattering center in the cell, will diverge even more on passage from the medium of high index of refraction in the cell to the outside air. The actual scattering volume and the solid angle of scattering measured will vary in function of the index of refraction of the liquid in the cell. The most important correction is that due to the spreading of the angle of 37~ T h e molecular weight of 37,000 reported for egg albumin 11 was recalculated on the basis of the corrected value of the p r i m a r y s t a n d a r d and was found to be in good agreement with t h a t of other measurements. 38 38 M. Halwer, G. C. Nutting, and B. A. Brice, J. Am. Chem. Soc. 73, 2786 (1951). .~9 C. I. Carr a n d B. H. Zimm, J. Chem. Phys. 18, 1616 (1950); M. Halwer, G. C. Nutting, and B. A. Brice, J. Chem. Phys. 21, 1425 (1953); B. A. Bricc and M. Halwer, J. Opt. Soc. Amer. 44, 340 (1954). 40 R. H. Blaker, R. M. Badger, and T. S. Gilmann, J. Phys. & Colloid Che~7. 53, 794 (1949). 40~ H. P. F r a n k and H. F. Mark, J. Polymer Sci. 17, I (1955).

[5]

LIGHT-SCATTERING MEASUREMENTS

163

centimeters. T h e difference in the index of refraction of solution and solvent, (n - no), for 1% solute concentration is for m o s t systems in the range of 0.001 to 0.002. As it is desirable to determine this value with b e t t e r t h a n 1% accuracy, i n s t r u m e n t s are required measuring An of 0.5 to 1 × 10-5. Pulfrich r e f r a c t o m e t e r s or interferometers m a y be suitable, if used with the proper precautions. 43 However, as absolute values of the indices of refraction are not required (the index of refraction of the solvent can be assumed as known), b u t only the difference of the indices between solution and solvent has to be established, differential refractometers are best suited for this purpose. The principle on which t h e y operate is simple. A narrow m o n o c h r o m a t i c b e a m of light is m a d e to traverse a prismatic t w o - c o m p a r t m e n t cell, one half of which is filled with the pure solvent, the other with the solution. T h e deviation of the b e a m of light on passing through the cell is linearly proportional to the difference in the index of refraction between the two c o m p a r t m e n t s . T h e i n s t r u m e n t and each cell h a v e to be calibrated with reference solutions of known instruments, as, for example, solutions of T1NO~44 or sucrose. 4~ Such refractometers are commercially available 33 or can be easily constructed. T h e a p p a r a t u s of Bier and Nord ~6 could be read to a b o u t 1 X 10 -8 An. T h e r e are several a d v a n t a g e s to the use of differential refractometers. Their sensitivity is of the desired order of magnitude, and t h e y are of relatively simple design. T h e y give directly in one m e a s u r e m e n t the desired value, n a m e l y the difference between the indices of refraction of two liquids. Applied to a solution and the pure solvent, t h e y are relatively insensitive to t e m p e r a t u r e , provided b o t h c o m p a r t m e n t s are at t h e r m a l equilibrium. T h e reason for this is that, although the index of refraction changes considerably with t e m p e r a t u r e , the increment for a solution changes v e r y little. I n practice the a u t h o r found it therefore a d v a n t a g e o u s to carry out all m e a s u r e m e n t s at room t e m p e r a t u r e r a t h e r t h a n in t h e r m o s t a t e d cells. For the actual values of the indices of refraction increments for the more c o m m o n proteins the readers are referred to the original literature. ~s,46-4s 43N. Bauer and K. Fajans, in "Physical Methods of Organic Chemistry" (Weissberger, ed.), 2nd ed., Vol. I, Part 2, p. 1141. Interscience Publishers, New York, 1949. 44A. E. Brodsky and N. S. Filippowa, Z. physik. Chem. B23, 399 (1933). 45C. A. Browne and F. W. Zerban, "Physical and Chemical Methods of Sug~tr Analysis," 3rd ed., Table 6. John Wiley & Sons, New York, 1941. 4GS. It. Armstrong, Jr., 1V[. J. E. Budka, K. C. Morrison, and M. ttasson, J. Am. Chem. Soc. 69, 1747 (1947). 47 G. E. Perlmann and L. G. Longsworth, J. Am. Chem. Soc. 70, 2719 (1948). 4s H. A. Barker, J. Biol. Chem. 104, 667 (1934).

164

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[5]

Experimental Procedure The ligh~scattering method requires the measurement of scattering data in solutions of different concentrations as well as in the pure solvent. The first step is therefore the preparation of a stock solution of the substance under investigation and of the solvent. Their preparation presents problems not encountered in usual physicochemical measurements. The most stringent requirement is that of the purity of the liquids, i.e., their freedom from any large-size particles, dust, lint, etc. In protein solutions, of course, this means also the freedom from any coagulated material, denatured protein, which is subject to easy aggregation, or surfacedenatured films. The solvents are best purified by distillation, which can be carried out in sealed glass vessels, the receiving vessel being rinsed by the distillate, which is then returned to the distilling flask. In protein solutions the solvent is usually a buffer solution, and, although carefully distilled water is used for its preparation, dust particles are introduced with the salts. These buffer solutions have therefore to be purified in a similar way as the protein solutions themselves, and, as a matter of fact, salt solutions can be frequently obtained in a colloidally purer state than salt-free water, owing to their coagulating activity. There is no single foolproof procedure which can be employed for the purification of protein solutions. Rather, for each system an individually developed procedure will have to be followed, using all the possible means of purification. These include notably a combination of ultracentrifugation and filtration through Seitz filters, Selas candle filters, sintered-glass filters, etc. The purity of solutions can be attested by several methods. One is the visual observation of the solution in the light-scattering apparatus from a forward direction. Any dust reveals itself in the form of small, brilliant particles. Frequently, a better test is to be found in the measurement of the dissymmetry of scattering, which is the first to increase in the presence of large particles. With solutions possessing an intrinsic dissymmetry of scattering, a good precaution is to ascertain that a repeated cycle of purification steps does not reduce the dissymmetry. In the experience of the author, gained with hydrophylic colloids, which are difficult to purify, or with egg albumin, known for its rapid surface denaturation, the dissymmetry can best be materially reduced to a fixed value by repeated filtration through the finest-size Seitz filters; this is followed by ultracentrifugation and final filtration through sintered-glass filters, to remove any dust coming from the Seitz filter pads. Fine gels, sometimes present in such solutions, invisible and not eliminated by centrifugation, are broken by the filtration and easily centrifuged afterwards.

[5]

LIGHT-SCATTERING

MEASUREMENTS

165

A particularly tedious problem is the one encountered with proteins subject to surface denaturation, to which serum proteins are not very prone, but of which the egg albumin is a good example. For the final filtration of such solutions the author has designed the filter presented in Fig. 3. 31 The solution to be filtered is introduced into compartment A and forced by nitrogen under pressure into compartment B through the ultrafine sintered-glass filter, C. Air can first be completely expelled from the filter element by forcing through it large volumes of distilled water, which also rinses the receiving compartment with dust-free water. This compartment is protected from dust by a glass timble. If this water is left in the filter element, and the protein solution is introduced into

A

B

£

F m . 3. Sintered-glass filter. 3~

compartment A and forced through the filter, it rises in the other compartment without any foaming or even inclusion of air bubbles. The water first contained in the filter element forms a well-visible layer on top of the protein solution and protects it from surface denaturation, as there is no protein-air interface. The same filter is also useful for the filtration of volatile liquids, as no evaporation takes place. The filters require good care for preservation of their filtering ability, and concentrated nitric acid was found to be the best cleaning agent. All the glassware employed in light-scattering measurements, notably the cells and pipets used in transfer of liquids, also has to be prepared with greatest care. They should be rinsed with dust-free solvents and dried in dust-free ovens, preferably in all-glass containers. Detergents are usually employed for their cleaning. For more energetic cleaning, the author has found concentrated nitric acid superior to the standard chromic acid cleaning solution, as the latter tends to coagulate proteins and is also washed out only with great difficulty. The determination of the exact concentration of the solutions is not

166

TECHNIQUESFOR CHARACTERIZATIONOF PROTEINS

[6]

always simple. Usually, the various dilutions employed in the measurements are prepared by progressive dilutions of a stock solution in the light-scattering cell. If measurements at very low concentrations are desired, increasing a m o u n t s of the stock solution are added to the pure solvent. I n m a n y cases the author has found it preferable to prepare every dilution in a separate light-scattering cell, as it avoids gradually increasing contamination of the solutions. Where the solute is available in dry state, as is the case with most polymers, the stock solution of desired concentration is directly prepared, with care t h a t no loss of concentration occurs in the process of purification. I n the case of protein solutions the usual methods of concentration determination are Kjeldahl nitrogen, d r y weight, index of refraction, etc., measurements. These methods are not unambiguous, however, and m a y give rise to discrepancies. 49 49p. L. Kirk, Advances in Protein Chem. 3, 155 (1947).

[6] F10w Birefringence

By W. F. H. M. MOMMAERTS The investigation of flow birefringence (double refraction of flow, anisotropy of flow, streaming birefringence) has not played a great role in enzymology. M y o s i n - A T P a s e is the only well-defined enzyme which is birefringent to a marked extent and which has been the subject of considerable investigation. 1-~ Recently, however, it has become technically possible to extend this m e t h o d of observation to less asymmetric molecules ~°-13 so t h a t several enzymes m a y become accessible to such 1F. Binkley, J. Biol. Chem. 174, 385 (1948). 2 M. Dainty, A. Kleinzeller, A. S. C. Lawrence, M. Miall, J. Needham, D. M. Needham, and S. C. Shen, J. Gen. Physiol. 27, 355 (1944). 3 j. T. Edsall and J. W. Mehl, J. Biol. Chem. 133, 409 (1940). 4 A. S. C. Lawrence, J. Needham, and S. C. Shen, J. Gen. Physiol. 27, 201 (1944). W. F. H. M. Mommaerts, Arkiv Kemi~ Mineral. Geol. 19A, No. 17 (1945). 6 W. F. H. M. Mommaerts, "Muscular Contraction, A Topic in Molecular Physiology." Interscience Publishers, New York, 1950. A. yon Muralt and J. T. Edsall, J. Biol. Chem. 89j 315 (1930). 8 A. yon Muralt and J. T. Edsall, J. Biol. Chem. 89, 351 (1930). 9 M. Joly, G. Schapira, and J. C. Dreyfus, Arch. Biochem. and Biophys. 59, 165 (1956). to j. T. Edsall and J. F. Foster, J. Am. Chem. Soc. 70, 1860 (1948). xl j. T. Edsall, J. F. Foster, and H. Scheinberg, J. Am. Chem. Soc. 69, 2731 (1947). 12j. T. Edsall, C. G. Gordon, J. W. Mehl, H. Scheinberg, and D. W. Mann, Rev. Sci. Instr. 15~ 243 (1944). la j. F. Foster and J. T. Edsall, J. Am. Chem. Soc. 67, 617 (1945).

166

TECHNIQUESFOR CHARACTERIZATIONOF PROTEINS

[6]

always simple. Usually, the various dilutions employed in the measurements are prepared by progressive dilutions of a stock solution in the light-scattering cell. If measurements at very low concentrations are desired, increasing a m o u n t s of the stock solution are added to the pure solvent. I n m a n y cases the author has found it preferable to prepare every dilution in a separate light-scattering cell, as it avoids gradually increasing contamination of the solutions. Where the solute is available in dry state, as is the case with most polymers, the stock solution of desired concentration is directly prepared, with care t h a t no loss of concentration occurs in the process of purification. I n the case of protein solutions the usual methods of concentration determination are Kjeldahl nitrogen, d r y weight, index of refraction, etc., measurements. These methods are not unambiguous, however, and m a y give rise to discrepancies. 49 49p. L. Kirk, Advances in Protein Chem. 3, 155 (1947).

[6] F10w Birefringence

By W. F. H. M. MOMMAERTS The investigation of flow birefringence (double refraction of flow, anisotropy of flow, streaming birefringence) has not played a great role in enzymology. M y o s i n - A T P a s e is the only well-defined enzyme which is birefringent to a marked extent and which has been the subject of considerable investigation. 1-~ Recently, however, it has become technically possible to extend this m e t h o d of observation to less asymmetric molecules ~°-13 so t h a t several enzymes m a y become accessible to such 1F. Binkley, J. Biol. Chem. 174, 385 (1948). 2 M. Dainty, A. Kleinzeller, A. S. C. Lawrence, M. Miall, J. Needham, D. M. Needham, and S. C. Shen, J. Gen. Physiol. 27, 355 (1944). 3 j. T. Edsall and J. W. Mehl, J. Biol. Chem. 133, 409 (1940). 4 A. S. C. Lawrence, J. Needham, and S. C. Shen, J. Gen. Physiol. 27, 201 (1944). W. F. H. M. Mommaerts, Arkiv Kemi~ Mineral. Geol. 19A, No. 17 (1945). 6 W. F. H. M. Mommaerts, "Muscular Contraction, A Topic in Molecular Physiology." Interscience Publishers, New York, 1950. A. yon Muralt and J. T. Edsall, J. Biol. Chem. 89j 315 (1930). 8 A. yon Muralt and J. T. Edsall, J. Biol. Chem. 89, 351 (1930). 9 M. Joly, G. Schapira, and J. C. Dreyfus, Arch. Biochem. and Biophys. 59, 165 (1956). to j. T. Edsall and J. F. Foster, J. Am. Chem. Soc. 70, 1860 (1948). xl j. T. Edsall, J. F. Foster, and H. Scheinberg, J. Am. Chem. Soc. 69, 2731 (1947). 12j. T. Edsall, C. G. Gordon, J. W. Mehl, H. Scheinberg, and D. W. Mann, Rev. Sci. Instr. 15~ 243 (1944). la j. F. Foster and J. T. Edsall, J. Am. Chem. Soc. 67, 617 (1945).

[6]

FLOW BIREFRINGENCE

167

studies. Further, the m e t h o d m a y become of use for the investigation of organized enzyme systems such as myofibrils 14 or mitochondria.15 Finally, some substrates of enzymic transformations are birefringent, e.g., starch 16 or nucleic acid, 17 and the measurement of this property during enzymic degradation m a y give information similar to t h a t more commonly obtained from viscosity or t u r b i d i t y measurements. For these reasons combined, a v e r y brief outline of the subject is in order, but it will be impossible to present any advanced theory or any actual detail of measurement. Recent reviews by Cerf and Scheraga 18 and by Signer, 34 as well as some older summaries ~9,2° can be consulted for further study. T h e most outstanding investigation on flow birefringence in a biochemical system is t h a t of von M u r a l t and Edsall on myosin; 7,8 this publication is strongly recommended to anyone planning to study this field. Additional references will be given under the different headings. The problem of flow birefringence has two entirely different aspects: the optical and the hydrodynamic. These will be dealt with separately.

Optical Principles Birefringence. The

optical phenomena are the same as those encountered in the observation of anisotropic objects such as fibers with the polarization microscope. 2~-23 In the simplest cases, the fiber constitutes a monoaxially birefringent body, the optical axis coinciding with the longitudinal axis. In such a body, a pencil of light traversing the fiber in the direction of the axis will not show any remarkable behavior, but a ray entering in a n y other direction will be refracted as if it were a mixture of two different rays, called the ordinary and the extraordinary, which are both polarized. The former, the electrical vibrations of which are perpendicular to the plane defined by its direction and the optical axis, follows 14 A. F. Schick and G. M. Hass, J. Expll. Med. 91, 655 (1950). 15 G. H. Hogeboom, W. C. Schneider, and G. E. Palade, J. Biol. Chem. 172, 619 ~1948). i~ j. F. Foster and I. H. Lepow, J. Am. Chem. Soc. 70, 4169 (1948). 17 W. J. Frajola, J. G. Rabatin, and H. C. Smith, Biochim. et Biophys. Acta 19, 540 (1956). 18 R. Cerf and H. A. Scheraga, Chem. Revs. 51, 185 (1952). 19j. T. Edsall, Advances in Colloid Sci. 1, 269 (1942). 2oj. T. Edsalt, in "Proteins, Amino Acids and Peptides as Ions and Dipolar Ions" (Cohn and Edsall, eds.), Chapter 21. Reinhold Publishing Corp., New York, 1943. 2~ H. Ambronn and A. Frey, "Das Polarizationsmikroskop." Akademische Verlagsgesellschaft, Leipzig, 1926. 22H. S. Bennett, in ' H a n d b o o k of Microscopical Technique" (McClung, ed.], Chapter 9. Paul B. Hoeber, New York, 1948. ~a M. Berek et al., "Anleitung zu optischen Untersuchungen mit dem Polarizationsmikroskop." E. Schweitzerbart'sche Verlagsbuchhandlung, Stuttgart, 1953.

168

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[6]

the classical laws of refraction. The latter, of which the electrical vibration vector is in the plane of the ray and the optical axis, has a variable refractive index dependent on the direction of its impact. The detailed explanation of the phenomena resulting from the differences between the refractive behavior of the two rays is quite impossible within the space allotted here, but can be obtained elsewhere. 24 For the understanding i~ OBSERVER

DIRECTION OF I,,~"--'--'------ POLARIZATION ~ OF ANALYZER

> i

OPTICAL AXIS OF OBSERVATION SYSTEM

~ ~ _ . . ~ . . O F --~

j,

DIRECTION OF f POLARIZATION POLARIZER

A NA LYZ E R ...._..._.~~ " ~ ' ~ PLANE OF ~ POLARIZATION ~ OF ANALYZER

ORIENTATION I DARK

I !

I

/ I

! I

ORIENTATION 2 MAXIMAL LIGHT

I

POLARIZER - . - ~ . . . ~ { ~ - - ~ PLANE OF

~

! -J

POLARIZATION " ' - OF POLARIZER LENS LIGHT SOURCE

I ,~ ~

,¥

t

ORIENTATION 5 DARK

I

Fro. 1. Left, scheme of a polariscope with vertical observation. In a plane between the polarizers and perpendicular to the system's axis (in practice: on a horizontal glass plate between the polaroids), a fiber is placed in different orientations (1-3). Right, schemes of the appearance of this fiber. In the two parallel positions (1 and 3, corresponding to the same positions on the left) the object remains dark. In other orientations it is light, maximally in the diagonal position (2).

of this chapter, however, it is quite sufficient to know that an elongated monoaxiaUy birefringent body, placed in the field between two crossed polarizers in a plane perpendicular to the line between the polarizers (the direction of observation), is dark when its optical axis is parallel to the direction of polarization of either the polarizer or the analyzer; it shows up light when it has any other orientation within that same plane, maximally when it is in the diagonal position. The schemes in Fig. i further e~.ucidate this summary statement. The object in these schemes is a short fiber, but 24 R. W. Wood, "Physical Optics." The Macmillan Co., New York, 1934.

[6]

FLOW BIREFRINGENCE

16(,)

we can think equally well of a rod-shaped macromolecule, except t h a t only the summated effect of a large n u m b e r of them would be observable. In both cases, fibers and molecules, we can frequently assume that the optical and the geometrical axes coincide. Two further properties of birefringent systems need be explained, viz.. the sign and the intensity of the double refraction. A body is called positively birefringent when the refractive index of the extraordinary ray in a direction not coinciding with the axis (he) is larger than t h a t of the ordinary ray (no) or, correspondingly, when the effective wavelength of the e-ray in t h a t direction in the object is smaller than t h a t of the o-ray. Negative birefringence is then the opposite case. Although the complete appreciation of these definitions demands further study, a simple experimental criterion can be introduced at this time. When a so-called gypsum plate red I (which, by itself, shows red between crossed polarizers) is inserted above the object., both being in the diagonal position and parallel to each other, the object will appear blue when it is positively birefringent, yellow when it is negatively birefringent. Tile intensity of the birefringence is defined by the magnitude of the difference ne - no. Dependent on the value of this difference, the e- and o-rays emerge from the object with a phase difference ,y = d(ne - no)/~o (in which d is the thickness of the object, h0 the wavelength in vacuo), giving rise to interference when, oll passage through the analyzer, the two perpendicularly vibrating rays are brought into the same plane of polarization. The value of 7 is measured b y means of instruments called compensators, such as the Sdnarmo~J or the Babinet compensator. ~,23 When the phase difference is sufficiently large, the emergent light is colored, but i~ biochemical systems this is usually not the case, and the light is gray or white. "-'~ Although the' intensity of birefringence is thus defined ill terms of a phase difference, the subjective intensity of the light m a y parallel the true a m o u n t of birefringence; in fact, photoelectric measurement has occasionally been employed.~7.:7 Intrinsic and F o r m Birefringence. In objects such as biological fibers, the double refraction is usually due in part to a fixed spatial orientation of molecules which are, in themselves, anisotropic. This is, however, not the only possible mechanism. Isotropic materials can, according to Wiener (see ref.2~), give rise to birefringence if they are arranged in a special manner, e.g., as rods of one isotropic substance of refractive index n~, embedded in a matrix of another, of different refractive index n~. T h e 55However, interference colors are strikingly shown in solutions of pure F-actin, at concentrations as low as 0.5%. 2~ ~ W. F. H. M. Mommaerts, J. Biol. Chem. 198, 459 (1952). ~7M. A. Lauffer and W. M. Stanley, J. Biol. Chem. 123, 507 (1938).

170

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[6]

requirement is t h a t the rods be v e r y long and v e r y thin in comparison to the wavelength (d << ), << 1). Such a body is positively anisotropic, whether nl is smaller or larger than n~. The presence of rod birefringence can be detected b y impregnating the object with media of varying refractive indices, provided t h a t these replace the embedding medium but do not modify nl. When n2 is made equal to nl, the form birefringence becomes zero, and increases according to a bell-shaped curve when I n 2 - n~ I increases; any remaining double refraction at the minimum of the curve indicates intrinsic birefringence of the material of the rods itself, which can be positive or negative. In biological objects this situation often prevails when rodlike micelles or filaments of submicroscopic thickness, embedded in an isotropic medium of lower optical density, have themselves an oriented molecular structure. In the muscle fibril, for example, form birefringence and the positive intrinsic birefringence of the actomyosin filaments are additive. ~s An additional cause of incidental birefringence m a y be in the directed adsorption of solvent. ~9 H y d r o d y n a m i c Principles Orientation i n Solution. In the structures discussed in the preceding section, orientation of the molecules or micelles is maintained b y structural factors. In solutions no p e r m a n e n t orientation can exist, but it can

v

FIG. 2. Orientation of a particle in a flow gradient. The dashed arrows signify the velocity of flow at different levels; the solid arrows indicate the magnitude of the forces acting on the ends of the particle. When the particle projects through different levels (position 1) the resulting couple of forces effects an orientation; this couple vanishes when the particle (position 2) is oriented in the streamlines. be induced b y an external influence such as the maintenance of flow. Particles in a flow field are subjected to an orienting influence which tends to align them parallel with the streamlines, perpendicular to the velocity gradient (Fig. 2). This orienting influence is counteracted b y Brownian movement, which tends to restore a random distribution of directions. The degree of orientation finally resulting depends on the balance of these opposed tendencies. In such intermediary cases, we can ~8K. Noll and H. H. Weber, Arch. ges. Physiol. 235, 234 (1934). 29D. Vermaas, Z. physik. Chem. B52, 131 (1942).

[6]

FLOW BIREFRINGENCE

171

assign an angle, ¢, signifying the average angle of orientation measured toward the streamline; for complete orientation, ¢ = 0. This orientation by flow can be brought about in various ways, the simplest being by rotational flow. This can be achieved with modest means by swirling the solution in a beaker (or by stirring it with an opaque rod) between crossed polaroids (Figs. 3A and B). More perfect observation can be done in a rotating cylinder apparatus (Fig. 3C; see section on apparatus). If it is assumed that in the simple apparatus, Fig. 3A, the regular flow pattern of Fig. 3B is obtained, it is easy to derive what observations will be made. If the orientation is complete (4 =.0), all the particles will be

ANALYZER

B

POLARIZER r....,.~....,.I

N ' CAN3~SCE~NT

C

FIG. 3. (A) A simp]e apparatus for the observation of flow birefringence, observation from above. (B) Schematic diagram of the flow patterns induced by stirring or swirling. (C) Schematic diagram of the flow pattern in a rotating cylinder apparatus, with stationary inner cylinder.

arranged parallel with the circular streamlines (Fig. 4A). This means that in four segmental positions in the beaker the particles will be parallel with the direction of vibration of either the polarizer or the analyzer. In these parts, the solution will appear dark between crossed polaroids, whereas elsewhere, the particles having other orientations, the solution will light up. As a result, there appears a dark cross, the cross of isocline, within a light field (Fig. 4B). In practice, since the flow is not regular, the cross will be irregularly twisted, but this does not change the principle. If the orientation is not maximal, but ~bhas a value of, say, 30 °, the orientation of the particles will be as in Fig. 4C, and the cross will appear at this angle (Fig. 4D). Therefore, measurement of the angle of isocline relative to the vibrational directions constitutes a direct measurement of the average angle of orientation of the individual molecules relative to the flow

172

TECHNIQUES

FOR CHARACTERIZATION

OF PROTEINS

[6]

lines. For more accurate measurement of ~b, one would employ a rotating cylinder type of instrument. Elementary Theory. A few explanations will suffice to indicate the type of information obtainable from quantitative measurements. In general, one studies the relation between the magnitude of the streaming field and the angle of orientation. Measurement of the inteHsity leads to additional information, which will not be considered here. ,

I

I

I

,

I

A

i

I

-

-

B

i

-I\\\\\\\\\~.y:

.

.

.

G

.

i

l

D

I

Fro. 4. (A) Complete orientation of particles in a rotating fluid, leading (B) to the

appearance of a sharp orthogonally placed isoclyne cross. (C) Incomplete orientation of particles, assuming an average angle, ~, with the streamlines, measurable (D) as the angle of the isocline cross. The velocity gradient is easily calculated in the rotating cylinder apparatus schematically indicated in Fig. 3C. If the gap is narrow, the velocity of the fluid increases linearly from the wall of the stationary inner cylinder, where it is zero, until the wall of the rotating outer cylinder, where it equals the speed of the latter. The velocity gradient, ~, is then given by ~R~ R2 R1 -

-

-

in which R1 and R,. are the radii of the inner and outer cylinders (in centimeters) and ~ is the angular velocity (in radians per second);/~ is thus derived from knowledge of the speed of rotation and the dimensions of the instrument. As stated before, the orientation angle ~ is measured optically.

[6]

FLOW BIREFRINGENCE

173

The orienting effect of the flow field on the particles increases with the length of the particles. This orienting effect has to compete with the randomization b y rotational Brownian movement. The tendency of molecules to rotate is expressed in terms of a rotational diffusion constant, DR. Values of DR are smaller for large than for small molecules and decrease notably when the molecules become asymmetric. Thus, longer particles will be b e t t e r oriented both because the flow affects them more and because the thermal disorientation is less effective. According to theory originated by Boeder, the orientation is determined b y a parameter a = ~/Dn. When orientation is at all observable, it starts with a value ~b = 45°; when a assumes larger values, ¢ approaches smaller angles and the intensity of the birefringence increases. F r o m experimental measurements, then, it is possible to obtain a and, since f~ is known, to measure DR. Knowledge of this constant, on the other hand, allows conclusions as to the length of the molecules. Several such measurements on proteins have been discussed by Edsall, ~9~.0and some determinations have been reported more recently. 9,'°,''.'3,18 Instrumentation

Qualitative Observations. For m a n y simple observations on strongly birefringent systems, a simple polariscope as pictured in Fig. 3A is most satisfactory. This can be easily constructed in Che laboratory, with two 2-inch Polaroid disks; glass beakers of, say, 30 ml. are used, which are selected for reasonably strain-free bottoms by inspection in the same apparatus. Systems like T M virus, 2m°,3~ fibrous insulin, '~-"actomyosin, ~,5 and F-actin 26 show light effects of a remarkable intensity. Qualitative observations can give a great deal of information about the disaggregation of such systems by added reagents ~ or about the occurrence of polymerization -"~,32 or other transformations. 2 When even at such lowvelocity gradients ¢ = 0, it can be concluded t h a t the particles are extremely long. 5 Rotating Cylinder Apparatus. Two different forms exist, in which the inner or the outer cylinder, respectively, rotate. The latter is theoretically preferable, and it is now generally preferred for instruments of moderate precision, used with systems of such strong birefringence t h a t a slight strain in the glass b o t t o m does not interfere. I n s t r u m e n t s of this nature have been described b y several authors;* recently a v e r y satisfactory design was published in full detail ~3 (this model is commercially available). 30 j. R. Robinson, Proc. Roy. Soc. A170, 519 (1939). 3, W. N. Takahashi and T. E. Rawlins, Science 77, 26, 284 (1933). a~ D. F. Waugh, J. Am. Chem. Soc. 68, 247 (1946). 33j. T. Edsall, A. Rich, and M, Goldstein, Rev, Sci, Instr. 25, 695 (1952).

174

TECHNIQUES FOR CHARACTERIZATION OF PROTEIN'S

[7]

In a similar instrument constructed in the author's laboratory, the rotating cylinder with a reasonably strain-free bottom is made of one piece of fused glass. High-Precision Instruments. When very high precision and sensitivity is needed, it appears impossible to use anything but a very small isotropic observation window, which is practical only when the outer cylinder is stationary. In order to obtain the necessary high velocity gradients, the annular gap is very narrow, e.g. 0.3 mm., which makes the alignment difficult. Several such instruments have been constructed,~4 but details of high-quality apparatus are not always available. 3~Fully described are the precision instrument of Snellman 36 and Fredericq and Desreux, ~7 which has an ingenious optical system to eliminate the effect of reflections, and that of Edsall et al.,12 with the aid of which even the orientation of relatively short macromolecules can be studied. In earlier years, orientation was often achieved by capillary flow, but this method is now held to be inferior except for special purposes.3~ 34 R. Signer, Determination of streaming birefringence, in "Physical Methods of Organic Chemistry" (A. Weissberger, ed.). Interscience Publishers, New York, 1954. 35 C. Sadron, J. phys. radium [7] 7, 263 (1936). 3~ O. Snellman and Y. BjSrnsthhl, Kolloid Beih. 52, 403 (1941). a7 E. Fredericq and V. Desreux, Bull. soc. chim. Belges 56, 403 (1948).

[7] Fluorescence Techniques for the E n z y m o l o g i s t

By DONALD J. 1~. LAURENCE

Light Sources for Excitation of Fluorescence Conditions for Excitation. The production of fluorescence by a fluorescent compound will occur only as long as the substance is illumihated and will cease abruptly when illumination is discontinued. To excite fluorescence, the wavelength of the exciting light must fall within a visible or ultraviolet absorption band of the substance. No fluorescence will result if the incident light is of a wavelength longer than that of the red edge of the absorption band of a compound. Compounds with an obvious fluorescence, e.g., fluorescein, rhodamine B, and 5-aminoacridine, are brightly fluorescent in daylight, as they absorb appreciably the region of visible light. Compounds without noticeable fluorescence in daylight may become brightly fluorescent when illuminated with ultraviolet light. As a general rule, substances with visible color may be excited by blue or even by green light, whereas substances without visible color require

174

TECHNIQUES FOR CHARACTERIZATION OF PROTEIN'S

[7]

In a similar instrument constructed in the author's laboratory, the rotating cylinder with a reasonably strain-free bottom is made of one piece of fused glass. High-Precision Instruments. When very high precision and sensitivity is needed, it appears impossible to use anything but a very small isotropic observation window, which is practical only when the outer cylinder is stationary. In order to obtain the necessary high velocity gradients, the annular gap is very narrow, e.g. 0.3 mm., which makes the alignment difficult. Several such instruments have been constructed,~4 but details of high-quality apparatus are not always available. 3~Fully described are the precision instrument of Snellman 36 and Fredericq and Desreux, ~7 which has an ingenious optical system to eliminate the effect of reflections, and that of Edsall et al.,12 with the aid of which even the orientation of relatively short macromolecules can be studied. In earlier years, orientation was often achieved by capillary flow, but this method is now held to be inferior except for special purposes.3~ 34 R. Signer, Determination of streaming birefringence, in "Physical Methods of Organic Chemistry" (A. Weissberger, ed.). Interscience Publishers, New York, 1954. 35 C. Sadron, J. phys. radium [7] 7, 263 (1936). 3~ O. Snellman and Y. BjSrnsthhl, Kolloid Beih. 52, 403 (1941). a7 E. Fredericq and V. Desreux, Bull. soc. chim. Belges 56, 403 (1948).

[7] Fluorescence Techniques for the E n z y m o l o g i s t

By DONALD J. 1~. LAURENCE

Light Sources for Excitation of Fluorescence Conditions for Excitation. The production of fluorescence by a fluorescent compound will occur only as long as the substance is illumihated and will cease abruptly when illumination is discontinued. To excite fluorescence, the wavelength of the exciting light must fall within a visible or ultraviolet absorption band of the substance. No fluorescence will result if the incident light is of a wavelength longer than that of the red edge of the absorption band of a compound. Compounds with an obvious fluorescence, e.g., fluorescein, rhodamine B, and 5-aminoacridine, are brightly fluorescent in daylight, as they absorb appreciably the region of visible light. Compounds without noticeable fluorescence in daylight may become brightly fluorescent when illuminated with ultraviolet light. As a general rule, substances with visible color may be excited by blue or even by green light, whereas substances without visible color require

[7]

FLUORESCENCE

TECHNIQUES

FOR THE ENZYMOLOGIST

175

ultraviolet excitation. Certain aromatic amines, phenols, and indole derivatives--e.g., tyrosine, tryptophan, and related metabolites-require short-wavelength ultraviolet light to excite a fluorescence which is entirely in the longer-wave (but invisible) ultraviolet region. Choice of Wavelength for Excitation. A knowledge of the absorption spectrum of the compound enables the best wavelength for excitation to be decided, viz., the absorption maximum of the compound. The presence of masking colors from impurities, reagents, etc., may preclude the use of this wavelength, however. An empirical method for determining the most efficient wavelength for excitation by means of a spectrophotometer is as follows. A suitable dilution of the fluorescent substance is placed in a spectrophotometer cuvette (no lid) with the photocell shutter closed and the exit slit opened wide. The room is darkened, and the cuvette is viewed from the top. The monochromator control is used to scan the solution with light of different wavelengths, and, commencing from the red, the wavelength is decreased. A more or less sharp onset of fluorescence occurs as the wavelength of the red edge of the absorption spectrum is reached, and the region of maximum excitation is easily observed. The fluorescence color does not depend on the wavelength of the exciting light. If a color changing with wavelength is obtained, this is probably due to scattered light, although the presence of more than one fluorescent substance in the solution must also be taken into account. If the fluorescence moves toward the illuminated face of the cuvette without noticeable quenching, the solution is too concentrated and should be diluted. If quenching is clearly marked, the presence of a masking color due to a nonfluorescent impurity must be suspected. The Medium-Pressure Mercury Lamp. This is the most generally useful source of exciting light. A lamp with a clear glass envelope is used unless a very diffuse beam is required. This lamp produces strong lines in the ultraviolet (365 m~), violet (405 m~), blue (436 m~), and green (546 mu) spectral regions. A yellow line is also present, but this is rarely required. If "corrected" by inclusion of metallic Cd, the lines of Cd at 468 and 480 mu will be present. Some red light due to the inert gas primer is always present. Advantages of this lamp are the high brightness value, the high intensity in a few well-separated lines which enables strong monochromatic light to be isolated, and the high ultraviolet output (2 to 3 % efficiency for wavelengths <400 m~). Disadvantages are the need for a choke or ballast transformer and the wandering of the arc inside the lamp. This last effect is small if a stabilized-arc lamp is used. The author has recently found that the Osram (Germany) spectral lamp type HgS is free from the usual instability and may be operated

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from a standard 230-volt 300-watt constant-voltage transformer with a series resistance. The Tungsten Lamp (Headlamp Bulb). This is also used to excite fluorescence, although the ultraviolet output is small (0.01% efficiency for wavelengths < 400 m~) and mainly in the near-ultraviolet ( > 370 m~) region. Used with the more sensitive detectors now available, the tungsten lamp is sometimes advantageous, as photochemical destruction is reduced and the low efficiency can be compensated by a high efficiency of the detector. A very stable power supply is required, as the ultraviolet output is sensitive to small changes in applied voltage. A large storage battery or a battery-stabilized power supply is recommended. The tungsten lamp may be over-run (10% voltage overload), with a considerable increase in ultraviolet output, but the life of the bulb is reduced about fivefold. For visual comparisons, the life of an over-run bulb may be extended by switching on only for the short time required for the estimation. A Hg lamp cannot be flashed in this way. The Low-Pressure (Germicidal) Mercury Lamp. This lamp can be used to supply short-wavelength ultraviolet "resonance radiation" at 254 m~. This light is not transmitted by glass, as is 365 m~, and can cause severe damage to the eyes if they are not protected by suitable screening. The main application of this wavelength in enzymology is to the examination of chromatograms. By coating the lamp bulb with an ultraviolet-emitting phosphor such as the 360 BL (peak output 350 m~) or the erythemal (peak output 320 m~) phosphors, the low-pressure lamp may be modified to emit at longer wavelengths of more general utility. Of special interest are the small "cathode glow" lamps adapted with these phosphors which may be operated from a 12- or 24-volt supply, with a power consumption of 3 to 4 watts.l Compact Light Sources. For measurement of fluorescence spectra a very intense line source is obtained from a high-pressure mercury lamp between point electrodes a few millimeters apart. Compressed source Hg arcs are available from many lamp companies and provide the most intense line source suitable for general laboratory use. These lamps are also indicated when instrument design requires a sharply defined point source. For measurement of the activation spectrum of a fluorescent substance, a compact continuous spectrum source is the xenon arc for which stabilizing transformers are available. The xenon arc is brighter, though somewhat less efficient, than the hydrogen arc in the wavelength region 2500 to 3200 A., and it emits an intense continuous spectrum throughout the longer-wavelength ultraviolet and visible regions of the spectrum. I W. E. Forsy~he and E. Q. Adams, "Fluorescent and Other Gaseous Discharge Lamps." Murray Hill Books, New York, 1948.

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Fluorescent Light Detectors Visual Comparisons. The simplest fluorimetric comparisons of solutions showing moderate intensity of fluorescence are made visually. An even, diffuse beam of ultraviolet light is used to illuminate the fluorescent solution under test, and also a suitable series of standard solutions, placed side by side in matched reagent glasses. Because of the difficulty of matching "luminous volumes" it is more convenient to arrange a fiat display of the intensities. The Photovolt Fluorescence Comparator, Model 60, uses this principle. Visual comparisons may also be made for paper chromatography. Visual Photometry. By means of a visual matching colorimeter, numerical estimates of fuorescence intensity may be obtained from the calibrated scales of the instrument. A Pulfrich step photometer is especially suitable, as the brightness of the field is very good. A fluorimeter using the Pulfrich photometer is shown in Fig. 1. A uniform diffuse beam of exciting light falls on the faces of the two prisms, P1 and P2, and is deflected through the cuvettes, K1 and K2, which are backed with mirrors, MI-M~. The internal width of the cuvettes must be at least 1 cm. to ensure that the field of the photometer is uniformly illuminated. This may be checked by adding the small lens, L, to the eyepiece. The standard solution is placed in the left-hand side of the photometer and should be at least as fluorescent as the unknown solutions, which are placed on the right-hand side. The right-hand control, C~, is set at a constant value, and adjustments made to the left-hand control, C1, to obtain equality of the fields in the eyepiece. The readings of C~ for different unknown solutions will be proportional to the fluorescence intensities, if the transmission scale is used. The Duboscq colorimeter can also be adapted as a fluorimeter.2 Advantages of the visual fluorimeter are the insensitivity to moderate fluctuations in the light source and the ease with which changes in the color of fluorescence may be observed. Disadvantages are the difficulty of choosing a standard solution for comparisons, both stable and of the correct color for matching, and of subtracting the blank, which is ge~erally of a fluorescence color different from that of the test substance. It is difficult to measure small differences between fluorescence intensities with proper objectivity, and visual matching becomes inaccurate if the light intensity is low. Photoelectric Methods. For general purposes a photoelectric detector is satisfactory. Barrier' layer cells are suitable for moderate fluorescence intensities but fail to give adequate sensitivity to meet all requirements. 2 W. Koch, Nature 154, 239 (1944).

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A v a c u u m photocell with a high-gain amplifier or a photomultiplier provides high sensitivity, and, of these, the latter is v e r y useful as a detector of fluorescence. The sensitivity of the photomu]tiplier m a y be varied over a range of 105 b y variation of the applied voltage. An o u t p u t ×H

i

I I

I

~2

F~ FIG. 1. Visual fluorimeter. H, light source; F1, primary filter; K1 and K2, cuvettes; P1 and P~, prisms; MI-M4, mirrors; F2, secondary filter; Q, heat trap and screen; C1 and C2, controls; L, lens. of 10 ~a. can be obtained without fatigue and m a y be recorded on a robust meter with pointer indication. A stable power supply of about 1000 volts direct current is required. This m a y be obtained from dry batteries (e.g., three Minimax batteries of 300 volts each). Alternatively, a simple mains-operated, neon-stabilized power pack m a y be constructed which is sufficiently stable if it is operated from the constant-voltage transformer

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used to operate the Hg lamp. The N.R.D. corona stabilizer supplies a stabilized high voltage from a single electronic tube. The detector is normally mounted so that it views the fluorescent light along an axis at right angles to the direction of the exciting light beam, to avoid an unduly high blank due to inadequate screening of the direct beam. Complementary filters are employed to reduce the direct participation of the exciting light in the measurement. R4

R

c

~

~

[-'z

x/ H?C

L4

(a)

R5

i'Xl

(b)

FIG. 2. Photoelectric fluorimeter. (a) Optical system. H, light source; F1, primary filter; K, cuvette; F2, secondary filter; S, shutter; D, photocell;LI . . . L4, lenses. (b) Electrical circuit. D, photocell; M, meter; R1 and S (R6), sensitivity controls; R2 and R~, zero setting controls; C, dry battery; Rs, dynode resistance chain; E, earthing point; V, corona stabilizer; RT, stabilizer ballast; C3, smoothing condenser; C1 and C:, X1 and X2, voltage-doublingcircuit; T, transformer; H, light source; R4, lamp ballast; Z, input from constant voltage transformer. Figure 2 shows a typical arrangement of a photoelectric fluorimeter using a photomultiplier. By varying the voltage across the multiplier or by the control of the intensity of the exciting beam, the appropriate range of sensitivity is selected. Defections of the meter are proportional to fluorescence intensities, provided the blank is subtracted. Commercial Fluorimeters. Fluorimeters are available commercially in a wide range of different types, both as attachments to spectrophotometers and as separate instruments. Apart from differences in light source and detectors, the main differences are in the use of constant-voltage stabilizers or of balanced photocells to reduce the consequences of fluctua-

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TECHI~IQUES FOR CHARACTERIZATION OF PROTEINS

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tions in the mains supply. According to Loofbourow and Harris,3 either type is of satisfactory stability. For general routine analysis the balanced instruments might be preferred, and for exploratory research the singlecell types would be more readily adaptable.

Choice of Light Filters

Complementary Filters. As a general rule, the wavelength of the exciting light is shorter than the wavelengths of the fluorescent light. The exciting light is selected by suitable filters (the lamp or primary filter), and a second filter can usually be found to position between the fluorescent solution and the light detector (the secondary or photocell filter) which is opaque to the exciting light but which allows the fluorescent light to pass. Two filters with the property that the light passed by one is absorbed by the other are said to be complementary. Primary Filters. The exciting light is selected b y means of a filter with a well-marked cutoff at the longer-wavelength side of its transmission band. The ultraviolet-transmitting glasses (Corning type 7 response) allow Hg 365 mg to pass, and in certain cases also Hg 405 m# or Hg 405 + 436 mg, but exclude the green and yellow lines of Hg. Similarly the blue filters are efficient in passing Hg 405 + 436 mg. By combining these filters with others with a sharp short-wave cutoff, the range of exciting wavelengths may be restricted to a single spectral line. Thus Corning 3-73 together with Corning 7-59 isolates the Hg 436-mg line. A didymium glass allows Hg 546 mg to pass but blocks the yellow Hg 578 m~. Wavelengths Less Than 365 M~. The natural cutoff of glass excludes wavelengths shorter than 365 m~. However, the resonance radiation of a germicidal Hg lamp at 254 m~ may be isolated, free from 365 m~ and visible lines, by using a few centimeters of a solution containing 300 g. of nickel sulfate (7H~O) and 100 g. of cobalt sulfate (7H~O) (both c.p. grade) in 1 1. of 0.01 N H2S04, placed in a quartz container. If only the visible radiation from the lamp is to be removed, a Chance OX7 or a Corning 7-54 will suffice. The medium pressure Hg lamp emits strong lines at 254 and 310 m~ and weaker lines between these wavelengths. In order to exclude 310 m~ a chlorine gas filter in a quartz bulb is often employed. Recent fluorimeters (e.g. by the Locarte Company, London) use quartz lenses and cuvettes and fluorescence may be measured for compounds requiring excitation by wavelengths shorter than 365 m~. The selection of primary filters for ultraviolet light of short wavelengths is still unsatisfactory although the investigation of quartz fused with various metallic salts J. E. Loofbourow and R. S. Harris, Cereal Chem. 19, 151 (1942).

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would uncover some useful results. For a secondary filter natural glass of various thicknesses and grades is suitable. Quartz is generally less fluorescent than glass and may be recommended for general purposes for construction of cuvettes and lenses. Interference Filters. Good discrimination between Hg lines is obtained with interference filters. As the cutoff of these filters is sometimes incomplete, and owing to the presence of harmonics, it is necessary to add an auxiliary glass filter. Some manufacturers choose a glass filter as the basis for construction of the interference filter. Interference filters are especially useful for lines such as the Hg 404 mu which are difficult to isolate in high intensity with colored glass filters. It is necessary to use a moderately parallel beam of light when interference filters are employed. Secondary Filters. These consist of a short-wavelength sharp cutoff glass such as the Corning type 3 response, which may be supplemented when fluorescent impurities are troublesome by a blue, blue-green, or green filter, matched to the fluorescence spectrum of the fuorescent substance. An approximate estimation of the spectrum of the fluorescence may be obtained with a hand spectroscope, placed close to the irradiated solution of the fluorescent substance. Certain yellow filters are themselves fluorescent and should be preceded by a protecting filter of nonfluorescent type. Thus Lowry4 employs a Wratten 2A (gelatine) filter to protect the Corning 3-73 used as a photocell filter. Yellow filters of chromium glass, such as the Chance OY12, 5 are nonfluorescent. Relation to the Fluorescence Detector. The wavelength of the stray light which is especially troublesome depends on the type of detector selected. With a Wood's glass primary, a secondary filter is often unnecessary for visual observation, since the eye is insensitive to moderate intensities of 365-mu radiation, but it is generally required for photoelectric methods. The red band of the Hg arc and infrared radiation are especially troublesome with barrier-layer cells but are of less importance with photomultipliers. The Coming 4-70 filter is widely used to remove scattered red light, and a 10% copper sulfate solution serves a like purpose. Both are transparent to wavelengths as low as 365 mu. Naturally the problem of filtering is more acute when very dilute solutions of fluorescent substances are being examined.

Practice of Fluorimetry The Inner Filter Effect. One of the chief factors to be controlled when fluorescent solutions are employed is the "inner filter" effect which can be 40. H. Lowry, J. Biol. Chem. 178, 677 (1948). 5 E. J. Bowen and F. Wokes, "Fluorescence of Solutions." Longmans, Green and Co., London, 1953.

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confusing if not anticipated. Any substance absorbing the exciting light, including the fluorescent substance and also impurities and reagents, will reduce the intensity of the exciting light as it passes through the cuvette. As long as the absorption of the exciting light is small, the intensity of light through the cell will be appreciably uniform and the fluorescence intensity will be proportional to the concentration of the fluorescent substance. If the concentration of the fluorescen~ substance is increased until the exciting light is appreciably attenuated on passing through the cuvette, the fluorescence will no longer be proportional to the concentration. Parts of the solution at a distance from the irradiated surface of the cuvette will receive increasingly small amounts of exciting light. At high concentrations of the fluorescent substance the exciting light will become so strongly absorbed that it will not penetrate more than a fraction of a millimeter into the cuvette, and the fluorescence will be confined to the irradiated surface of the vessel, apparent only as a thin surface film of fluorescent light. In early work it was often necessary to work in the region of nonproportionality between fluorescence and concentration, owing to the insensitivity of the available light detectors. With more sensitive detectors the solution may be diluted until light absorption across the cell is small and the fluorescence intensity proportional to the concentration. Any light-absorbing substance added along with the fluorescent substance contributes to the nonproportionality effect. Any lightabsorbing substance of constant concentration will produce a quenching of the apparent fluorescence, when the standard is a pure solution of the fluorescent substance. An alternative method of overcoming the inner filter effect is to select an internal standard of the pure substance, added to the test solution. In this method it is assumed that the added standard does not contribute appreciably to the light absorption. Reduction of the Blank. Dilution of the fluorescent solution to reduce the inner filter effect may result in the emergence of the " b l a n k " as the limiting factor in the determination. The reduction of the blank provides an exercise with both chemical and physical aspects. The possibility of stray light from the room entering the fluorimeter must first be excluded. This can be done by covering the whole instrument with a black cloth and noting whether a change in deflection of the measuring instrument results. By interpolation of an opaque screen between the light source and the instrument it can be ascertained whether the blank is optical or electrical in nature. If a deflection of the meter remains after exclusion of light but disappears when the power supply is disconnected, an electrical leakage must be suspected. With photomultipliers there is inevitably a leakage of about 0.01 ~a at an applied potential of

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TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

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be used, as it is brilliantly fluorescent. Certain grades of filter paper (e.g., Whatman No. 42) are said to contain soluble fluorescent substances; others (e.g., Whatman No. 30) are without such impurities. Rubber stoppers may not be used. Perhaps one of the most important properties of a cuvette holder is that it should be readily removable for cleaning. Contamination with fluorescent substances is very likely to occur through occasional carelessness and can considerably reduce the efficiency of an instrument. Other Considerations. Apart from the inner filter effect and the blank, there are other aspects of fluorescence which require special attention. The fluorimetric measurement is usually independent of the volume of solution within the cuvette over a moderate range of volumes, but tests should be made to confirm this. Cuvettes should be kept free from impurities absorbing ultraviolet light and be cleaned with nitric acid rather than with chromic acid which deposits absorbing films. Certain fluorimeters rise in temperature some 5° or 10° above the room temperature, s owing to heating from the light source. As fluorescence is often temperature-dependent, this can lead to errors if the samples are left in the apparatus for variable periods of time and an unstandardized rise in temperature is permitted. Certain experimental conditions may result in a quenching of the fluorescence due to the composition of the medium. The chloride ion is a well-known quencher of fluorescence, and for this reason standard solutions of quinine sulfate are made in sulfuric, not in hydrochloric, acid. Sulfuric acid is also recommended in the partition of porphyrins between organic solvents and acid solutions. Atmospheric oxygen 8,9 quenches the fluorescence of certain compounds, e.g., quinine sulfate and thiochrome, and more noticeably the fluorescence of carcinogens. For most assays it is sufficient to equilibrate the solution with the atmosphere, but with the carcinogens deoxygenation of the solutions is required. The fluorescence of many compounds is sensitive to the pH of the solution, and this must be controlled by means of buffers. Finally, special care must be taken with solutions used in fluorescence assay. The concentrations are so much smaller than those usually met in quantitative work that the possibilities of atmospheric oxidation, adsorption by glass surfaces, and photochemical destruction during the examination are greatly increased. Adaptors for Filter Paper. Modifications of fluorimeters for evaluation of fluorescent intensity on filter paper have been described. For the conventional 90° arrangement, the paper is mounted at 45 ° to the light source 8 D. 1~. Clausen and R. E. Brown, Ind. Eng. Chem. Anal. Ed. 16, 572 (1944). 9 H. Weil-Malherbe and J. Weiss, Nature 149, 471 (1942).

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and viewed at an equal angle by the detector. Alternatively, the light and detector are mounted in line as in a colorimeter, the paper being placed between so that it is illuminated on one surface and viewed from the opposite surface, using suitable complementary filters. All the usual precautions for fluorimetry are required and, in addition, uniformity of illumination and detector response over the field of observation. The linear response with concentration characteristic of suitable fluorimetric procedures obviates the need for scanning and integration which is associated with the evaluation of colored substances on filter paper, the fluorimetric method being less affected by differences in spot size than the corresponding colorimetric methods. Scanning of fluorescent spots is generally required only when spatial resolution is inadequate or the fluorescent response noticeably non-linear. Applications of Fluorimetry. When proper precautions are taken, fluorimetric methods can be extremely sensitive analytical procedures. The work of Lowry on the microestimation of flavines (Vol. III [141]) and of malic acid 1° and the method of Leonhardi and von Glasenapp 11 for the estimation of acetoacetic acid (Vol. III [51]) illustrate the usefulness of the technique for the assay of metabolic products. Robinson 12 has described an enzyme assay with a substrate (4methylumbelliferone E-glycoside) which is nonfluorescent but which gives rise to a fluorescent product (4-methylumbelliferone) after enzymatic hydrolysis. In this case the enzyme activity could not be followed continuously as the fluorescence of the product was in a pH region outside the region of enzyme activity, but the method was very sensitive. For a number of coenzymes the fluorescence is decreased on reduction by substrate. An excellent review of the many fluorescent substances found in plants is given by Goodwin. TM A number of metal activators of enzymes give soluble fluorescent complexes (e.g. Ca ++, 5Ig ++ and Zn++ with 8-hydroxy-quinoline-5sulphonic acid) which may be used to detect and estimate the metals and may also act as enzyme inhibitors.

Measurement of Activation and Fluorescence Spectra Recently quantitative measurement of fluorescence intensity wavelength curves has become an established technique in metabolic work. Bowman et al. have described a fluorimeter with a " l a m p " mono10 F. 11 G. 12 D. 13 R. 14 R.

A. Loewus, T. T. Tchen, and B. Vennesland, " ~iol. Chem. 212, 787 (1955). Leonhardi a n d I. yon Glasenapp, Z. physiol .~nem. 286, 145 (1951). Robinson, B w c h e m . J . 63, 39 (1956). H. Goodwin, A n n . Rev. P l a n t Physiol. 4, 283 (1953). L. Bowman, P. A. Caulfield, and S. Udenfriend, Science 122, 32 (1955).

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chromator between the exciting light and the cuvette and a second "photocell" monochromator between the cuvette and the detector. The wavelength controls of the monochromators are coupled to the chart drive of an instrument recording detector response, and a continuous graph of fluorescence intensity at various wavelengths is obtained. By varying the wavelength of the exciting light (a xenon arc) by the " l a m p " monochromator an "activation spectrum" results which corresponds to the absorption spectrum of the fluorescent compound. The fluorescence spectrum is the distribution of the fluorescent light with respect to wavelength and is obtained by using the "photocell" monochromator. The activation spectrum may be obtained when absorption is too small to measure directly, and the fluorescence spectrum confirms the identity of the activated molecule. The method is well suited to examination of material eluted from chromatograms in microgram quantities, and its specificity allows estimations in crude extracts. The variation of activation and fluorescence spectra with pH increases the specificity further (cf. ref. 13). Spectrophotofluorimetry is especially useful for estimations of fluorescence intensity for substances which fluoresce in the ultraviolet region, for which filter systems are still less than adequate. Unless the method is used for concentrated solutions the problem of fluorescence transfer is unlikely to be important. If solutions are concentrated in at least one fluorescent component, light energy absorbed by a component may transfer to another with the absorption band overlapping the fluorescence band of the first. The activation and fluorescence spectra in this case would relate to different chemical species, and the spectrochemical analysis might become complex. As with all difficulties of this kind, the analyst will be able to use fluorescence transfer to his advantage; the study of such transfers in vivo is of great importance.

The Polarization of Fluorescent Light For small molecules in a medium of low viscosity the light from a fluorescent solution is emitted indiscriminately in all directions; the fluorescence is unpolarized. For molecules of colloidal dimensions or for smaller fluorescent molecules in a viscous medium it is found in contrast that there is a preferred direction or plane of emission of the fluorescent light; the fluorescence is polarized. The polarization in any given direction of observation is described as follows. Consider the light from a fluorescent solution observed along a direction Oz (Fig. 3). The intensity components of the light can be represented by displacements at right angles to Oz, and there will generally be two directions, mutually perpendicular, in which the components are maximum (Ox) and minimum (Oy). In the

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experimental procedures to be described, with a horizontal beam of exciting light and a horizontal direction of observation (Oz), Ox is vertical and Oy is the corresponding horizontal direction (parallel to the direction of the exciting beam). , x (Vertical)

L:

0

,

r

( Direction y ~of exciting ( beam

Horizontal plane

z

~Direction of tobservation

FIG. 3. Fluorescence polarization. Oz, the direction of observation; Ox and Oy, directions of maximum and minimum components of light, intensities Ix and Iy, respectively. If I~ and Iv are the intensity components, maximum and minimum with respect to observation along Oz, the polarization of the fluorescent light is given by the equation Ix - I,, (1) P - Ix--k ly where I~ -{- I~j is the total light intensity observed along Oz. The variation of the polarization of the fluorescent light with molecular size and viscosity of the medium can be used with considerable ease and certainty to observe changes occurring in colloidal systems, as will be described. To provide a qualitative account of these relationships the following description is given. Origin of Fluorescence Polarization. It is known that molecules have electronic oscillators responsible for absorption of light and emission of fluorescence, rigidly bound in the molecular structure. Absorption of the exciting light is a maximum for those molecules with the oscillators of absorption parallel to the electrical components of the exciting light. These components a~e always in a plane at right angles to the direction of propagation of the exciting beam (light is a transverse vibration). If the exciting light is polarized, the components are further restricted to

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F L U O R E S C E N C E T E C H N I Q U E S FOR T H E ENZYMOLOGIST

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condenser lens, L1, converts the light from the light source, H (usually a medium-pressure Hg lamp), into a parallel beam, and the light may be polarized in a vertical plane by means of the large Nicol prism, N1. The light is collected by the second lens, L2, and enters the cuvette, K, through a suitable light filter, F1. The cuvette is placed in a small metal tank, T, which can be used as a constant-temperature bath. Mirrors M1 and M2 are placed behind the cuvette to increase the brightness of the field. The window Wi allows light to enter the cuvette, and observations are made through a second window, W~, and the light filter F2, complementary to F1. The glass windows may be sealed onto the brass tank with Picien wax.

L1

H ×

i

NI

L2

D I

I

AL 3

(Oxvertical)

z

FIG. 4. Apparatus for measuring fluorescencepolarization. H, light source; L~-L3, lenses; N, and N~, Nicol prisms; El, primary filter; F~, secondary filter; W~ and W~, windows; M, and M2, mirrors; K, cuvette; A, Arago compensator; S, Savart plate; T, metal tank; Ox, Oy, Oz, see Fig. 3.

Compensator and Detector. Observations of the fluorescent light are made along an axis accurately at right angles to the direction of the exciting beam. The Savart plate, S, and the small Nicol prism, N2, serve as a detector of polarization. If polarization exists in the fluorescence beam, light and dark fringes are seen in the field of the Savart plate on looking through the Nicol prism, N.,. They are more readily detected if the Savart plate is mounted on a horizontal pivot and can be rocked through a small angle by means of a rocking arm. A small lens, L3, may be introduced to increase the brightness and the angular aperture of the fluorescence beam. The Arago compensator, A, consists of a pile of glass plates (usually four plates) mounted on a turntable with a vernier goniometer scale, and turned by a knurled knob, geared to the table by a

190

TECHNIQUES

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OF PROTEINS

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friction drive. The purpose of the compensator is to introduce into the fluorescence beam a polarization opposed to that in the fluorescent source. The zero of the compensator scale should correspond to a position of the compensator plates at right angles to the fluorescence beam. The Savart plate and Nicol prism serve as a polarization detector to indicate the null point at which the compensator removes the polarized component of the fluorescence. The apparatus must be set up in a room from which daylight may be excluded during the measurements. The compensator scale is provided with a small light which may be switched on to read the scale. Savart Plate (S). This is made by cutting a plate (of thickness t cm.) 2 X 1 cm. at 45 ° to the optic axis from a quartz crystal. The faces are polished on either side to parallel optical flats, cut across to give two pieces 1 X 1 cm. The pieces are rotated mutually through 90 ° and mounted one upon the other, using Canada balsam to give a block 1 X 1 X 2t cm. There is some doubt concerning the proper value of t. The author has found that t = 0.6 cm. gives good results if the rocking arm is spring-loaded and provided with an adjustable stop. In the original apparatus of Weber, t = 0.3 cm., and a free rocking arm was satisfactory, as the fringes were broader and less sensitive to angular displacements of the plate. Arago Compensator (A). The plates may be microscope cover glasses and are mounted in a metal frame. There is some justification for providing thin spacers to separate the plates. Nicol Prisms (N1 and N~). The provision of N1 is optional, and N2 may be a small prism of about 1-cm. aperture. Cuvettes (K). These should be parallel-sided. Internal dimensions of 4 X 2 X 1 cm. are convenient. Procedure for Measuring Polarization of Fluorescence. It is assumed that the Arago compensator has been calibrated. A table for converting the angle of the compensator to polarization will therefore be available. The Nicol prism, NI, will normally not be used except for polarizations, measured by natural light excitation, less than 0.12 and should otherwise be removed. Measurements made with and without the Nicol prism, N1, are related by the equation p~v = p/(2 -- p)

(2)

where p~ is the polarization with natural (unpolarized) excitation, and p is the polarization with polarized excitation. Removal of N1 will increase the intensity of the exciting light, and a shorter light path may be used. The light source is switched on, and, after it has become stabilized (5 to 10 minutes), the cuvette containing the fluorescent solution is placed

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in the corner of the metal tank. The cuvette is viewed from the top to ascertain whether the fluorescent light is uniformly excited across the cell. If the solution is concentrated, the light beam will become attenuated across the cell. In this case the cell should be viewed in an inclined position so that the illuminated face is visible (see below). The beam passing through the cell should not converge or diverge by more than 10° from the central axis of the beam. 1~The mirrors, M1 and M2, are put in place, and the room light is switched off. When the eye has become adapted to the dark, the fluorescence is viewed through the eyepiece with the Arago compensator set at zero (perpendicular to the fluorescence beam). Fringes will be seen if there is a polarization in the source and may be more clearly seen by rocking the Savart plate. By rocking the plate with one hand and rotating.the turntable of the Arago compensator by means of the knurled knob with the other, the fringes will become indistinct and finally disappear. This is the null point, and the reading of the compensator scale will enable the polarization to be obtained from the calibration table. It is usual to make readings on either side of the turntable zero to correct for errors in the setting of the zero. The average of the two readings is taken as the angle for reference. On closer examination it will be seen that the fringes move out of the field of view at the null point and reappear on the opposite side of the field. At moderate polarizations ( > 0.12), both incoming and outgoing fringes can be seen simultaneously and the fringeless space between may be adjusted to be central in the field of view. Whatever convention is adopted for representing the null point must also be adopted in calibration of the instrument. Recently an improved method of obtaining the null point has been described by Harrington et al.16 Between the Arago compensator and the Savart plate is placed a biplate of quartz followed by a glass plate (P) which rotates about an axis at right angles to the joint in the biplate, and parallel to the rotation axis of the compensator. With P normal to the incident light, compensation is carried out in the usual way. P is then inclined to the light, and fringes appear, owing to the small polarization introduced by P. If the compensation has been exactly made, the fringes introduced by P are equal in intensity and continuous across the junction of the biplate. Any failure to compensate is shown by differences in intensity and position of the fringes on either field of the biplate, and further adjustment can then be made to correct the compensator position. Calibration of the Arago Compensator. 17 Principle. The compensator is calibrated by using the light reflected from the surface of a glass prism as a source of known polarization. is W. F. Harrington, P. Johnson, and R. It. Otterwille, Biochem. J . 63, 349 (1956). 17 E. Gaviola a n d P. Pringsheim, Z. Physik 24, 24 (1924).

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Control of Temperature. The temperature of the cuvette may be controlled by filling the metal tank, T (Fig. 4), with water and using a small heating spiral inside a Pyrex tube containing mineral oil. 15 This is conveniently operated from a small (60-watt) transformer with a series of output voltage tappings from 2 to 30 volts. By experiment, the steady temperature difference with respect to room temperature obtained with any given voltage input can be measured and a working chart provided

Oi

~Y

T

F Fla. 5. Observation for concentrated fluorescentsolutions. K, euvette; E, exciting beam; F, fluorescencebeam; T, metal tank; see Fig. 3.

Oy,Oz,

so that any temperature may be immediately obtained. The bath may be rapidly heated at the highest voltage and stabilized at the required temperature with the appropriate voltage for that temperature. For temperatures below room temperature, a steady low temperature may be obtained by using ice and water, and fairly steady temperature (within 15° below room temperature) by the natural inertia to change of small temperature differences.

The Perrin Equation and Choice of Conjugating Groups The equation of Perrin is describes how the fluorescence polarization changes as a function of temperature and the viscosity of the solution for a spherical molecule excited by polarized light: 1_ p

~)(~)T

1 ~(1 p0

-

(5)

where p is the polarization observed, p0 is a constant, R is the gas constant (8.314 X 107 erg per degree centigrade per mole), T is the absolute temperature (°K.), v is the viscosity (poise), r is the lifetime of the activated state (seconds), and V is the molecular volume of the fluorescent rotational unit (cubic centimeters), r is a characteristic for the fluorescent substance under examination. In order to obtain molecular volumes of protein molecules, a conjugate must be formed between the protein and a fluorescent group with such a value of r that the second term on the right-hand side of the equation is appreciable for aqueous solutions at is F. Perrin, J. phys. radium 7~ 390 (1926).

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room temperature. For proteins of molecular weights in the range 10,000 to 200,000, this restricts the choice of a conjugating group to one with r - 10-8 second. For 1-dimethylaminonaphthalene-5-sulfonamides~9,2° (DNS conjugates) r = 1.2 × 10-8 second, which is therefore of the right order for this range of protein sizes. Other requirements for a suitable fluorescent group have been enumerated by Weber, 19 viz., that the group should form a single type of bond with the protein, stable under a wide range of conditions of temperature and pH, that the protein must remain unaffected in properties after conjugation, and that the fluorescence of the conjugate group must not be greatly quenched after conjugation, nor may the fluorescence properties of the conjugate vary with the pH. These requirements are met by DNS conjugates for many proteins.

The Perrin Equation and Conditions of Excitation Effect of Wavelength of the Exciting Light. If the wavelength of the exciting light is changed, changes are observed in the polarization of the fluorescent light. The polarization is usually greatest when the light cuts the longer-wavelength absorption band of the substance (as does Hg 365 m~ for DNS conjugates). As the wavelength is decreased, the polarization may decrease to zero or even become negative. It has been shown by experiment that these variations in polarization are due to a variation in p0 (equation 5) and that r is not dependent on wavelength. Weber ~5 has shown that, when this is so, the Perrin graph remains unchanged in form and the molecular volume determined from the empirical value of p0 is unaffected by wavelength. In particular, monochromatic light is not necessary in order to obtain reliable molecular volumes. Effect of Polarization of the Exciting Light. The Perrin equation is appropriate in experiments with polarized excitation. When natural (unpolarized) light is used in experiments to determine molecular volumes, the Perrin equation is suitable if polarizations observed with natural light are transformed to the corresponding values expected for polarized excitation from equation 2. More simply, equation 2 can be used to transform the Perrin equation to the form appropriate to natural light. When this is done the result is

1

1 + (~N0 _t_1) (_~) ~T

p-~ = pN~

(5a)

where PN is the polarization observed with natural excitation and pNo is 19 G. Weber, Biochem. J. 51, 155 (1952). 20 G. Weber, Advances in Protein Chem. 8, 4!5 (1953),

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the corresponding constant. It will be noticed that this equation differs from the Perrin equation only in the substitution of a positive for a negative sign in the second term.

Determination of Molecular Sizes and Asymmetries from Fluorescence Polarization ]~xperiments

Spherical Molecules and HomogenousPopulations. The Perrin equation suggests how the fluorescence polarization technique should be used for measuring molecular volumes. The experimental variables which may be controlled are the temperature and the viscosity of the solution. The fluorescent conjugates are studied in media of differing viscosity 19 (and temperature), and the polarizations observed are recorded together with the temperature and composition of the medium. The values of 1/p are plotted against corresponding values of T/~, and the Perrin equation shows that for the simplest case (homogeneous population of spherical molecules) a straight-line relationship should be obtained. Control of T/y. A moderate range of T/~ may be obtained by heating and cooling aqueous salt solutions of the protein conjugates. Between 0 ° and 50 °, T/~ changes from about 2 X 104 to 5 X 104 °K. per poise. The control of temperature has already been considered. If it is required to extend the range of the determinations to lower values of T/~, the viscosity of the solutions may be further increased by adding glycerol or sucrose to the solutions of the conjugates. Sucrose is recommended, as it is easier to weigh out than is glycerol and the range of viscosities obtained is adequate for most purposes. For aqueous salt solutions the viscosity of pure water at the appropriate temperature should be used in the calculations. The points obtained with the sucrose solution should fit onto the line obtained with salt solutions. If this is not found, interaction of the sucrose with the configuration of the protein may be suspected. Significance of po. The intercept at the 1/p axis (T/v = 0) will represent the condition p = P0. If the conjugated fluorescent group is rigidly bound to the protein structure, the value of p0 obtained by extrapolation from finite values of T/~ will represent the polarization obtained in the absence of all Brownian rotational diffusion, i.e., when the molecule remains nonrotational between absorption of the exciting light and emission of the fluorescence. The value would be expected to be identical for molecules of all sizes, as the term containing the molecular volume of the protein is absent from the situation defining po(T/v = 0). Determination of Molecular Volume. From Perrin's equation the slope (s) of the graph is givela by =

(1

-

-?-

(6)

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As p0 is known (from the intercept at the axis), the molecular volume, V, may be calculated if r is known. An estimate of r is available for DNS conjugates (r = 1.2 × 10-8 second) based on experimental work with bovine serum albumin and ovalbumin. An absolute determination of T is possible with specialized apparatus, and absolute values should soon become available. Meanwhile the values of V obtained from the estimate of r depend on certain measurements of dielectric dispersion (Weber19). If natural excitation is used, a positive sign should replace the negative sign in equation 6. Note that for any two proteins (both spherical and homogenous), coupled with the same conjugating group such that p0 is the same for both proteins, S1 V2 (7) S~ Vi where S1 and $2 are the slopes of the (1/p:T/~) graphs and V1 and V~ are the molecular volumes of the proteins. The slope of the graph is inversely related to the molecular volume of the protein. Relation of Molecular Volume to Molecular Weight. In order to relate molecular volume to molecular weight 19 the equation

M ( H + 6) = V

(8)

is used, where M is the molecular weight, H is the hydration of the protein in solution (grams of water per gram of anhydrous protein), and ~ is the partial specific volume (usually 6 - 0.75 for proteins; lipoproteins are an exception). Nonspherical Molecules and Mixed Populations; Dissociation and Intramolecular Rotation

The situation described for a homogeneous population of spherical molecules is an ideal one, rarely met in practical work. In real systems, however, the same practical methods are used, viz., the construction of a graph relating 1/p to T/n. Effect of Asymmetry. At constant molecular volume, the spherical molecule can be considered to become deformed by a simple extension or compression along a single axis. The result will be either a flattened discus-shaped (oblate) ellipsoid or an extended cigar-shaped (prolate) ellipsoid. The resulting asymmetry can always be represented in such cases by the axial ratio, i.e., the ratio of the longest to the shortest axis of the ellipsoid..ks the axial ratio increases at constant molecular volume, the polarization under any conditions always increases and the (1/p: T/v) graph approaches closer to the axis of T/~ (s decreases). For prolate ellipsoids of axial ratio < 3 and for oblate ellipsoids of all axial ratios, the

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verge for high axial ratios~ and correspondingly ph/po is insensitive to the axial ratio for high values of axial ratio with elongated ellipsoids (Fig. 6). In this case Fig. 7 should be used, where the 1/p scale has been normalized b y subtraction of ~ and division b y (l/p0 - 1/~), and the T/V axis has been transformed b y the equation T _ Rr T p0 - 3V' ~/

(t0)

where V' is the true molecular volume. T h e experimental curve is plotted on these transformed axes and the axial ratio determined b y interpolation. 5

8 12

4

1B 30

TIT 3

2

I

0

I

i

I

I

1

2

3

4

5

r/p o

FIG. 7. Determination of axial ratios. Theoretical curves for prolate ellipsoids. The number of each curve gives the axial ratio. [After G. Weber, Biochem. J. 51, 145 (1952), but using equation 32b of G. Weber, Advances in Protein Chem. 8, 415 (1953).] If natural excitation is used, the ordinate should become (1/pN + 1/~)/ (1/pN0 + 1~). An example of the application of this m e t h o d is given b y Weber. 1° Effect of Heterogeneity. Apart from molecular a s y m m e t r y , a curvature toward the T/v axis m a y be obtained 15if the protein is heterogeneous with respect to molecular volume. This effect m a y be superposed on a similar curvature due to a s y m m e t r y . H o m o g e n e i t y of the protein solution m a y be tested b y any of the methods available for this purpose and experiments made with purified fractions of the mixture.

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Freedom of the Conjugated Group. A similar curvature might be expected if the conjugate contains traces of free fluorescent impurities of low molecular weight or if the fluorescent conjugating group is allowed vibrational or rotational freedom relative to the protein, i.e., is not rigidly attached. The difference in molecular sizes between the fluorescent conjugating group and the protein is so great, however, that curvature is apparent only in solutions of extremely high viscosity. In aqueous salt solutions, or even in sucrose solutions, the practical effect of free fluorescent substance or partially restricted conjugating groups is to reduce the polarization uniformly and in such a manner that a straight-line relationship is still obtained, passing through a value of 1/po greater than that expected for rigid binding of the conjugating group. Weber 15 has shown that, if the experimentally determined value of p0 is used in the Perrin equation, the true values of molecular volumes are still obtained. The only disturbance in the method is the displacement of p0 from its expected value. When the presence of free/luorescent substance is excluded, an estimate of the average angle, 8, through which the attached fluorescent group can oscillate can be made by the equation 15 (1_1)=(~

2 ~)3eos~0_

1

(11)

where p0 is the displaced value of the limiting polarization observed by experiment, and P0' is the value expected for rigid binding of the conjugate. There is good evidence that rigid binding of DNS conjugates is obtained with serum albumin where strong secondary forces bind the fluorescent molecule to the protein surface. From experiments with this protein ~9 it is found that 1/pNOp = 3.90 _ 0.06 for DNS conjugates, and so 1/po p = 2.45 __+ 0.03 by equation 2. Curvature away from the T/~ Axis. It remains to inquire what significance may be attached to a curvature of the ( l / p : T / ~ ) graph away from the T/~ axis (convex to this axis) with increase in T/~. This type of behavior will be obtained only when the restraints imposed on the rotational degrees of freedom of the fluorescent group or of the entire conjugate are themselves temperature-dependent. 2~ If the protein dissociates into smaller units or becomes less asymmetric (decrease in Ph/Po) with increasing temperature, a decrease in polarization may be expected, greater than that predicted from the Perrin equation. The graph will then curve away from the T/~ axis with rise in temperature. A similar result may be expected if the freedom of rotation of the conjugating group relative to the protein increases with increasing temperature. Apart from the dissociation of the protein molecule with temperature which is easily confirmed by sedimentation and diffusion if the units

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become completely independent, the changes in configuration of the conjugate leading to an upward curvature are probably more easily observed by the fluorescence polarization method than by any other. It may be necessary to use this method alone to elucidate the changes. Rotational Dissociation. Of special interest is the possibility of a rotational dissociation of the molecule~° with rise in temperature. The molecule becomes divided into subunits which rotate relative to one another but are denied the possibility of separation. Such a situation may arise if the subunits are joined by strong secondary forces of a nonspecific nature, the activation energy of rotation being small. This phenomenon may be of importance with some enzymes. 21 Discontinuities in the Perrin Curve. Finally, discontinuities may be observed in the ( l / p : T/~) graph. 21 These would seem to correspond to a kind of microscopical "melting point" of the colloidal structure with a sudden increase in rotational degrees of freedom at a given temperature. The reversibility of the changes of polarization with temperature should always be tested.

The Range of Application of the Fluorescence Polarization Method With DNS conjugates the method is applicable to proteins in the molecular weight range 10,000 to 200,000 or to subunits of larger proteins falling within this range. Other possible conjugating groups for other molecular weight ranges have been discussed by Weber. z° As the fluorescence properties of the conjugates are independent 19 of pH in the range pH 1 to 12, the method may be used to study changes in configuration or dissociation within this range. This may not apply to proteins with isoelectric points below pH 4, as stability of the fluorescence in acid solution depends on the protective effect of the positive charge of the protein in acid solution. In the case of certain proteins or degradation products of a very expanded structure, open to the aqueous environment, the fluorescence of DNS conjugates is rather weak and yellow in color. For most globular proteins the fluorescence is green and of a satisfactory intensity. Conjugates of bovine albumin with anthracene isocyanate have recently been studied. 22 This conjugate carries no titratable group, and the lifetime of the activated state (~ - 4 X 10-8 second) is very suitable for proteins in the molecular weight range 100,000 to 300,000. The fluorescence polarization method is very robust in practice, and the method of observation has some similarities with simple optical microscopy. Knowledge of protein concentration is not necessary for the evaluation of the results, although an estimate would normally be re21V. Massey, in Weber, ref. 20. ~2 W. F. Harrington, P. Johnson, and R. H. O tterwille, Biochem. J. 62, 569 (1956').

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quired for the record. Apart from associated changes of the protein molecule, the polarization is independent of dilution. The method is insensitive to small turbidities in the solution, though large turbidities may cause a depolarization. This may always be checked by placing a filter consisting of the solution under test in the path of the fluorescent beam. If depolarization is occurring, the effect will be increased by this control; if depolarization is not important, the added filter will have no effect. The method can be used in wide ranges of salt concentration and in the presence of other proteins lacking the fluorescent conjugating groups. 2° In this last respect the method is of unusual specificity. Fluorescence polarization is unsuitable for proteins containing colored groups which absorb wavelengths in the fluorescence spectrum of the conjugating group. For such proteins transfer of the fluorescence energy to the colored groups followed by thermal degradation leads to complete fluorescence quenching in some cases. Criticism of the Fluorescence Polarization Method

Recent critical studies 22 have confirmed the results of Weber 19 for changes in polarization of DNS-serum albumin conjugates in acid solution, but have shown that the results of the largely established methods to which these changes were referred were subject to possible reinterpretation. The polarization method appears to be less sensitive to changes in pH, salt concentration, and protein concentration than the older methods, and independent checks by direct methods of the lifetimes of the activated state throughout the range of application of the method will soon become available from Weber's laboratory. An important possibility is deviation from the assumption used in deriving Figs. 6 and 7 of random orientation of the fluorescent conjugating groups with respect to axes of an ellipsoidal molecule. For some enzymes the conjugating group may enter the active center and random orientation of the group may not be assumed. Any change in orientation of the group due to local changes in configuration would alter the weighting to be given to the various rotational diffusion constants of the molecule, and changes in polarization might be due to this cause alone. Massey and collaborators ~3 have shown that for chymotrypsin the conjugate may be excluded from the active center by use of inhibitors and that the active center conjugate may involve a histidine rather than a lysine residue. By varying the distribution and number of conjugating groupings the assumption of random orientation may be checked. ~a V. Massey, W. F. Harrington, and B. S. ttartley, Discussions Faraday Soc. 20, 24 (1955).

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cent light, and a rotating half-wave plate driven by a synchronous motor together with a stationary Polaroid disk provides a signal whenever polarization is present in the light. The signal (5 c.p.s, or 5~ revolutions of the plate per second is convenient) is amplified by low-frequency selective amplifiers of narrow band width. Compensation is indicated by the decrease of the signal to zero. The use of narrow band-width amplifiers considerably reduces the contribution of multiplier "shot noise" and the 100-c.p.s. component of the a.c.-operated mercury lamp to the final signal. Photoelectric methods are capable of considerable development with the replacement of the partially polarizing elements of the Arago compensator by linear fully polarizing components (Weber, to be published). Dr. Weber has kindly sent details of the apparatus prior to publicatioI~ from which the following details have been taken. The apparatus is a simple double-beam null-point fluorimeter (Fig. 8) containing adjustable fully polarizing elements G1 . . • G, (1.27-cm.-aperture Glan Thompso~l prisms). Light from a mercury lamp (Hg) (either a compressed source or a medium-pressure lamp with arc horizontal and stable arc position) is converted to a parallel beam by the lens, L, and passes through a prism, G1, which can be set by fixed stops with its polarizing axis either vertical or horizontal. The fully polarized light then falls on the cuvette, C, in a holder maintained at steady temperature by a water jacket. The fluorescent light is viewed at right angles to the direction of the exciting light by the photomultipliers PR and Pv (E.M.I.6095) which are bridgeconnected. 28 FI are a pair of similar filters for the photocells, and F~ is a filter for the exciting light. In the left-hand fluorescence beam is a prism, G2, with polarizing axis horizontal and an adjustable self-centering slit, A. In the right-hand beam is a prism, G3, with polarizing axis vertical and another (G4) mounted in a vernier goniometer scale. A stop, B, restricts the beam to within the face of G4. The components are mounted oa a~ optical bench and covered by a light-tight box with access to the filters and to the cuvette holder and vernier scale. Controls for the slit and goniometer scale allow adjustments to be made to slit width and the orientation of G, from outside the box. To adjust the orientation of the prisms, they are mounted in closefitting brass sleeves within an outer mounting and can be rotated only on application of moderate pressure. A glass plate rotating about a true vertical axis is placed on the optical bench, and an illuminated diffusing screen placed so that a reflection of the screen in the glass plate can be seen through G4, the screen subtending an angle of 1° or 2 ° at the eye. The position of the screen is adjusted until a part of it is reflected into 28 j . p. Dowdall and H. Stretch, Analyst 79, 651 (1954).

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[~]

the prism at the polarizing angle. G4 is then adjusted until the dark shadow at this part of the screen is central in the field of the prism, when the vernier scale reads 90.00 °. G1, G~, and G~ are now adjusted to extinction, with G4 set to 0 or 90 ° as required. Finally Hg is set so that the axis between the lamp and the aperture of the cuvette holder is horizontal. \1/

]al q f~

B

1.5V FIG. 8. Photoelectric measurement of fluorescence polarization (due to Dr. G. Weber). Hg, light source; L, lens; Fe, primary filter; F], secondary filters; G1 . . . G4, Glan-Thompson prisms; Pa and Pv, photomultipliers; C, cuvette; 7', cuvette holder; A, slit; B, stop; G, galvanometer.

To measure fluorescence polarization the sensitivity of the photomultipliers is adjusted to an appropriate value by varying the voltage on the dynodes. The exciting light is obscured by a screen, and the difference in dark current of the multipliers is compensated by a current supplied from the small 1.5-volt battery until the galvanometer, G, indicates balance. The screen is then removed and with G1 polarizing

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horizontally and G4 set to 0.00 ° the photocurrents are balanced by adjusting A. G1 is now rotated to give vertical polarization, and balance is again obtained by rotating G4. The angle 0 given by the scale of G4 allows the polarization to be calculated by the equation sin s 0 P = i -i- cos 2 0 cos 20

(15)

If negative polarization is encountered, rotation of G4 will increase the extent of out-of-balance with G1 vertical. Negative polarization may be measured by repeating the cycle of operations, but with G1 polarizing vertically before adjusting A and horizontally before rotating G4. Then: P =

sin 2 0 2 - - s i n s0

(16)

Advantages of this apparatus are high sensitivity and accuracy, uniform response over a wide range of Wavelengths (3500 to 6500 A.), and absence of need for any calibration procedure. An alternative method of measurement uses fixed optical components and variable resistances in the anode bridge. 28 This method is more suitable for automatic recording but assumes that photocell response is linear with the light intensity. For rapid-reaction techniques using fluorescence polarization, an electronic computer for the ratio of difference and sum of photocurrents in the anodes of the photomultipliers could be used (cf. equation 1) if the photoelectric response is linear. Preparation of the Photomultipliers. With end-window multipliers of the E.M.I. type operated with the anode near earth potential, the manufacturers recommend that the sides of the glass envelope for about 5 cm. from the cathode should be coated with a conducting film of colloidal graphite which is connected to the negative cathode potential by a well insulated cable. The part of the envelope not coated with graphite may be treated with insulating water-repellent varnish. Control of Temperature. A large water-bath controlled by a Variac transformer may be used for supplying water at a steady temperature to the water jacket of the cuvette holder. In order to facilitate observation the temperature of the cuvette may be measured by thermometers placed in the inlet and outlet streams of the water jacket. With suitable lagging of the holder and leads the temperature difference between inlet and outlet streams may be reduced to 0.4 ° for a temperature 60 ° above the surroundings. So that the temperature of the cuvette may be exactly intermediate between these temperatures the cuvette holder should be made water-tight by windows flush with the inside wall of the holder

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fitting the optical parts. These are conveniently attached to brass bushes using a Picein wax rendered non-fluorescent by dissolving 5% Sudan Black in the molten wax. The brass bushes slide into the optical ports with some silicone grease and are keyed for easy removal for cleaning. A second window on the outside of the lagging prevents condensation when working at temperatures below the dew point. A little water is placed in the cuvette holder so that when the cuvette is inserted a thin water film joins the cuvette and the brass walls of the holder. With this film in place the cuvette reaches a temperature within 0.1 ° of the average temperature of inlet and outlet streams and thermal equilibration requires less than 10 minutes; without the water film, equilibration requires about three times as long and the temperature reached is lower than for either inlet or outlet. Reading the Vernier Scale. It is difficult to read the vernier scale inside the box unless a suitable optical system is employed. A mirror at 45 ° opposite the scale reflects light from the vernier vertically. A large shortfocus lens is mounted above the mirror and illumination is provided by a suitably screened tungsten lamp shining down through the edge of the lens nearest the observer. The deviation of light by the lens and partial reflection by the mirror give suitable diffuse illumination of the vernier without highlights. The vernier may then be read through the lens without difficulty. It is advisable to place screens across the box between the cuvette holder and photocells in order to avoid intense illumination of the cells when the box is opened for reading the vernier. The voltage across the multipliers is always switched off before opening the box but even when the photocells are not connected to the supply, the dark current may be increased by excessive illumination. There is little advantage in placing the vernier outside the box when measurements are being repeated at various temperatures, as delay in thermal equilibration gives time to take a previous reading. Also it is advisable to inspect the cuvette from time to time in order to remove air bubbles. For following isothermal reactions a readier method of taking readings would be useful. For connection to a scale outside the box, pairs of gear wheels cross-meshed under tension may be used to eliminate backlash in the connections.

Preparation of l-Dimethylaminonaphthalene-5-sulfonic Acid from l-Aminonaphthalene-5-sulfonic Acid The method of Fussgiinger~9 may be used: 12.3 g. of 1-aminonaphthalene-5-sulfonic acid is introduced into a thick-walled soda glass tube together with 40 ml. of methanol, 17 g. of methyl iodide, and 12 ml. of s9 V. Fussg~nger, Ber. 35, 976 (1902).

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which the acid is insoluble. The acetone solution is diluted with 6 vol. of water from which the chloride crystallizes as yellow or orange crystals. The crystals are dried and stored over CaCl.~ (ammonia-free). They are stable for several months. Yield, 25 to 40 %; melting point, 69 ° . Alternatively the sulfonyl chloride may be made in a thick-walled boiling tube with a glass rod as pestle. At the end of the reaction the melt is smeared round the inside of the tube and the acid neutralized by adding ice-cold M dipotassium phosphate solution, the outside of the tube being cooled in an ice-water mixture. The sulfonyl chloride is extracted into diethyl ether which is separated and dried with calcium chloride followed by calcium sulfate. The dry ether solution is filtered and the ether evaporated at a pump when the sulfonyl chloride crystallizes as the last traces of solvent are removed. It is then recrystallized from a small volume of diethyl ether with the temperature range 34 ° to - 10°, washed with n-heptane, and dried at a pump. The sulfonyl chloride may now be obtained from the California Foundation for Biochemical Research. Preparation of ~-Anthramine 3° 50 g. of 2-aminoanthraquinone are heated under reflux for 8 hours with 80 g. zinc dust and 2 1. of 5 % aqueous NaOH solution. The solution first becomes red and later fine yellow plates of/~-anthramine separate out. After 8 hours the solution is filtered while hot and thoroughly washed with hot water. The amine is extracted from the filter cake with hot toluene and recrystallized from the same solvent. Yield 35 g. m.p. 238 °. Repeated recrystallization from a number of different solvents gives m.p. 2440. 31 Preparation of ~-Anthrylisocyanate 31 T o a solution of 3 g. of ~-anthramine in dry benzene is added an excess (20 g.) of phosgene in dry toluene. A white flocculent precipitate of the amine hydrochloride is obtained and on refluxing the solution for 30 minutes, this redissolves. The resulting solution is evaporated at 100 ram. pressure with dry nitrogen gas until an opalescence is obtained. The solution is then filtered and transferred to a v a c u u m desiccator. The isoeyanate crystallizes as further solvent is removed and m a y be recrystallized from dry benzene and carbon tetrachloride. Yield 70%, m.p. 207.5 - 208 °. 80 p. Ruggli and E. Henzi, Helv. Chim. Acta 13, 409 (1930). 31L. F. Fieser and H. J. Creech, J. Am. Chem. Soc. 61~ 3502 (1939).

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Preparation of Conjugates of Proteins with 1-Dimethylaminonaphthalene-5-sulfonyl Chloride 19 I t is convenient to introduce one to three groups of the fluorescent residue into each protein molecule (molecular weight, 50,000). An amount of sulfonyl chloride is weighed out equal to 1 to 2 % of the weight of the protein. To a solution of the protein in 0.1 N phosphate buffer, pH 7.5, or in 1% NaHCO~ (10 ml.), cooled to 0 to 3°, is added rapidly with stirring a solution of the sulfonyl chloride in acetone (0.5 ml.). The acetone solution is filtered through a sintered-glass disk if traces of the insoluble free acid have been formed on storage. The chloride forms an emulsion, and the solution is put away in an icebox until the reaction is complete. After about 3 hours and at intervals the solution is inspected. When the reaction is complete the solution clears and loses the orange color of the chloride. Excess of unhydrolyzed chloride may be removed by centrifugation. Excess of hydrolyzed chloride must then be removed. It is convenient to do this by dialysis, by ion exchange resins, or by a combination of both. The solution is placed in a dialysis sac and dialyzed against an appropriate buffered solution. If the protein has a high binding affinity (e.g., serum albumin), 5% sodium sulfate can be used to displace the free acid. The diffusate is examined and is changed until no fluorescence appears outside the sac when illuminated by a Hg lamp with a nickel oxide glass filter. Free acid may also be removed by alcohol precipitation of the protein, 19 by passing the protein down an ion exchange column, 3: e.g., IRA 400, or by dialyzing the solution into a medium containing ion exchange resin. To test the absence of free acid mixed with the conjugate, chromatography or eleetrophoresis on filter paper 2°,32 may be employed. A test sample of the conjugate is placed on the filter paper, and adjacent to it a sample of the free acid (1-dimethylaminonaphthalene-5-sulfonie acid). The free edge of the paper is dipped in a salt solution of a concentration that will permit migration of the free acid but not of the conjugate. After the solvent front has traveled a few centimeters, the paper is removed and examined by a Hg lamp, with a Wood's glass filter. The free acid present will show a separate fluorescent spot in advance of the brighter spot of the conjugate. With paper electrophoresis, the same principles may be applied. A pH is selected near the isoelectric point of the conjugate (but >,pH 4 below which the free acid is present as a zwitterion). The free acid will migrate to the positive side of the eleetrophoresis cell and will separate from the conjugate. With buffers of higher pH the protein can be shown to be free of traces of free sulfonamide a2 G. Weber, Discussions Faraday Soc. 13, 33 (1953).

212

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[8]

(formed f r o m traces of a m m o n i a in the original sample) which do n o t m i g r a t e in an electric field. T o calculate the n u m b e r of conjugating groups bound to a unit weight (e.g., to 1 g. molecule) of the protein, the absorption s p e c t r u m in the region 300 to 450 m~ should be measured 19 for a solution of k n o w n protein content. D N S conjugates show an absorption m a x i m u m at 330 to 360 m~. T h e concentration of conjugating groups in the solution m a y be calculated f r o m the extinction coefficient of 4.3 X 10 e cm.2/g, mole for the absorption m a x i m u m . A b l a n k determination should be m a d e for u n c o n j u g a t e d protein so t h a t the observed absorption of the conjugate m a y be corrected for the absorption of the protein at the same wavelength. T h e correction will usually be small.

Coupling of ~-Anthrylisocyanate This is done as for the D N S conjugate except t h a t r a t h e r more organic solvent is used. ~2 F o r r e m o v a l of uncoupled fluorescent material, organic solvent precipitation would a p p e a r to be essential owing to the limited solubility of the hydrolysis products and their strong interactions with proteins. T h e molecular extinction coefficient is 3.04 × 10 e cm.2/mole. 22

[8] The Solubility Diagram as a Criterion of Protein Homogeneity By ROGER ~/[. HERRIOTT Introduction T h e solubility d i a g r a m is a powerful tool for detecting the presence of impurities in a n y t y p e of soluble material. T h e essential feature of this m e t h o d is simply t h a t in a s a t u r a t e d solution of a single solute the concentration of dissolved solute does not v a r y with the q u a n t i t y of s a t u r a t ing solid phase present.i This fact had been appreciated for a long time, b u t because of the complex nature of their protein p r e p a r a t i o n s the early workers 2-4 failed to produce convincing evidence of the value of the solu1The well-known effect of extreme differences in the size of the solid particles on the solubility is reported to be due to surface energy differences and is not considered in the present discussion, since it is probably a second-order difference. See the following references for a review of the effect of the size of solid particles: A. Hulett, Z. physik. Chem. 37, 385 (1901); 47, 357 (1904); M. L. Dundon and E. Mack, Jr., J. Am. Chem. Soc. 45, 2479 (1923); M. L. Dundon, ibid. 45, 2658 (1923). 2 G. Galeotti, Z. physiol. Chem. 40, 492 (1903); 42, 330 (1904); 44, 461 (1905); 48, 473 (1906). s W. B. Hardy, Brit. J. Physiol. 33, 251, 338 (1905).

212

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[8]

(formed f r o m traces of a m m o n i a in the original sample) which do n o t m i g r a t e in an electric field. T o calculate the n u m b e r of conjugating groups bound to a unit weight (e.g., to 1 g. molecule) of the protein, the absorption s p e c t r u m in the region 300 to 450 m~ should be measured 19 for a solution of k n o w n protein content. D N S conjugates show an absorption m a x i m u m at 330 to 360 m~. T h e concentration of conjugating groups in the solution m a y be calculated f r o m the extinction coefficient of 4.3 X 10 e cm.2/g, mole for the absorption m a x i m u m . A b l a n k determination should be m a d e for u n c o n j u g a t e d protein so t h a t the observed absorption of the conjugate m a y be corrected for the absorption of the protein at the same wavelength. T h e correction will usually be small.

Coupling of ~-Anthrylisocyanate This is done as for the D N S conjugate except t h a t r a t h e r more organic solvent is used. ~2 F o r r e m o v a l of uncoupled fluorescent material, organic solvent precipitation would a p p e a r to be essential owing to the limited solubility of the hydrolysis products and their strong interactions with proteins. T h e molecular extinction coefficient is 3.04 × 10 e cm.2/mole. 22

[8] The Solubility Diagram as a Criterion of Protein Homogeneity By ROGER ~/[. HERRIOTT Introduction T h e solubility d i a g r a m is a powerful tool for detecting the presence of impurities in a n y t y p e of soluble material. T h e essential feature of this m e t h o d is simply t h a t in a s a t u r a t e d solution of a single solute the concentration of dissolved solute does not v a r y with the q u a n t i t y of s a t u r a t ing solid phase present.i This fact had been appreciated for a long time, b u t because of the complex nature of their protein p r e p a r a t i o n s the early workers 2-4 failed to produce convincing evidence of the value of the solu1The well-known effect of extreme differences in the size of the solid particles on the solubility is reported to be due to surface energy differences and is not considered in the present discussion, since it is probably a second-order difference. See the following references for a review of the effect of the size of solid particles: A. Hulett, Z. physik. Chem. 37, 385 (1901); 47, 357 (1904); M. L. Dundon and E. Mack, Jr., J. Am. Chem. Soc. 45, 2479 (1923); M. L. Dundon, ibid. 45, 2658 (1923). 2 G. Galeotti, Z. physiol. Chem. 40, 492 (1903); 42, 330 (1904); 44, 461 (1905); 48, 473 (1906). s W. B. Hardy, Brit. J. Physiol. 33, 251, 338 (1905).

[8]

SOLUBILITY DIAGRAMS

213

bility measurements as a criterion of purity. Sorenson and HSyrup ~ gave very careful consideration to phase rule aspects in their extensive studies on crystalline egg albumin. Landsteiner and Heidelbergeff made excellent use of solubility principles to differentiate hemoglobins of different species. The general subject received relatively little attention, however, until Northrop and Kunitz 6 re-examined and extended it to cover the various theoretically possible systems. In addition, these latter workers prepared proteins of such homogeneity that virtually ideal solubility diagrams were obtained experimentally which brought about an increased interest and confidence in the method. Perhaps even more important, this work demonstrated that proteins respond to thermodynamic principles and methods in a manner similar to that observed for small molecules, a concept not generally accepted in the early 1930's. It may be worth noting at this point that, although this solubility method originally was designed and applied almost exclusively to protein preparations, it is equally applicable to all types of molecules. General Procedure Briefly, the solubility method consists in (1) mixing a series of increasing quantities of the material under investigation with equal or known volumes of solvent until equilibrium is attained; (2) separation of the solid phases from the solutions; (3) determination of the concentration of the material dissolved in aliquots of the various solutions; and (4) plotting the concentration of dissolved material against the total (solid and dissolved) added per unit volume. A plot of the analytical results will take the form of one of the three general type curves shown in Fig. 1. Interpretation of Results

In those systems containing no solid phase at equilibrium, the dissolved concentration equals the total added, and therefore the points fall on the line O A in Fig. 1. This line has a slope of 1. As the total concentration is increased, a point will be reached where a small amount of solid will remain after equilibration. The key to any interpretation of the solubility diagram depends on the variation of the solubility (dissolved concentration) beyond the point where the first solid phase persists at equilibrium. As the total material increases beyond the point of first persistence of solid phase, the solubility may vary in any of three ways. 1. It may remain unchanged, as represented in Fig. 1 by curve O A E . 4S. P. L. SCrenson and M. H6yrup, Compt. rend. tray. lab. Carlsberg 12, 213 (1917) ; see also ibid. 18~ No. 5 (1930). s K. Landsteiner and M. tteidelberger, J. Gen. Physiol. 6, 131 (1923). 6j. H. Northrop and M. Kunitz, J. Gen. Physiol. 15, 781 (1930).

214

TECIIN'IQUES FOR CHARACTERIZATION OF PROTEIN'S

[8]

Such a curve represents a constant solubility and is obtained when the material is (a) a single component (i.e., pure); (b) a solid solution of two or more materials having identical solubilities; or (c) a mixture of two or more materials which are present in the preparation in direct proportion to their solubilities. Cases b and c are not often encountered, and they m a y be resolved b y performing the solubility determination in solvents of a different pH, temperature, kind of salt, or organic solvent composition. In general the ratio of the solubilities of materials would be expected to change under variations in conditions and the second component will be revealed. If the material under s t u d y has a constant solubility in several different solvents, it is probably a single component. However, a 1 : 1 racemic mixture of d and l isomers or perhaps a mixture of isotopic isomers would behave as a single component even in several solvents.

e 3P~ o~-

A

C

E

e~ex

~3

Total

concentration

FIG. 1. General types of solubility curves. See text for explanation of curves. 2. F r o m the point where the solid phase first persists the solubility m a y increase linearly at a different rate from before as the total increases and change a b r u p t l y to a still different linear positive slope or to one of zero. This t y p e of result is obtained when the components form simple mixtures in the solid phase and their solubilities are independent of one another. Each time there is an a b r u p t change in slope, a new solid phase appears. Curve O B C E represents such a system with pure solid phases appearing first at B and C. A mixture of d and 1 optical isomers in a n y proportion other than 1:1 would be expected to produce a curve of this type. F r o m certain algebraic considerations N o r t h r o p and Kunitz e,7 have shown t h a t the value of the intercept of the line B C with the ordinate is the solubility, S, of the pure component which first appears as a solid phase. The q u a n t i t y of this pure component, as a fractional part of the M. Kunitz and J. H. Northrop, Cold Spring Harbor Symposia Quant. Biol. 6~ 325 (1938).

[8]

SOLUBILITY DIXGRA~S

215

total, is given by 1 minus the slope of the line BC. The corresponding values of the second component are obtained by difference. Thus, the solubility of the second component equals the total less S, the value of the first component. The relative amount of the second component will be equal to the slope of line BC. Thorp 8 found that known mixtures of isomers of DDT respond quantitatively to this method of analysis, as Kunitz and Northrop 7 had shown for proteins. 3. Third, and finally, the solubility may vary continuously as the total is increased (see curve ODE), asymptotically approaching a constant value. Such a result is obtained with solid solutions. In such cases no quantitative predictions are possible. In general one may expect the first precipitate appearing along this curve to be richer than the starting material in one of the components. Similarly, the solution in equilibrium with a large excess of solid will be richer in the other components than is the starting material. It follows from what has been said above that in distinguishing between eases 2 and 3 it is only necessary to run a solubilitydiagramon the first or smallest solid phase to persist at equilibrium. In both eases the solubility of this fraction will be less than that of the original material. The key to the differentiation lies in the fact that a material behaving like a single component should be obtained from a mixture (ease 2), whereas another rounded curve similar in shape to ODE in Fig. 1 will be obtained if the material is derived from a solid solution (ease 3). Application of the Phase Rule. The phase rule is a useful thermodynamic generalization of heterogeneous equilibria relating the number of components, phases, and degrees of freedom. Use has been made of it in establishing the validity of the solubility method. It is not necessary, however, to apply the phase rule or even to understand it to make considerable use of the solubility method which generally is applied to decide whether a preparation consists of one or more components. The simple concept that in a saturated solution (equilibrium) the solubility of the solute is independent of the quantities of any of the phases will in most cases serve as a test of homogeneity, for, if the solubility value is independent of the quantity of solid phase, it is a constant solubility denoting a single component. For those interested in a proof of the various possible cases, the following is a brief description based on the phase rule. This rule in its abbreviated form is P + F = C + 2, where P equals the number of phases (physically separable portions which differ in structure), F equals the number of degrees of freedom (variables such as temperature, pressure, and concentration of components), and C equals the number of D. Thorp, J. Soc. Chem. Ind. 65, 414 (1946).

216

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[8]

c o m p o n e n t s (the s m a l l e s t n u m b e r of i n d e p e n d e n t c o n s t i t u e n t s t h a t will define t h e s y s t e m ) 2 I n s o l u b i l i t y e x p e r i m e n t s t w o of t h e d e g r e e s of f r e e d o m , t e m p e r a t u r e a n d p r e s s u r e , a r e h e l d c o n s t a n t , so t h e e q u a t i o n simplifies t o P Jr F ' = C, w h e r e F J e q u a l s F - - 2. T h e m a t e r i a l in t h e t a b l e s h o w s h o w t h i s r e l a t i o n s h i p c a n be u s e d t o d i f f e r e n t i a t e t h e v a r i o u s k i n d s of s y s t e m s . PHASE RULE ANALYSES OF SOLUBILITY DIAGRAMS

Number of Region of curves of Fig. 1

Phases

Components Degrees of Solutes b freedom ~ Solvents

OB OAE

1

1 or more

1

1 or more

2

0

1

1

OBC

2

1

l

2

OBCE

3

0

1

2

ODE

2

1 or more

1 (liq.)

2 or more

Systems satisfylng these conditions A solution (no solid phase) Single pure solid in contact with a saturated solution Single pure solid in a solution of two components Mixture of two pure solids in a saturated solution of both Solid solution of two or more components in contact with liquid solution

a The degree of freedom observed is the variation in concentration of dissolved solute. Temperature and pressure are held constant. b Deduced from the phase rule. Such components as buffer, salts, etc., are considered as part of the solvent, since presumably they do not vary. I t s h o u l d be c l e a r t h a t t h e p h a s e rule is n o t c o n c e r n e d w i t h t h e t i m e r e q u i r e d to g e t t o e q u i l i b r i u m , n o r d o e s i t i n d i c a t e t h a t t h e s o l u t e in solut i o n is i d e n t i c a l w i t h t h a t in t h e solid p h a s e . I n s o m e i n s t a n c e s s o l u t e a n d solid a r e k n o w n t o differ. T h u s , t h e solid p h a s e m a y b e a c r y s t a l l i n e s a l t of a p r o t e i n a n d a p a r t i c u l a r ion, a n d in s o l u t i o n t h a t i o n m a y n o t be associated with the protein. A r e v e r s i b l y d i s s o c i a b l e s o l u t e d o e s n o t p r e s e n t a n y d i f f i c u l t y in t h e s o l u b i l i t y m e t h o d . I n s u c h a s y s t e m t h e r e is o n l y one m o r e c o m p o n e n t , e v e n if t h e s o l u t e d i s s o c i a t e s i n t o t w o p a r t s , for t h e c o n c e n t r a t i o n s of t h e u n d i s s o c i a t e d s o l u t e a n d one of t h e d i s s o c i a t e d p a r t s will define t h e c o n c e n t r a t i o n of t h e s e c o n d p a r t . I n t r o d u c t i o n of t h e a d d i t i o n a l c o m p o n e n t For more details regarding the phase rule and the definitions, see A. Findlay, "The Phase Rule and Its Applications." Longmans, Green, New York, 1938; J. A. V. Butler, J. Gen. Physiol. 24, 189 (1940).

[8]

SOLUBILITY DIAGRAMS

217

will be balanced by an additional degree of freedom; hence the number of phases will remain unchanged. The concentration of the dissociated components can also be considered as fixed by the concentration of undissociated material and the particular solvent used. Thus, reversible phenomena do not jeopardize the value of the solubility method by limiting it to nondissociating systems or requiring that the existence of such dissociation be known. If the dissociation is not reversible, then an equilibrium cannot be established and the method is not applicable.

Quantitative Limitations of the Method The resolving power of this method is dependent on the relationship of the components in the solid phase, i.e., whether mixtures or solid solutions, the difference in solubilities of the individual components, and the precision of the analytical method. If the solid phase is a solid solution, no quantitative prediction of the resolving power is possible. In general, however, the greater the difference in solubility of the individual components, the greater will be the resolution. No one analytical method can be followed with all materials, but the dry weight of solute has a very wide range of usefulness and can be a very precise measurement. It is open to a serious objection, however, if the solvent is a strong salt solution. For proteins the nitrogen content and the absorbancy at 280 mu are convenient measurements.

Conditions and Certain Details of Technique

Solvent. Although few definite rules apply to all proteins, it is obvious that certain general aspects of the problem must be kept in mind. Thus, it is important that the protein be stable under the conditions of the experiment. A solvent (usually a buffered aqueous salt solution) should be chosefi that will permit precise estimation of the concentration of dissolved protein and yet not so high that too large a proportion of the total protein is in solution in a concentrated suspension. Amorphous and Crystalline Forms. With some proteins the solubility experiment can be performed on either the crystalline or amorphous forms. Each has certain advantages, but other things being equal a solubility experiment on the crystals is preferred, for the exact nature of the amorphous state is not well understood. Preliminary Equilibration. Usually protein preparations contain additional ions sufficiently different from those of the solvent to be used that some preliminary adjustment is necessary before making the final distribution of suspension. In bringing the solid into equilibrium with the solvent much time can be saved by first washing the preparation in question on a Biichner funnel with small aliquots of the complete solvent

218

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[8]

or by dialyzing the suspension in collodion or Visking tubing against an excess of solvent. To determine whether adjustment is complete a portion of the solid may be stirred or precipitated with successive aliquots of the solvent until the filtrates from several successive treatments contain the same amount of dissolved protein. If the amount dissolved is not constant after a number of washings, it may be the result of a change in the protein composition due to removal by dissolution of a small amount of a relatively soluble component and not because equilibrium of the salt is not reached. When several successive aliquots do contain equal amounts of dissolved protein, it may be presumed that equilibrium exists between the solid and solution. Criteria of Equilibrium. The equilibrium point may be approached from either the supersaturated or the undersaturated side. Both approaches should be made when the amount of material and other considerations permit. It is rather important that at least one or two points be obtained from both sides to establish that a true equilibrium value is being observed. Approach from the supersaturated side of the equilibrium can be conveniently carried out with amorphous preparations by using a split solvent. This is accomplished by dissolving the solid phase or suspension in water or buffer and adding the concentrated salt portion. It is difficult with crystalline preparations of proteins to get reproducible solubility figures when approaching the equilibrium solubility from the supersaturated side. This is particularly true for those systems in which the excess of solid phase is small. Equilibrium is usually approached from the undersaturated side for both amorphous and crystalline forms by stirring the solid phase with the complete solvent. The time required to attain equilibrium at any given temperature depends on a number of factors paramount among which are the quantity of solid, the size of the solid particles, and the rate of stirring. This time, therefore, must be determined experimentally in each case. To ensure against an unforeseen mistake the solubility value should be determined at two different time intervals. Distribution of Solute. The solvent and solid solute having been brought into salt equilibrium, the suspension is distributed in varying amounts in a number of glass or plastic test tubes and the solvent added. The amount of suspension in each tube is adjusted so that at equilibrium the series of tubes ranges from complete solution of the protein to a large excess of solid. It should be emphasized that the greatest interest is in those points which fall near the " b r e a k s " in the diagrams, in the regions where new solid phases appear. With a limited amount of material it is, therefore, important to adjust the concentrations of suspension with this in mind.

[8]

SOLUBILITY DIAGRAMS

219

It is possible to test for a one-component system with a minimum of effort and material by using only two points. From the preliminary equilibration data the solubility in the presence of an excess of solid is known. It is then only necessary to dilute such a suspension with solvent to the calculated point where there should be only a slight (10%) excess of solid present at equilibrium. In such a case, if the concentration of dissolved solute is the same as that observed when the excess of solid is large, the diagram is one of constant solubility and therefore indicates the presence of a single component. If, however, the solubility proves to be lower, the investigator will be better prepared to design the distribution of the remainder of the samples to get the maximum information. For reasons already discussed in the earlier sections of this paper, it is more important to have more experimental points in the region of small excess solid than of great excess solid. Final Equilibration. In stirring the protein solution and suspensions, the temperature should be kept constant and foaming should be avoided as it may represent considerable denaturation. It has been found that glass beads, marbles, or stainless-steel ball bearings in tubes that are completely filled, stoppered, and then rocked or rotated suffice to stir the suspensions. The time of stirring (necessary to attain equilibrium) must be determined experimentally and should be based on the time necessary to equilibrate the system with a minimum of excess solid phase. Separation of the Solid and Solution Phases. After equilibrium has been attained, the solid is separated from the solution by centrifugation or filtration. Care must be taken in solubility studies of amorphous material, when the approach is from the supersaturated side, that equilibrium is reached before the solid phase is removed. It has usually been assumed that a protein solution cannot be supersaturated with the amorphous protein, but certain experiments with pepsin seem to indicate that such supersaturation is possible. Filtrates from amorphous solids should remain clear for several hours unless crystallization occurs; in the event of cloudiness crystals usually may be detected by microscopic examination. Should the preparation crystallize in the complete solvent, one may depress this tendency by allowing the protein to stand in solution in the dissolving part of the split solvent for several hours before adding the precipitation part of the solvent. This treatment dissolves the last traces of crystals which would otherwise act as seeding crystals. The supernatant solution or filtrate must, therefore, be crystal clear. It has been found that filtration through Whatman No. 42 filter paper gives uniformly good results. ,If the amount of dissolved protein is under 0.5 mg./ml., the filter paper takes up a significant amount of the protein from the first portion of solution passing through so that the first few

220

TECHN'IQUES FOR CHARACTERIZATION OF PROTEIN'S

[8]

milliliters of filtrate may be low in dissolved protein and, therefore, are usually discarded. Estimation of Dissolved Protein. The amount of dissolved protein may be determined by any of a number of methods such as Kjeldahl nitrogen, turbidity, biological activity, color test for tyrosine and tryptophan, biuret, copper phenol value, 1° or any other easy and precise method. In the event that an unidentified substance having a specific property such as a biological activity is being examined, one can learn a great deal about the class of substances to which the unknown belongs by making as many different examinations on the test solutions as possible. For example, one may find that protein, carbohydrate, nucleic acid, specific absorbency, or another property parallels or separates from the specific biological property being studied, and this information should prove useful whether the solubility diagram is that of a preparation consisting of single or multiple components.

Separation of Protein Components by Methods Based on the Solubility Diagram From the discussion in the section on Interpretation of Results it follows that, if the system under examination is shown by the solubility diagram method to consist of two or more components, a procedure for a separation can be developed from this diagram. Thus, if the components occur as a simple mixture (case 2), the first solid phase appearing, represented in Fig. 1 by the region enclosed in the triangle BAC, will be a pure component having the solubility indicated by S. If the components form solid solutions (case 3), the first solid phase persisting at equilibrium will be richer in one of the components than was the starting material. The solution in equilibrium with an excess of solid phase will be enriched in respect to another component. Preparations of diptheria antitoxin H and swine pepsin ~2 yielded solubility curves showing a high degree of inhomogeneity. These materials were purified by procedures based on extraction under conditions which dissolved only a fraction of the total, precipitation of the dissolved protein from the supernatant solution, and re-extraction of this last precipitate. With both proteins the solubility curves of the dissolved fractions were improved (i.e., approached more nearly that of a single component) with each extraction step, being essentially constant after three extractions. For full details see refs. 11-13. 10 M. Heidelberger and C. MacPherson, Science 97, 405 (1943). H j . H. Northrop, J. Gen. Physiol. 25, 465 (1941). 18 R. M. Herriott, V. Desreux, and J. H. Northrop, J. Gen. Physiol. 24, 213 (1940). 13 R. M. Herriott, Chem. Revs. $0, 413 (1942).

[9]

DNP METHOD FOR AMINO ACID SEQUENCE IN PROTEINS

221

Summary The solubility method is a theoretically sound, highly selective procedure for detecting the presence of impurities associated with any kind of molecule. In general it can be made as accurate as the available methods of analysis permit and requires no special apparatus. When the results reveal additional components, it is possible to design a method of isolating one, and in some instances two, components as homogeneous fractions.

[9] Determination of Amino Acid Sequence in Proteins by the Fluorodinitrobenzene Method

By R. R. PORTER The complete amino acid sequence has been determined in insulin and in ACTH, and considerable progress has been made with ribonuclease. Extension to other proteins and polypeptides may be expected at an increasing rate. The procedure followed is: 1. Amino acid analysis. 2. Estimation of N terminal and C terminal amino acids. 3. Separation of individual peptide chains if more than one is present. 4. Determination of the sequence in the peptides produced by selective partial hydrolysis in order that the total sequence of one chain may be deduced. Only the application of the fluorodinitrobenzene technique to these procedures will be described here.

Principle1 If 1,2,4,-fluorodinitrobenzeue (FDNB) in solution in ethanol is shaken with a solution or suspension of a protein in aqueous NaHCO~ at room temperature it will react with the amino, phenolic, imidazole, and sulfhydril groups of the protein. Only the substitution of the amino groups gives rise to colored derivatives which are relatively stable to drastic acid hydrolysis of the proteins. The DNP amino acids obtained by hydrolysis may be fractionated, characterized, and estimated, and hence the N terminal amino acids and the lysine residues with a free NSNH2 group F. Sanger, Biochem J. 39, 507 (1945).

[9]

DNP METHOD FOR AMINO ACID SEQUENCE IN PROTEINS

221

Summary The solubility method is a theoretically sound, highly selective procedure for detecting the presence of impurities associated with any kind of molecule. In general it can be made as accurate as the available methods of analysis permit and requires no special apparatus. When the results reveal additional components, it is possible to design a method of isolating one, and in some instances two, components as homogeneous fractions.

[9] Determination of Amino Acid Sequence in Proteins by the Fluorodinitrobenzene Method

By R. R. PORTER The complete amino acid sequence has been determined in insulin and in ACTH, and considerable progress has been made with ribonuclease. Extension to other proteins and polypeptides may be expected at an increasing rate. The procedure followed is: 1. Amino acid analysis. 2. Estimation of N terminal and C terminal amino acids. 3. Separation of individual peptide chains if more than one is present. 4. Determination of the sequence in the peptides produced by selective partial hydrolysis in order that the total sequence of one chain may be deduced. Only the application of the fluorodinitrobenzene technique to these procedures will be described here.

Principle1 If 1,2,4,-fluorodinitrobenzeue (FDNB) in solution in ethanol is shaken with a solution or suspension of a protein in aqueous NaHCO~ at room temperature it will react with the amino, phenolic, imidazole, and sulfhydril groups of the protein. Only the substitution of the amino groups gives rise to colored derivatives which are relatively stable to drastic acid hydrolysis of the proteins. The DNP amino acids obtained by hydrolysis may be fractionated, characterized, and estimated, and hence the N terminal amino acids and the lysine residues with a free NSNH2 group F. Sanger, Biochem J. 39, 507 (1945).

222

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[9]

will be determined. This procedure may be expressed as follows: NO~ N O ~ ~ F

R

L

-[- NH2CH--CO--protein

?

N02

--~ N 0 , ~ N H C H - - C 0 - - p r o t e i n Ethanol

D N P protein ~- H F

NaHCO3

NO2 NO~~NHCHCO--protein DNP protein NO2

t~

RI

--* N O 2 ~ N H C H C O O H -t- NH2CHCOOH Boiling DNP amino acid -t- amino acids 5.7 N HC1 The error of the method arises chiefly from the breakdown of the D N P amino acids during hydrolysis. As this breakdown is in certain cases considerable and may vary from protein to protein, correction is difficult, but it is sufficiently exact to determine accurately the number of terminal residues per molecule if this is less than ten.

Technique 1. Preparation of 1,2,$-Fluorodinitrobenzene. 2 101 g. (0.5 mole) of 1,2,4-chlorodinitrobenzene is dissolved in 101 g. of dry nitrobenzene, and to the mixture is added 60 g. (1 mole) of finely powdered dry KF. The mixture is heated on an oil bath at 190 to 195 ° for 5 hours under reflux and with very vigorous stirring. The mixture is cooled and filtered through sintered glass, the inorganic salts remaining being washed with hot toluene. The combined filtrates are dried over anhydrous Na2SO4 overnight, filtered, and the toluene distilled over in vacuo. The residue is fractionally distilled at 1 ram. pressure, and the fraction boiling at 60 ° (nitrobenzene) discarded. The second fraction, boiling at about 130 °, is collected and if necessary purified by redistillation. The F D N B thus obtained is a pale-yellow liquid which crystallizes on cooling (melting point 24°). The yield is about 70 g. 2. Preparation of D N P Amino Acids. D N P amino acids are required as standards for colorimetric estimation and for chromatographic identi2 H. G. Cook and B. C. Saunders, Biochem. J. 41, 558 (1947).

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DNP METHOD FOR AMINO ACID SEQUENCE IN PROTEINS

223

fication. Most of the derivatives to be expected from the hydrolysis of a D N P protein have been prepared and examples of the methods used are as follows. (It has been reported from several laboratories 3,~ that quantitative or near quantitative yields of D N P amino acids are obtained if only a slight excess of bicarbonate and F D N B is used.) a. DNP-L-valine: This m a y be taken as typical of all the simple D N P amino acids. Three-tenths gram (0.3 mole) of L-valine and 1.1 g. of NaHCO3 are dissolved in 14 ml. of H20, and to this is added 1.1 g. (0.6 mole) of F D N B dissolved in 28 ml. of ethanol. The mixture is shaken for 2 hours at room temperature, concentrated to remove ethanol, dissolved in water, and the excess F D N B extracted with ether. The aqueous solution is acidified with N HC1, and the DNP-L-valine, which precipitates, is filtered off. I t can be crystallized from aqueous methanol and recrystallized from ether-ligroin. Yield, 0.45 g. b. NS-DNP-L-lysine: 5 To prepare NS-DNP-L-lysine and N4-DNI)-L ornithine, the NUamino group is blocked in a copper complex before reacting with F D N B . One-half gram of L-lysine is dissolved in 10 ml. of H20, and CuC03 is added slowly to the boiling solution until no more dissolves. The excess CuCO3 is filtered from the dark-blue solution and washed with 2 to 3 ml. of H20. To this solution excess NaHCO3 and 1.1 g. of F D N B dissolved in 20 ml. of ethanol are added. The mixture is shaken for 2 hours at room temperature, and the precipitate filtered off and washed with water, ethanol, and ether. I t is suspended in 5 ml. of H20, and just sufficient N HC1 is added to produce a clear solution. This is cooled in ice, H2S bubbled through for 2 minutes, a little charcoal added, and the mixture filtered immediately. The filtrate is rapidly taken to dryness in vacuo and the NS-DNP-L-lysine hydrochloride crystallized from water and recrystallized from 20% HC1. Yield, 0.45 g. c. N~-DNP-L-lysine: ~ 1.25 g. of NS-benzoyl-L-lysine is dissolved in 10 ml. of water containing 1.0 g. of NaHCO~, and to it is added 1.0 g. of D N C B which has been dissolved in 20 ml. of ethanol. The mixture is refluxed on a water bath for 4 hours. The ethanol is removed by evaporation i n vacuo, the residue dissolved in water, and after filtering to remove excess D N C B it is acidified with HC1. On standing, an oil separates which weighs 2.0 g. Of this N1-DNP-NS-benzoyl-L-lysine, 0.5 g. is boiled under reflux for 3 days with a mixture of 5 ml. of acetic acid and 5 ml. of concentrated HC1. The mixture is evaporated to dryness, taken up in water, and filtered 3 W. A. Sehroeder and J. LeGette, J. Am. Chem. Soc. 76, 4612 (1953). 4 A. L. Levy and D. Chung, J. Am. Chem. Soc. 77, 2899 (1955). R. R. Porter and F. Sanger, Biochem. J. 42, 287 (1948).

224

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[9]

to remove unchanged substance. On neutralization with pyridine the N1-DNP-L-lysine crystallizes out. Yield, 0.2 g. d. O-DNP-L-tyrosine: 1 0.55 g. of N-acetyl-L-tyrosine 6 is heated with 2.0 g. of D N C B for 4 hours as in the p r e p a r a t i o n of N~-DNP-L-lysine. On acidification an oil forms which later crystallizes. I t could be recrystallized from aqueous ethanol as white needles. This is N - a c e t y l - O - D N P - L tyrosine. Yield, 0.1 g.; melting point 194 °. The p r o d u c t is boiled for 3 hours under reflux with 2 0 % HC1, cooled, and e v a p o r a t e d to small volume. The precipitate obtained is filtered off, dissolved in warm, dilute H N 0 3 , and neutralized with pyridine while hot. O-DNP-L-tyrosine crystallizes in white needles containing 1 mole of water. Crystallization of D N P Amino Acids Other D N P derivatives are prepared according to one of the a b o v e methods; convenient solvents for crystallization are listed in Table I. Thc melting points of crystalline D N P derivatives will be found in Table II. TABLE I SOLVENTS FROM WHICH D N P AMINO ACIDS HAVE BEEN CRYSTALLIZED

Amino acid derivative DNP-L-alanine DNP-L-arginine DNP-D~-aspartic Di-DNP-L-cystine DNP-D~-glutamic acid DNP-glycine Di-DNP-histidine DNP-D~-isoleucine Di-DNP-L-lysine DNP-DL-methionine N~-DNP-~ornithine N'-DNP-L-ornithine DNP-~-phenylalanine DNP-~-proline DNP-DL-serine DNP-D~-threonine DNP-L-tryptophan Di-DNP-DL-tyrosine

Solvents Ethanol and water Dilute and strong HCI Water 1. Ethylene glycol monoethyl ether and water 2. Acetic acid and water Water Acetic acid and water Methanol and water 1. Formic acid and water 2. Methanol and water Dry ether and ligroin Water Water and dilute HC1 Methanol and water 1. Ether and ligroin 2. Acetic acid and water Methanol Methanol and water Methanol and water Methanol and water

e V. du Vigneaud and C. E. Meyer, J. Biol. Chem. 98, 295 (1932).

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DNP METHOD FOR AMINO ACID SEQUENCE IN PROTEINS

225

TABLE II M E L T I N G POINTS OF D N P

Amino acid derivative DNP-DL-alanine DNP-L-arginine DNP-DL-aspartic Di-DNP-~cystine DNP-DL-glutamic DNP-glycine Di-DNP-L-histidine DNP-nL-isoleucine DNP-DL-leucine Di-DNP-L-lysine N~-DNP-L-lysine N1-DNP-b-lysine DNP-nL-methionine

Melting point 178 ° 252 ° 195 ° 109 ° 170 ° 205 ° 250 ° 168 ° 131 ° 173 ° 186 ° 260 ° 117 °

(d.) (d.)

(d.) (d.)

(d.)

DERIVATIVES OF AMINO ACIDS a

Amino acid derivative

Melting point

N4-DNP-L-ornithine N1-DNP-L-ornithine DNP-nL-phenylalanine DNP-L-phenylalanine DNP-L-proline DNP-DL-serine DNP-Db-~hreonine DNP-L-tryptophan Di-DNP-DL-tyrosine O-DNP-Tyrosine (1 H20) DNP-Db-valine DNP-L-valine

223 ° (d.) 227 ° (d.) 217 ° 188 ° 137 ° 198 ° 152 ° 180 ° 84 ° 202 ° (d.) 185 ° 130 °

There are some discrepancies in the literature; these figures are based on data generously supplied by Dr. H. Neurath, Dr. F. Sanger, and Dr. T. S. Work, as well as personal observation.

Preparation of DNP Proteins T h e p r o t e i n is d i s s o l v e d or s u s p e n d e d in t e n t i m e s i t s w e i g h t of N a H C O 3 s o l u t i o n ( 5 % w / v ) . T o t h i s is a d d e d t w i c e t h e v o l u m e of a n e t h a n o l i c s o l u t i o n ( 1 0 % w / v ) of F D N B a n d t h e m i x t u r e is s h a k e n for 2 h o u r s a t r o o m t e m p e r a t u r e . T h e D N P p r o t e i n , w h i c h is u s u a l l y i n s o l u b l e , is c e n t r i f u g e d a n d w a s h e d t h r e e t i m e s e a c h w i t h w a t e r , e t h a n o l , a n d e t h e r . I t is a i r - d r i e d for s e v e r a l d a y s . I f a s o l u b l e D N P p r o t e i n or D N P p e p t i d e is e n c o u n t e r e d , t h e d i f f i c u l t y of r e m o v i n g t h e s a l t m a y be a v o i d e d b y u s i n g a v o l a t i l e b a s e s u c h as t r i m e t h y l a m i n e i n s t e a d of N a H C O 3 t o k e e p t h e reaction mixture alkaline. 7 T o e s t i m a t e t h e c o n t e n t of t h e o r i g i n a l p r o t e i n in t h e a i r - d r i e d D N P p r o t e i n i t is n e c e s s a r y t o e s t i m a t e t h e c o n t e n t of a g r o u p or residue, which is u n a f f e c t e d b y t h e r e a c t i o n , in e a c h s a m p l e . E s t i m a t i o n of t h e a m i d e group has been found to be a simple and accurate method. ~ Amide estim a t i o n s a r e c a r r i e d o u t b y h y d r o l y s i s in b o i l i n g 2 N HC1 for 4 h o u r s , f o l l o w e d b y d i s t i l l a t i o n of t h e a m i d e a m m o n i a in a m i c r o - K j e l d a h l a p p a r a t u s a t 95 °, u s i n g a b o r a t e b u f f e r a t p H 9.5. A n a p p r o x i m a t e figure for t h e r a t i o s c a n be c a l c u l a t e d f r o m t h e m o i s t u r e c o n t e n t a n d t h e n u m b e r of D N P g r o u p s t o be e x p e c t e d f r o m t h e a m i n o a c i d c o n t e n t . I n m o s t cases t h e a i r - d r i e d D N P p r o t e i n c o n t a i n s a b o u t 75 % of t h e o r i g i n a l p r o t e i n . 7 F. Sanger and E. O. P. Thompson, Biochem. J. 53, 353 (1953).

226

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[9]

Hydrolysis of D N P Proteins The weight of D N P protein required depends on the number of a-amino groups present, but for preliminary examination 50 mg., is normally taken; this should be sufficient to identify one end group per molecular unit of 100,000. Owing to the lability of some D N P amino acids, it is necessary to carry out the hydrolysis under three sets of conditions. 1. Hydrolysis in boiling 5.7 N HC1 for 24 hours will destroy all peptide bonds and leave sufficient of all the D N P amino acids, except the derivatives of glycine, proline, and hydroxyproline, for identification. Under these conditions, DNP-tryptophan is converted to a compound which no longer gives a color with Erlich's reagent but which behaves chromatographically in the same way as DNP-tryptophan. 2. Hydrolysis in 12 N HC1 (sealed tube) at 105 ° for 24 hours enables DNP-proline and DNP-hydroxyproline to be identified. The yellow substances remaining are not DNP-proline and DNP-hydroxyproline, however. As with DNP-tryptophan, they behave chromatographically as the original compounds, but have a different absorption spectra, s 3. Boiling in 5.8 N HC1 for 4 hours will leave sufficient DNP-glycine for identification. Under the last two conditions D N P peptides may be present, but these can readily be recognized by submitting the colored derivatives to further hydrolysis. It has been observed 9 that if DNP-glycylglycine is hydrolyzed in a mixture of 90% w / v formic acid (10 vol.), acetic anhydride (5.5 vol., i.e., enough to combine with the water in the formic acid), and 60% w / v HC104 (1.4 vol.) for 2 hours at 100 °, there is complete hydrolysis of the peptide with very little destruction of the DNP-glycine. Such a medium may be of value also for hydrolyzing D N P proteins. For the estimation of D N P amino acids in a hydrolyzate it is clearly desirable to use the least drastic conditions of hydrolysis which are sufficient to break the adjacent peptide bonds. The approximate recoveries after hydrolysis in HC1 are given in Table III, but as the presence of the protein influences the amounts lost it is essential to carry out a direct estimation in the presence of the protein being investigated and under the conditions being used. Fractionation of D N P Amino Acids The D N P method has been used extensively during the last ten years, and it is noteworthy that the many modifications suggested have been 8 H. Fraenkel-Conrat and B. Singer, J. Am. Chem. Soc. 76, 180 (1954). 9 C. S. Hanes, F. J. R. Hird, and F. A. Isherwood, Biochem. J. 51, 25 (1952).

[9]

DNP METHOD FOR AMINO ACID S E Q U E N C E IN P R O T E I N S

227

confined a l m o s t e n t i r e l y to the p r o c e d u r e for t h e f r a c t i o n a t i o n of the D N P a m i n o acids. 1°-1~ This i n d i c a t e s where the g r e a t e s t difficulty has been met, b u t in this article a d e s c r i p t i o n will be given only of the m e t h o d s with which t h e a u t h o r is familiar. P a p e r c h r o m a t o g r a p h y is now used in preference to columns, as smaller a m o u n t s can be h a n d l e d successfully, t h o u g h in certain circumstances columns m a y still be used w i t h a d v a n tage. The p r o c e d u r e w i t h b o t h m e t h o d s will be described. TABLE III APPROXIMATE BREAKDOWN OF DNP AMINO ACIDS ON ACID HYDROLYSIS Hydrolysis in boiling 5.7 N HC1 Time, Amino acid derivative hours DNP-alanine DNP-arginine DNP-aspartic acid Bis-DNP-cystine DNP-glutamic acid DNP-glycine DNP-hydroxyproline DNP-isoleucine DNP-leucine Di-DNP-lysine NS-DNP-lysine DNP methionine DNP-phenylalanine DNP-proline DNP-serine DNP-threonine DNP-tryptophan DNP-tyrosine DNP-valine

12 12 24 12 12 8 4 12 12 8 I2 12 12 2 12 24 12 12 12

Hydrolysis for 16 hours in 12 N HC1 at 105°

Amount Amount unchanged (minimum), unchanged, % % 80 90 60 25 75 40 40 80 80 95 95 75 70 10 90 90 90 75 80

75 75 75 0 75 50 50 75 75 75 75 75 50 50 75 75 0 50 75

Paper Chromatography A description of the complete s e p a r a t i o n of D N P amino acids b y twow a y p a p e r c h r o m a t o g r a p h y has been described b y Levy. 13 This was d e v e l o p e d as a m e t h o d of amino acid analysis, a n d t h e p r o b l e m of endgroup analysis where only one or two different D N P amino acids are to be s e p a r a t e d a n d identified is m u c h simpler I n L e v y ' s m e t h o d the 10 F. C. Green and L. M. Kay, Anal. Chem. 24, 726 (1952). 1~S. M. Partridge and T. Swain, Nature 166, 272 (1950). l~ S. Blackburn, Biochem. J. 45, 579 (1949). 19A. L. Levy, Nature 174, 216 (1954).

228

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[9]

toluene-chloroethanol-pyridine-0.8 N ammonia solvent (Biserte and Osteux ~4) is run first, and 1.5 M aqueous phosphate buffer second. We have had difficulty in getting satisfactory results with the solvent of Biserte and Osteux and prefer to use the tertiary amyl alcohol solvent described by Blackburn and Lowther, 16 followed by phosphate buffer if necessary. The procedure is as follows: The protein hydrolyzate is diluted several times with water and extracted three times with ether, the ethereal extracts being washed three times with small quantities of water. If large quantities of water are used for washing the extract, some loss of DNP-serine and DNP threonine may occur. This extract, which contains all the acidic DNP amino acids, is then taken to dryness i n vacuo, water added, and again taken to dryness. This is repeated until the HC1 has been removed. The DNP amino acids are now taken up in a few drops of ethanol for application to the paper for chromatography. The paper (Whatman No. 4) is washed, by irrigation, with 0.05 M phthalate, pH 6, before use. The tertiary amyl alcohol, which should be free of peroxide, is equilibrated with a small quantity of buffer. Known DNP amino acids are spotted onto the paper as markers (about 20 ~, gives a satisfactory color), together with the unknown. The paper is now hung in the chromatographic tank in which stands a beaker with the tertiary amyl alcohol saturated with buffer. It is allowed to equilibrate with the solvent vapor for several hours before addition of solvent to the trough. If this equilibration is omitted the spots may tail badly. In an overnight run satisfactory resolution should be obtained of most of the DNP amino acids except DNP-aspartic acid and DNP-glutamic acid. These may be resolved if the ehromatogram is continued for a longer period. After the paper is dried, the spots are cut out and eluted by standing with occasional shaking in 4 ml. of 1% NaHCO3 for 15 minutes at 60 °. The extract is read at 3600 A. in a spectrophotometer. In order to compensate for any losses during chromatography, comparison is made with the absorption of known aliquots of standard solution run on the same paper. If the identity of the unknown is in doubt, the solution is acidified and the DNP amino acid extracted into ether and again chromatographed with 1.5 M phosphate buffer (containing 1 M NaH2PO4 and 0.5 M Na~HP04). This procedure is considered to be more satisfactory than running a two-way chromatogram directly when only one or two unknowns are expected. 14 G. Biserte and R. Osteux, Bull. soc. chim. biol. 83, 50 (1951).

j6 S. Blackburn al~d A. G. Lowther, Biochem. J. 48, 126 (1951).

[9]

DNP METHOD FOR AMINO ACID SEQUENCE IN PROTEINS

229

Fractionation with Silica Gel Columns

1. Preparation of Silica Gel. 16 One volume of commercial water glass. 1 vol. of H20, and 1 vol. of ice are well stirred in a large vessel; 12 N HCI is slowly poured in with continual stirring until the mixture of precipitated silica is acid to thymol blue. During the precipitation the mixture becomes very stiff, but it thins again as the correct pH is reached. It is allowed to stand for 3 hours, HC1 being added to maintain the acidity if necessary. The silica is filtered off under suction on a large funnel and washed well (approximately 2 1. of water per 250 g. of gel). This precipitate is suspended in 0.5 N HC1 for 2 days, filtered, and vcashed until free of chloride. It is then dried at about 100°. Only distilled water should be used throughout this preparation. There is some variation in the chromatographic behavior of different preparations, but in no case has an unusable batch been obtained. Generally the silica is prepared in large batches (say 5 kg.) in order that its characteristics may be fully understood and used to advantage. Samples are kept of any batch which proves to be particularly effective in certain fractionation steps. The variability of different batches of gel may be reduced if it is allowed to stand m HC1 solution of known concentration before washing and drying.17 The commercial water glass may be bought in 5-gallon drums from Joseph Crosfield Ltd., Warrington, England. 2. Preparation of Silica Gel Columns. The wet gel is prepared in several ways according to the solvent system in conjunction with which it is to be used. In most cases one-half its weight of water or buffer is well stirred into the dry gel. If intended for fractionation of basic DNP amino acids one-half its weight of N HC1 is used instead of the water, as N 5DNP-L-lysine is more stable in acid solution. If mixed organic solvents with a water-miscible component are used, two-thirds its weight of the aqueous phase is ground into the dry gel. To prepare the column, the wet gel (6 to 10 g.) is well mixed with wet organic solvent to give an even, freely running suspension. This is poured into a vertically held glass tube (30 X 1 cm. is convenient) whose lower end has been turned to support a perforated silver disk and filter paper. The column is rotated as it settles to remove air bubbles, and the supernatant solvent is allowed to drain through, care being taken that the bottom of the column is always immersed in the solvent in the receiving vessel. Unless this is done, the column will crack and have to be discarded. Despite this precaution frequent cracking of columns occurs if the gel has too high a water content. If a small positive pressure is used in the prepa,8 A. H. Gordon, A. J. P. Martin, and R. L. M. Synge, Biochem. J. 37, 79 (1943). ~7 p. Desnuelle, M. Rovery, and G. Bonjour, Biochim. et Biophys. Acta 5, 116 (1950).

230

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[9]

ration of the column, more even packing and, therefore, sharper bands are obtained. The compositions of the solvent systems, and abbreviations used to denote them, are given in Table IV. All the solvents are kept in contact with a small a m o u n t of aqueous phase, and sufficient time for equilibration should, of course, be allowed. The only exception to this procedure is the glycol-benzene system, in which case the glycol replaces the H~O and the gel is prepared by grinding the d r y gel with an equal weight of glycol. TABLE IV COMPOSITION OF SOLVENT 1VIIXTURES

Abbreviation

Composition

Chloroform Chloroform saturated with H~O CB1 99 % chloroform, 1% n-butanol, saturated with H~O 97 % chloroform, 3 % n-butanol, saturated with H~O CB3 95% chloroform, 5% n-butanol, saturated with H~O CB5 85% chloroform, 15% n-butanol, saturated with H20 CB15 70% chloroform, 30% n-butanol, saturated with H~O CB30 1 vol. I-I~O, 2 vol. ethanol, 10 vol. petroleum ether (b.p. 90-100 °) AL 1 vol. ether, 2 vol. petroleum ether (b.p. 90-100°), saturated with H~O EL 1 vol. H20, 1 vol. acetone, 10 vol. cyclohexane AC 1 vol. propanol, 20 vol. cyclohexane, saturated with H20 PC 1 vol. H20, 1 vol. methanol, 15 vol. carbon tetrachloride MC 2 vol. methyl ethyl ketone, 1 vol. ether, saturated with H20 M66 Benzene saturated with glycol GB Chloroform should be well washed before use, as the 1% ethanol, frequently added by manufacturers as a stabilizer, greatly alters its behavior. 3. Fractionation of D N P Protein Hydrolyzate. The mixture of D N P amino acids obtained b y ether extraction as described previously is dissolved in 1 to 2 ml. of the desired solvent (wet chloroform in the first instance) and run onto the prepared column. A bent-tipped pipette is used to avoid disturbing the gel surface. Quantitative transfer is effected by washings of the same volume added after the previous extract has sunk into the gel. With certain solvents a considerable number of washings m a y be necessary, though taking to dryness after addition of a few drops of 12 N HC1 often assists subsequent solution of the D N P derivatives. After transfer has been completed, the head space of the tube is filled with solvent and an indication of the D N P amino acids present can be obtained b y measuring the R values. (The R value is the rate of m o v e m e n t of the front of the band relative to the rate of movement of the surface

[9]

DNP METHOD FOR AMINO ACID SEQUENCE IN PROTEINS

231

of the solvent.) Synthetic D N P derivatives are run on parallel columns with the same solvent to give standard R values. In the hydrolyzate of a D N P protein it is usual to find a yellow band moving faster than any known D N P amino acid on a chloroform column. This is an artifact band of uncertain origin and should be ignored. If parallel columns of synthetic D N P amino acids are used, further fractionation according to the scheme in Fig. 1 should enable the components of an unknown mixture to be identified. To test the conclusions, the unknown is shown to be separable from other synthetic D N P derivatives, on an appropriate column, and inseparable from the identical derivative with any solvent system. It is most important in this work of fractionation and identification that only sufficient D N P derivatives are put on the column to make them clearly visible (50 to 100 ~ of D N P amino acid on a column 1 cm. in diameter). Greater amounts of color increase their R values and sometimes make separation impossible. Fractionation of the basic D N P amino acids, left in the aqueous residue after extraction with ether, follows the scheme shown in Fig. 2. In addition to the artifact band moving fast on a chloroform column, already referred to, other artifact bands may appear. Thus DNP-tyrosine and DNP-methionine on acid hydrolysis give rise to second colored bands stationary on a chloroform column, and DNP-tryptophan to a brown compound which is not soluble in ether. These artifacts are small relative to the DNP amino acids from which they arise and can be recognized by further fractionation. The possible presence of small amounts of D N P peptides must also be considered if uncharacterizable bands are found, as some of the D N P peptides are remarkably stable to acid hydrolysis. During work with D N P derivatives it is important that they be protected from direct sunlight, which causes discoloration and decomposition. If difficulty in fractionation is met, recourse to buffered columns with phosphate buffer in the range pH 3.5 to 6.5 may be helpful. 12 The rate of movement of the amino acids is greater with more acid buffers. Variation in the chromatographic behavior of different silica gel preparations still occurs with buffered columns, but perhaps less than when water is the stationary phase. 4. Estimation of DNP Amino Acids. In the quantitative assay of the end group of proteins it is desirable to use rather larger amounts of material (200 to 500 ~/ on a column 1 cm. in diameter) than are needed for identification because if only small quantities are taken the traces of D N P amino acids occasionally adsorbed onto the gel become a significant loss. The acidic D N P amino acids are collected as they run from the column, the organic solvent is removed in vacuo, and the acids are dissolved

232

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

~?

[9]

~. O

O

i

O CD ¢J

CD

O

__

.

[9]

DNP METHOD FOR AMINO ACID S E Q U E N C E IN P R O T E I N S

233

in 1 % N a H C O 3 solution for estimation in a photoelectric colorimeter at 3600 A. Calibration with s t a n d a r d solutions is, of course, necessary. The basic D N P amino acids are t r e a t e d similarly but are dissolved in N HC1 and estimated at 3900 A. Their m a x i m a l absorption is at 3600 A., as with the other derivatives, b u t c o n t a m i n a t i o n with colorless O D N P - t y r o s i n e , which also absorbs in t h a t wavelength, occurs. Original Mixture

t M66 I

1

t

1

I

2

i

3

4

[ N1-DNP-arginine N6-DNP-lysine NI-DNP-lyslne CB17

t

i

1.1 di-DNP-histidine

1

1.2 S-DNP-eysteine

FIG. 2. Scheme of fraetionation of water-soluble DNP amino acids. T a b l e V 18 gives the optical densities of some D N P - a m i n o acids and peptides, and Figs. 3 and 4 show their absorption curves. D N P - p r o l i n e is exceptional in t h a t its maximal absorption is at 3850 A. OPTICAL DENSITIES OF

TABLE V 20-~M. SOLUTIONS OF DNP AMINO ACIDS AND PEPTIDES Optical density (log Io/I)

DNP derivative DNP-glycine DNP-phenylalanine DNP-glyeyl-glycine DNP-phenylalanyl-valine NS-DNP-lysine O-DNP-tyrosine

Solvent

3500 A.

3900 A.

1% NaHCO3 1% NaHCO.~ 1% NaHCO~ 1% NaHC03 N HC1 N HC1

0. 309 0.313 0.316 0.310 0. 296 0. 058

0. 210 0. 214 0. 173 0. 178 0. 204 0.0

5. Determination of Amino Acid Sequences of Terminal Peptides of Proteins. I n a s t u d y of terminal peptides with the D N P technique it is preferable t h a t the work should be done with a single polypeptide chain; otherwise the confusion arising f r o m different sequences in different chains m a y p r e v e n t a d e q u a t e interpretation of the results obtained. ~sF. Sanger, Biochem. J. 45, 563 (1949).

234

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[91

Insulin is the only protein where the different polypeptide chains have been separated from each other. In this case the disulfide bridges, apparently the only stable interchain bonds, were split by oxidation with performic acid. 19 The oxidation products could then be separated into two main fractions which corresponded to the two peptide chains. Other proteins made of dissimilar chains would no doubt present special problems.

0.4

Z

.t..,

0.2

o"

\

/ \

' ~,

/

%,

0.I 0 2500

"', I

f

3000

3500

4000

4500

5000

Wavelength (A) Fro. 3. Spectral absorption curves in 1 cm. cell of 23.7-~M. solution of D N P phenylalanine ( ) and DNP-phenylalanylvaline ( ....... ) in 1% NaHCO3.

If end-group analysis has shown the protein to contain only one open chain, or if the different chains can be fractionated, this material is treated with F D N B and subjected to partial hydrolysis. The terminal D N P amino acid and the terminal D N P peptides of increasing size may be separated from NLDNP-lysine and NS-DNP-lysyl peptides by extraction of the hydrolyzate with ethyl acetate and then separated from each other by chromatography in a similar way to the fractionation of D N P amino acids. Complete hydrolysis of these peptides should release the same terminal D N P amino acid and an increasing number of free amino acids to which it was bound. These amino acids may be identified by paper chromatography and hence the order of terminal sequence deduced. This method has been usefully applied to determine the structure of terminal penta- and hexapeptides. Larger D N P peptides are so little soluble that fractionation becomes impossible. 19 F. Sanger, Biochem. J. 44, 126 (1949).

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DNP METHOD FOR AMINO ACID SEQUENCE IN PROTEINS

235

PARTIAL HYDROLYSIS. The best conditions of hydrolysis to produce a series of D N P peptides of increasing size can only be found by trial with the particular protein or peptide to be investigated, as the stabilities of different peptide sequences vary greatly (cf. Sanger2°). Hydrolysis with HC1, with the strength, temperature, and time varied, may give the desired maximum yield of smaller peptides without too complete hydrolysis. FRACTIONATION. The partial hydrolyzate is diluted if necessary and treated as follows: (1) The hydrolyzate is extracted three times with ethyl acetate. (2) The ethyl acetate extracts are combined and extracted in countercurrent manner three times with 1% NaHC03 solution. (3) The

0.4

0.3

._~

0.2

o 0.1

0 .... 2500

I 3000

I ' 3500

~

1 4000

I 4500

5000

Wavelength(A) Fro. 4. Spectral absorption curves in 1 cm. cell of 22-~M. solutions of N L D N P lysine ( ) and O-DNP-tyrosine ( ....... ) in N HC1.

combined NaHCOa solution extracts are acidified with HC1 and extracted as above with ethyl acetate. This final extract should contain all the acidic D N P peptides and very few nonacidic D N P peptides. Some N L DNP-lysyl peptides may, however, persist in the ethyl acetate fraction, and, though none have been met, it is possible that terminal peptides containing a basic residue would remain in the aqueous fraction. For fractionation of the peptide derivatives oil silica gel columns, chloroform containing up to 30% by volume n-butanol has been found to give a most useful series of solvent mixtures. Thus, if the crude ethyl acetate extract is run on a CB15 column (see Table IV), all but the NLDNP-lysyl peptides, which have been extracted into the ethyl acetate, and perhaps very large terminal peptides, move fast. This fast~0 F. Sanger, Adxances in Protein Chem. 7~ 1 (1952).

236

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

.[9]

moving band is then put on a CB5 column (see Table IV), and the new fast-moving band again transferred to a chloroform column. The peptide bands which run slowly are developed, if necessary, with intermediate chloroform-n-butanol mixtures. Buffered gel systems have also been used to advantage on occasion, but some induce considerable tailing of the bands. In general, the more acid the buffer used, the more quickly will the DNP amino acids and peptides move down the column. Tailing is a difficulty met more frequently with DNP peptides than with DNF amino acids and varies with the batches of gel used. The running of the colored bands on different columns to establish homogeneity is particularly necessary when pronounced tailing occurs. In the partial hydrolysis products of both DNP-insulin and DNP rabbit ~,-globulin, small amounts of color, which are stationary on a chloroform column but which move on a CB1 column to split into a number of fractions, have occasionally been observed. They contain a complex mixture of amino acids which may arise from esterification occurring during the procedure. Other anomalous behavior, such as the splitting on a column of a homogeneous component to give identical fractions which subsequently behave similarly, rarely occurs with DNP amino acids but has at times been observed during work with DNP peptides. This fractionation procedure may have to be adapted considerably to meet the requirements of the great variety of peptide mixtures which can occur, but it has proved successful in the applications so far made. Estimation of the DNP peptides is carried out as for the DNP amino acids; the absorption is read at 3500 A., where it is uninfluenced by adjacent amino acids in peptide linkage. 6. Identification of D N P Peptides. The DNP peptides are collected as they run from the column, the solvent removed and the residue dissolved in 5 to 6 drops of 5.7 N HC1 by warming over a microburner. The solution is sucked into a capillary tube which is sealed and kept at 105° for 24 hours. The hydrolyzate is dropped into a small tube for extraction with ether and the DNP amino acid in the ethereal fraction identified as previously described. The aqueous residue is transferred by the capillary tube to a polythene strip and taken to dryness in a desiccator to remove excess HC1. A drop of water is added to dissolve the amino acids and the drop " p r i n t e d " on to a filter paper sheet so that the acids may be identified by partition chromatography. This method is based on that described by Consden et al. 21 for the identification of peptides. In any partial hydrolyzate a mixture of terminal peptides will result, and should the original material have only one polypeptide chain with, consequently, one terminal amino acid, the colored derivatives obtained ~ R. Consden, A. H. Gordon, and A. J. P. Martin, Biochem. J. 41, 590 (1947).

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DNP METHOD FOR AMINO ACID SEQUENCE IN PROTEINS

237

under appropriate hydrolytic conditions will be constituted as follows: (1) D N P A, (2) D N P A + B, (3) D N P A -b B -[- C, (4) D N P A q- B ÷ C q- D, where A, B, C, and D correspond to different amino acids. If it is possible to account for the total amount of D N P A known to be originally present in terms of these peptides and to show that, say, peptide 4 will, on further partial hydrolysis, give only peptides 3 W 2 and D N P A, it is reasonable to conclude that the terminal sequence of amino acids in the chain is ABCD. Clearly the same method may be used to determine the terminal sequence of small or large peptides obtained by the hydrolysis of a protein. In this case fractionation of the hydrolyzate is the principal difficulty, and it is essential that the purity of the peptide to be investigated is established before partial hydrolysis of the D N P derivatives is undertaken. The presence of only one terminal amino acid would be good enough though not conclusive evidence of the purity of the peptide to be examined.

NS-DNP-Lysyl Peptides If only one or two lysyl residues are present in the peptide or protein being studied, it is possible to identify the adjacent amino acids in an analogous manner. These DNP-lysyl peptides will be in the aqueous residues from the ethyl acetate extraction together with the unsubstituted peptides. There is a possibility that the latter will not be separated from the DNP-lysyl peptides during chromatography. Sanger 17 overcame this by absorbing the D N P peptides onto talc from acid solution, other amino acids and peptides passing through. After the talc was washed with N HC1 the D N P peptides were eluted with 4 parts of ethanol and 1 part of N HCI. There is still some risk of the N~-DNP-lysyl peptides being contaminated by O-DNP-tyrosyl peptides which are colorless. Such contamination could be detected from the shape of the absorption curve of the lysyl peptides, as mentioned earlier. Fractionation of the NS-DNP-lysyl peptides may be effected with CB15, CB30, and M66 columns. It should perhaps again be stated that the methods of fractionating D N P peptides need to be varied according to the protein being investigated and the special problems the resulting partial hydrolysis mixture offers.

238

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

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[10] The Pipsyl Method for Amino Acid Sequences in Proteins B y MILTON LEVY

Principle A reagent, p-iodobenzenesulfonyl chloride (pipsyl chloride), reacts with amino and phenolic hydroxyl groups to form stable derivatives. The amino derivatives have a molar absorption coefficient of approximately 16,000 at 250 m~. The reagent can be prepared with 1131 or S ~5 label. If the reaction between the reagent and the functional groups mentioned can be made stoichiometric, quantitative analyses of the derivative mixtures can be made on the isotopic dilution principle. Two forms of this method have been proposed. These are the carrier method,~ in which the diluent is the unlabeled compound of interest, and the indicator method, ~ in which the diluent is the compound of interest labeled with a different isotope than the unknown. These methods differ from the classical isotope dilution method by introducing a detectable label in the unknown rather than in the cartier. They can be used to demonstrate the absence of a particular derivative with a certainty not possible by any other method. The key to adequate quantitative work by this method is the initial derivative formation. Methods for quantitative derivative formation are available for most amino acids (see below) and for some peptides. The reactions of proteins require individual study2 In spite of the great resistance of the sulfonamide bond to hydrolysis and the use of internal control in the hydrolysis, the reagent is not yet firmly established as an end-group label for proteins. Qualitative but not quantitative agreement with the DNBF method has been demonstrated, s Independence of quantitative yield in the pipsylation reaction can be achieved by adding an isotopically labeled sample of the substance to be determined (i.e., C14-1abeled alanine) to the unknown and isolating the derivative from the mixture. 4 This method is closely related to classical isotopic dilution. Thus the absence of the substance is difficult to establish because the dilution of the C 14label is zero and the errors of counting and spectrophotometry obscure the result. 1A. S. Keston, S. Udenfriend,and R. K. Cannan, J. Am. Chem. Soc. 71, 249 (1949). A. S. Kestoa, S. Udenfriend, and M. Levy, J. Am. Chem. Soc. 72, 748 (1950). 3S. Udenfriend and S. Velick, J. Biol. Chem. 190~ 733 (1950). ' A. S. Keston and J. Lospalluto, Federation Proc. 11, 239 (1952).

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PIPSYL METHOD FOR AMINO ACID SEQUENCES IN :PROTEINS

239

Preparation of Labeled Pipsyl Chloride Standard radiological safety precautions are necessary t h r o u g h o u t all procedures but will not be described here. The methods described have given radiochemical yields of 70 to 80 %.

Sulfanilic Acid (S 35) HOAC C H 3 C O - - N H C 6 H 5 + H2SO4 ' > CH3CONHC6H4SO~H (AcO)20 1 N HCI CHaCONHCsH4SO3H -t- H20 > CH3COOH -t- NH2C6H4SO~H The procedure is based on the method of SSll and Stutzer ~ as modified b y Lichtin 6 for high conversion of sulfur to the derivative. Isotopic sulfuric acid is obtained carrier-free from the Oak Ridge National Laboratories in lots of 100 to 200 mc. E v a p o r a t e the solution to 0.5 ml. in a 25-ml. flask under vacuum. (All glass apparatus referred to is standard-taper "Micro Organic. ") Add 80 rag. (0.8 millimole) of sulfuric acid, and heat the open flask until dense white fumes appear. Cool. Add 1.4 ml. of glacial acetic acid and 0.6 ml. of redistilled acetic anhydride. R e m o v e these solvents in vacuo at 55 ° (bath). Again add the same a m o u n t of acetic acid and the anhydride to the residue. Add 100 mg. (0.82 millimole) of acetanilid, and heat in a bath at 60 ° for 2 hours under a 100-cm. aircooled condenser. Let the mixture stand at room temperature for 16 hours. A copious white precipitate will be present. Add 15 ml. of 3 N HC1, and reflux for 2 hours. Remove all solvents b y distillation in sacuo to dryness. Add 10 ml. of water, and evaporate. The d r y residue contains a b o u t 0.8 millimole of SSS-labeled sulfanilic acid. Dissolve it in 1.5 ml. of 1 N N a O H for the next step.

Sodium Pipsyl (S ~5) HC1 NH2--CsH4SO3H + H N O 2 > + +N2C6H4SO~- -/- H20 +N2CsH4SO~ ~- I---~ IC6H4SO3H + N2 Add 55 mg. (0.8 millimole) of sodium nitrite. Cool on ice. Add 3.2 ml. of 3 N HC1. After 30 minutes add a solution of 34 rag. of sulfanilic acid (0.2 millimole) in a minimal a m o u n t of 1 N NaOH. Test for nitrous acid with starch iodide paper. If present, add sulfanilic acid. After 0.5 hour add a solution of 250 rag. of K I in water. Let stand overnight at room temperature, and warm to 60 °. Neutralize with solid NaHCO3, and J. SSll and A. Stutzer, Ber. 42, 4539 (1909). e R. A. Lichtin, M. S. thesis, New York University, 1945.

240

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

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evaporate by letting the solution stand in an oven at 90 to 95 °. Sodium Pipsyl (I 131)

+N~C6H4SO~

-{- I---~ IC6H~SO3H

~ N~

Carrier-free I TM (I00 to 200 inc.) is obtained from the Oak Ridge National Laboratories in alkaline solution containing bisulfite. Transfer the main portion to a 25-ml. pear-shaped distilling flask. Rinse the remaining I TM to the flask using 10 mg. of K I in 1 ml. of water and t h e n 1 ml. of water. Evaporate the solution in vacuo to about 0.5 ml. Remove the still head, and wash the capillary inlet with 0.5 ml. of water. Dissolve 57 rag. (0.3 millimole) of sulfanilic acid in 0.5 ml. of 1 N NaOH. Add 21 rag. of NaNO~, cool on ice, and add 1 ml. of 3 N HC1. After 10 minutes a white precipitate appears. Add i ml. of 3 N HC1 to the cooled " i o d i d e " solution. Add 0.4 ml. at the diazo slurry. Shake until all has dissolved, and warm to 60 ° for 3 minutes. Cool. Add 10 rag. of KI, dissolve, and add 0.4 ml. of a diazo slurry. Mix and warm to 60 °. Cool, add 10 mg. of KI, dissolve, and add 0.1 ml. of diazo slurry. Warm to 60 °, let stand for an hour, and then add solid NaHCO3 cautiously until neutral. Evaporate overnight in an oven at 90 to 95 ° . Pipsyl Chloride (S ~5 or I TM) POCl3 4IC~H4SO3Na q- PC15

~ 4IC6H4SO~C1 q- NaC1 q- Na3PO4

Add 1 ml. of POC13 to the dried sodium pipsyl, and break up the solid with a glass rod. Add a pea-sized portion of PC16, and crush with the rod. Warm to 60 ° for a few minutes. Add the same amount of PC15 twice more and additional POC13 to keep the mixture liquid except for the NaC1 present. Fill a 250-ml. separatory funnel half full of crushed ice. Transfer the mixture in the flask to the separatory funnel with four 5-ml. portions of benzene. Shake to destroy the phosphorus chlorides, and allow the ice and benzene to melt. Add solid sodium bicarbonate cautiously until the aqueous layer is neutral to congo red paper. Discard the aqueous layer, and wash the benzene with 20 ml. of ice water. This washing should not be acid. Transfer the benzene layer to a 125-ml. Erlenmeyer flask, and dry it with a minimal amount of anhydrous Na~SO4. After an hour transfer the benzene solution to a distilling unit, and remove the benzene in vacuo. Wash the Na2SO4 residue with benzene containing 50 mg. of ordinary pipsyl chloride. Repeat this washing a second time. Evaporate all the benzene solutions to a volume of about 0.5 ml., and transfer to a cold-finger vacuum sublimation apparatus, using a little benzene. Remove the benzene in the sublimation apparatus at room temperature with no

[10]

PIPSYL METHOD FOR AMINO ACID SEQUENCES IN PROTEINS

241

water flow in the condenser. Sublime the pipsyl chloride in a bath at 150 ° and at a pressure of about 3 mm. The white crystalline deposit on the cold finger is washed with benzene into a 20-ml. vial containing 1 g. of ordinary pure pipsyl chloride. Bring all material into solution with as little benzene as possible, using gentle heat. About 2 ml. should be sufficient. After cooling add 8 ml. of petroleum ether to precipitate the pipsyl chloride. Evaporate the solvents with a stream of dry N2. Add another portion of petroleum ether, and again evaporate. Nonisotopic pipsyl chloride is prepared by the methods described by Weygand. 7 It should be sublimed at 150 ° and 3 mm. to ensure purity. It can be readily recrystallized from benzene. Nonisotopic pipsyl chloride does not discolor over long periods, but the radioactive samples do. The I131-1abeled material discolors more quickly than the S aS. The discoloration does not interfere with the utility of the reagents.

Carriers Dissolve about 30 millimoles of amino acid in 50 ml. of N sodium bicarbonate in a flask provided with an efficient stirrer. Add 6 g. (20 millimoles) of pipsyl chloride, and heat to about 90° to melt the chloride. With efficient stirring the pipsyl chloride goes into solution quite rapidly. Cool the solution, and add 1 g. of KC1. A small amount of impurity precipitates. Add activated charcoal, filter, and acidify the filtrate. Crystals of the derivative generally appear. If not, extract the aqueous layer with ether or ethyl acetate, extract the organic layer with a small volume of NaOH solution, heat to drive out solvents, and acidify. The materials are readily recrystallized from hot water or from water-acetone mixture.

Indicators These are used in much smaller amounts than the carriers. They are prepared from one of the isotopic pipsyl chlorides, usually S% Weigh 6 to 10 mg. of S 3s pipsyl chloride on a bit of aluminum foil, and transfer to a dry Folin-Wu blood sugar tube. Measure into the tube 0.1 m]. of amino acid (or other pure compound) solution containing 50 micromoles of amino group, and add 0.4 ml. of saturated borax solution. Clamp the tube on a ring stand to which is attached an electric massage unit of the motor-driven type. The object is to produce a vibration of 5- to 10-mm. amplitude at about 3600 cycles. Bring a hot water bath about the tube, and when the pipsyl chloride is molten start the vibration. At 90 ° the pipsyl chloride disappears in a minute or so. Stop, cool, and add a glass bead to the tube and 2 ml. of ether. Shake, remove the ether with a C. Weygand, "Organic Preparations," p. 112. Interscience, New York, 1945.

242

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

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Pasteur pipet, and repeat the extraction twice more. Transfer the aqueous layer to a 3 g. column of Dowex I (2 % cross-linked in the chloride form, 200 to 400 mesh before cycling), and elute with a solvent made of 350 ml. of 95% alcohol, 10 ml. of 1 N HC1, and water to make 1 1. The rate of elution may be 3 to 6 ml. per hour, and hourly collections are made. The compound may be located by ultraviolet absorption (250 m~) or by radioactivity. The combined fractions containing the derivative may serve as indicator stock, or the solutions may be evaporated and the compound dissolved in a small volume of alkali.

Preparation of Radioactive Standards Pure DL-alanine obtained by hydrolysis of benzoyl alanine is pipsylated with the iodine or sulfur reagent and purified by the method described above. Evaporate the solution to dryness, and recrystallize the pipsyl alanine from 1 ml. of hot water by cooling on ice. After two recrystallizations, discard the supernatants and dissolve the residue in about 1 ml. of water with a few drops of dilute ammonia. Accurately dilute 25 ~l. of this solution to 25 ml. with 0.2 N HC1, and measure the absorption of the resultant solution at 250 m~ in a 1-cm. cell on the Beckman spectrophotometer. The absorption coefficient for pipsyl alanine at this wavelength is 15.8 per micromole per milliliter. Planchettes are prepared by evaporating suitable amounts of a dilution of the stock in water. The amounts of material on the planchettes are so small as to make self-absorption corrections unnecessary. The fresh reagents prepared as above have shown 1 to 2 X 10e (for S 35) and 8 × 10e (for 1131) counts per minute per micromole under mica window (1.7-mg./cm. 2) Geiger tubes. The counting apparatus must be rigorously standardized for geometry, dead-time losses, etc.

Preparation of Unknown The mlknown in 0.1 ml. of solution should contain about 1 micromole of total amino acompounds, but the amount may be increased simply by providing additional pipsyl chloride in about stoichiometric amounts. The water and amino compound in the mixture compete for the pipsyl chloride so that the hydrolysis at an absolute amount of pipsyl chloride (about 20 micromoles) is accompanied by the formation of pipsyl derivative from about 0.7 to 0.9 of the amino compound present. If attempts are made to drive the reaction further by addition of more pipsyl chloride, some of the product may be lost by "dipipsylation" or decomposition of amino acid. 8 To obtain quantitative conversion, the product is removed and the reaction is carried out in three steps. s E. Slobodian, in preparation.

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PIPSYL METHOD FOR AMINO ACID SEQUENCES IN PROTEINS

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Weigh 7 mg. of radioactive pipsyl chloride (generally the I TM reagent) into a Folin-Wu tube, add the sample in 0.1 ml. of water, and then 0.5 ml. of saturated borax (standardize by titrating with standard HC1 to methyl red). It is about 0.3 N in available alkali. Clamp to the vibrator and water bath unit, and carry out the reactions as indicated for preparation of indicators at a bath temperature of 90 to 95 °. Cool and acidify with 1 ml. of 3 N HC1. Add a glass bead. Extract three successive times with 3-ml. volumes of ether. Place the extracts in a 250-ml. Erlenmeyer flask. Connect a rubber-stoppered outlet tube to the top of the Folin tube, and evaporate while vibrating in the hot-water bath with an aspirator vacuum. Add 1 ml. of water, and evaporate again. To the residue in the tube add 10 mg. of the same pipsyl chloride preparation as previously, then 0.5 ml. of 0.3 N NaOH, and repeat the processes as described. A final reaction using 15 mg. of pipsyl chloride is carried out to ensure nearly quantitative conversion. To the combined ether or ethyl acetate extracts add a few drops of concentrated aqueous ammonia to react with any residue of pipsyl chloride. The process described above is suitable for amino acids. Peptides react more completely in Na2HP04 solutions. Instead of saturated borax add 28 mg. of Na2HPO4 and 0.5 ml. of water. Use 15 mg. (50 micromoles) of pipsyl chloride. The yield for tested peptides is 85 to 95 % in a singlestage reaction. After cooling add 1 ml. of 3 N HC1 and a glass bead. Extract with three portions of 3 ml. each of ethyl acetate. If desired, the aqueous residue may be evaporated and 0.5 ml. of 2 M NaOH added with additional pipsyl chloride (15 mg.). The process is repeated and the additional ethyl acetate extracts added to the others.

Analysis It will be assumed that the indicator method is to be used. The carrier method allows crystallization to be used in the purification, but this seems to be less powerful as a separation device than are the egraphic* (chromatographic) methods to be described. Add measured volumes of one or more of the indicator stock solutions to the organic layers; make sure that homogenization of the isotopic compounds is complete by adding sufficient NH3 and water to ensure solution. From this point on quantitative recovery of compounds is not necessary. The object is to obtain demonstrably pure samples for determination of isotope ratio. Separation techniques which have been used are countercurrent distribution between solvents (for distribution coefficients see Table I), ion exchange egraphy (chromatography) on Dowex 1 X 2 C1- with 35 % or 20 % alcoholic solvents * Egraph: a writing out; Latin e (ex) out, and graphus, written. The meaning is synonymous with chromatography b u t does not suggest color.

244

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[10]

TABLE I DISTRIBUTION COEFFICIENTS OF PIPSYL DERIVATIVES (Systems: I, CC14-0.2 N HC1; II, CHC18-0.2 N HC1; III, ethyl ether-0.2 N HC1; IV, ethyl acetate-0.2 HCI; V, n-butanol-0.2 N HC1; VI, n-butanol-0.2 N NHs. Values entered are concentration in organic phase/concentration in aqueous phase) Pipsyl derivative of Alanine Aspartie acid Glutamic acid Glycine Hydroxyproline Isoleueiae Leucine Methionine Phenylalanine Proline Serine Threonine Valine Alanylglyeine Alanylalanine Glycylalanine Glycylglyeine Glyeylserine Glycylalanylglycine Alanylglyeylglycine Glycylalanylglycylalanine

I

II

0.04 0.00 0.00 0.01 0.01 1.60 1.24 0.26 0.75 0.67 0.00 -0.48 0.00 -0. 004 0.01 -----

2.9 O. 01 0.01 0.63 0.11 50 100 . . 47 0.05 0.16 22 0.06 0.23 0.15 0.05 ---0.20

III 100 15 17 43 4.7 100 100 . .

. . 56 0.42 7.9 100 2.4 4.8 4.2 1.5 0.34 0.34 0.20 0.41

IV

V

VI

100 90 100 100 30 100 I00 . . 100 22 45 100 31 39 37 30 6.9 6.9 3.5 4.5

83 54 74 78 28 100 -. . 33 8.8 43 -32 100 48 26 18.3 21 19 20

3.2 0.02 0.02 1.15 -6.5 9.5

T A B L E II RETARDATION OF PIPSYL COMPOUNDS ON DOWEX 1 X 2 CI- COLUMN (Eluant 350 ml., 95% ethanol, 10 ml. 1 N HC1, water q.s. 1 1.; 3 g. Dowex 1 X 2 CI-, 200 to 400 mesh) Pipsyl derivative of Alanine Aspartic acid Glutamic acid Gly(.ine Hydroxyproline Isoleucine Leucine Phenylalanine Proline Serine N-Tyrosine Glyeylalanylglyeylalanine

35% eluant, ml. 66 155 107 93 82 5t 57 128 56 78 210 53

-0.56 1.8 8.6 -1.13 ----0.09 --

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PIPSYL METHOD FOR AMINO ACID SEQUENCES IN PROTEINS

245

containing 0.01 N HC1 as eluent, and paper egraphy using such solvents as butanol equilibrated with 1 N NH3 or tetrachloroethane-butanol mixtures equilibrated with 0.2 N HC1. Some data suggesting the possibilities are given in Tables II and III. TABLE III SOME RS VALUES FOR PIPSYL AMINO ACIDS ON WHATMAN NO. 1 PAPER (Eluants: I, butanol saturated with 0.1 N NH.~; II, 0.1 N HC1; III, 85 ml. propanol-15 ml. 1 N NH.~)

Pipsyl derivative of Alanine Aspartic acid Glutamic acid Glycine Hydroxyprolinc Isoleucine Leucine Methionine Methionine sulfone Phenylalanine Proline Serine Threonine Valine

I

II

III

0.5 0.03 0.03 0.40 0.30 0.75 0.75 0.65 0.40 0.65 0.40 0.40 0.50 0.70

0.7 .

0.7 0.13 0.13 0.60 0.53 0.90 0.86 0.82

0.60 0.60 0.60 0.35 0.70 0.60

0.60 0.50 0.63 0.90

Special Purification Technique, Washout This special technique is employed in the indicator method when separation in the countercurrent distribution on the limited basis used (10 plates) is not sufficient for the mixture of compounds. Thus combined plates of a couutercurrent distribution to obtain group I amino acids (glutamic and aspartic acids, serine, threonine, and hydroxyproline) contain a considerable a m o u n t of pipsylglycine. 2 To the mixture add nonradioactive pipsylglycine in q u a n t i t y (2 to 3 mg.) dissolved in alkali, acidify to precipitate it, and remove b y filtration or centrifugation. To the supernatant add additional pipsylglycine and repeat. The precipitated pipsylglycine removes the radioactive analog in proportion to the fraction precipitated. However, losses of other compounds b y adsorption m a y limit the extent to which this purification m a y be carried.

Paper Egraphy Any suitable arrangement for paper egraphy m a y be used. I t is well to spread the sample along a 20-mm. line about the origin. The spots

246

TECHNIQUES FOR CHAR,kCTERIZ~kTION OF PROTEINS

[10]

may be located by their radioactivity by a scanning method, by autography on X-ray film, or by observation under an ultraviolet lamp which shows the pipsyl compounds as darkened areas. Any suitable spot is cut perpendicular to the direction of development into three or more strips. Each strip is separately eluted with dilute NH3 and planchetted. A constant ratio of S 35 to I TM activity in the successive strips is an excellent assurance of purity. A trend indicates impurity. A second egraphic development with the same or a different solvent is often useful.

Ion Exchange Egraphy The ion exchange egraphy of the pipsyl compounds on Dowex 1 × 2 C1- is carried out on 0.9-cm.-diameter columns using various dilutions of 95% alcohol containing 0.01 N HCI. The resin is cycled from the commercial 200- to 400-mesh material through hydroxide and chloride forms. Three grams of air-dried resin are placed in the columns as a slurry in the 50% eluent (500 ml. of 95% alcohol, 10 ml. of 1 N HCI diluted to 1 1.). After settling, 50 to 100 ml. of the 50% eluent is forced through as rapidly as possible (5 to 6 seconds per drop). The sample is then added and allowed to flow into the resin by gravity, and elution is carried out with a suitable alcoholic eluent (35% for the amino acids, 20% for peptides) at a rate of about 3 ml. per hour (40 to 50 seconds per drop). The fractions are followed for radioactivity or ultraviolet absorption or both. Some retardation values are given in Table II. Pipsyl acid seems never to be eluted from the columns, but the higher the peptide molecular weight the earlier it comes out. With an 8 % cross-linked resin the bands are much wider and more retarded than with 2% cross-linked resin.

Counting Samples are mounted on metal planchettes from solutions in NH3. They are centered as accurately as possible. It is desirable to have sufficient counts on a planchette to reduce the time required but not so many as to produce dead-time losses. The samples will be obtained as successive fractions from columns or as elutions of the successive strips of paper egraphic separations. For final analysis it is necessary to count the planchettes without a filter (sin count, S) and with a filter (cum counts, C). A suitable filter is made from 0.003-inch thick aluminum foil mounted to be placed without disturbing the sample. The filter factors (f) are determined by measuring C and S for pure I TM pipsyl alanine (f~) and pure S 35 pipsyl alanine (f~). Our filters give about f, = 0.003 and f~ = 0.60. The calculations are based on the simultaneous equations S -- Ci - C,, where C~ and C, are the counts due to iodine and sulfur,

Ill]

METHODS FOR INVESTIGATING THE ESSENTIAL GROUPS

247

respectively, from the unfiltered sample, and C = f ~ C i - f8C8. Thus C , = (c -

f~S)/(f,

C8 =

-

(AS

C)/(f~

-

fs) -

f,)

The pipsyl alanines from the two reagents used are standardized by ultraviolet absorption, planchetted, and counted both with and without filters. Ci for the sample divided by the molar radioactivity of the standard gives the number of moles of I TM compound on the planchette. When this is multiplied by the ratio of total S 35 counts (added as indicator) to C,, the result is the number of moles of the unknown in the original derivative mixture and if derivative formation was complete is the desired analytical figure.

Sequences The determination of any peptide in a mixture derived from hydrolysis of a protein is pertinent data for sequence studies. The determination of the amino end group in pipsyl peptides follows the methods used for D N B peptides (see Vol. IV [9]) with the advantage that the sulfonamide bond is very stable (1% loss in I N HCI for 4 hours at 110 °) and can be internally controlled (in part) by adding the S35-1abeled pipsyl derivative of the end group to the I131-1abeledpipsyl peptide before hydrolysis. The isolated pipsyl amino acid will have both isotopes, and a quantitative estimate is made on the basis described above. W e have applied the hydrazinolysis method of Akabori et al2 directly to pipsyl peptides to find the C terminal acid. The value of nearly quantitative estimations of the amounts of particular peptides in determining sequences in proteins like silk where a few amino acids make up the greater part of the protein is illustrated in papers by Levy and Slobodian.1°,H 9 S. Akabori, K. Ohno, and K. Narita, Bull. Chem. Soc. Japan 25~ 214 (1952). ~0 M. Levy and E. Slobodian, J. Biol. Chem. 199, 563 (1952). H E. Slobodian and M. Levy, J. Biol. Chem. 201, 371 (1953).

[II] M e t h o d s

for Investigating the Essential G r o u p s for E n z y m e Activity By

H. FRAENKEL-CoNRAT

Introduction

The customary approach in an investigation of the essential groups of enzymes and other biologically active proteins consists in studying the effect of various chemical modifications on the specific biological activity. Thus the first step is the preparation of chemically modified enzymes.

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METHODS FOR INVESTIGATING THE ESSENTIAL GROUPS

247

respectively, from the unfiltered sample, and C = f ~ C i - f8C8. Thus C , = (c -

f~S)/(f,

C8 =

-

(AS

C)/(f~

-

fs) -

f,)

The pipsyl alanines from the two reagents used are standardized by ultraviolet absorption, planchetted, and counted both with and without filters. Ci for the sample divided by the molar radioactivity of the standard gives the number of moles of I TM compound on the planchette. When this is multiplied by the ratio of total S 35 counts (added as indicator) to C,, the result is the number of moles of the unknown in the original derivative mixture and if derivative formation was complete is the desired analytical figure.

Sequences The determination of any peptide in a mixture derived from hydrolysis of a protein is pertinent data for sequence studies. The determination of the amino end group in pipsyl peptides follows the methods used for D N B peptides (see Vol. IV [9]) with the advantage that the sulfonamide bond is very stable (1% loss in I N HCI for 4 hours at 110 °) and can be internally controlled (in part) by adding the S35-1abeled pipsyl derivative of the end group to the I131-1abeledpipsyl peptide before hydrolysis. The isolated pipsyl amino acid will have both isotopes, and a quantitative estimate is made on the basis described above. W e have applied the hydrazinolysis method of Akabori et al2 directly to pipsyl peptides to find the C terminal acid. The value of nearly quantitative estimations of the amounts of particular peptides in determining sequences in proteins like silk where a few amino acids make up the greater part of the protein is illustrated in papers by Levy and Slobodian.1°,H 9 S. Akabori, K. Ohno, and K. Narita, Bull. Chem. Soc. Japan 25~ 214 (1952). ~0 M. Levy and E. Slobodian, J. Biol. Chem. 199, 563 (1952). H E. Slobodian and M. Levy, J. Biol. Chem. 201, 371 (1953).

[II] M e t h o d s

for Investigating the Essential G r o u p s for E n z y m e Activity By

H. FRAENKEL-CoNRAT

Introduction

The customary approach in an investigation of the essential groups of enzymes and other biologically active proteins consists in studying the effect of various chemical modifications on the specific biological activity. Thus the first step is the preparation of chemically modified enzymes.

248

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

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This is in most instances very easy and requires no special techniques or apparatus. It is usually desirable to attack only one type of protein group at a time, however, and reagents or reaction conditions of high selectivity are rather scarce. Much research needs yet to be done to supply us with a specific reagent for each type of protein group. The fact that each of the various kinds of polar residues may show a wide range of reactivity within a given protein, as well as from one protein to another, makes it appear unlikely that absolutely selective reagents will ever become available. At present, certainly, thorough analytical characterization is necessary for most protein derivatives to ascertain to what extent the desired reaction has proceeded and side reactions involving other types of groups have been excluded. This analytical work is usually harder and more time-consuming than the preparation of the derived protein; however, the analyses are of great importance if any conclusions are to be drawn from this type of study. Actually, the drawing of valid conclusions concerning the chemical nature of the enzymatic site is generally more problematical than either the preparative or the analytical work. Only in the few instances where one single group per molecule is found essential for enzyme activity is there some justification in assuming that this group is located at or near the active site; and even then its direct implication in the mechanism of enzyme action cannot be regarded as definitely proved. ~ Inactivation produced only after extensive modification of presumably one type of group should be interpreted with the greatest caution because of the imperfect selectivity of all methods and the danger of nonspecific inactivation which may be due to such factors as denaturation, change in net charge, or solubility of the enzyme. In contrast, retention of enzymatic activity after chemical modification of all reactive groups of a certain type appears to be of much greater interpretive value in suggesting the noninvolvement of the modified group. One must not overlook the fact that one or a few groups per protein molecule are within the error of most protein analytical methods; but the assumption appears justified (until disproved) that the polar groups associated with the enzymatic site are at least as available to chemical reagents as the average group of its type. Thus the fact that various enzymes retain their activity after maximal acetylation of their amino groups ~-4 strongly suggests that amino groups do not occur in the active site of these enzymes. 1T. P. Singer, J. Biol. Chem. 174, 11 (1948). 2 R. M. tterriott, J. Gen. Physiol. 19, 283 (1935-1936). 3I. W. Sizer, J. Biol. Chem. 160, 547 (1942). 4 H . Fraenkel-Conrat, R. C. Bean, and H. Lineweaver,J. Biol. Chem. 177, 385 (1949).

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METHODSFOR INVESTIGATING THE ESSENTIAL GROUPS

249

In the following brief description of some of the more useful methods for the preparation of chemically modified enzymes, analytical techniques available for the evaluation of each reaction will be indicated but no further discussion concerning the interpretation of the data will be included. For reviews of the literature and more detailed and critical evaluation of the methods of protein modification, the reader is referred to various publications. 5-8 Reactive Protein G r o u p s The chemical structure of apoenzymes, as of all proteins, is primarily determined by the order of amino acid residues along the peptide chains. I t appears probable that enzymatic specificity and, in the absence of coenzymes, enzymatic activity is largely a function of this arrangement of the R groups (formula I). However, the specific folding of chains, which R2

/

NH

\

/ CH

I

R1

CO

R4

I

\

/ NH

CH

\

/ CO

NH

\

/ CH

CO

I

\

/ NH

CH

\

/ CO

I

R3

can bring nonadjacent R groups into close spatial position, also probably plays an intrinsic role i n the shaping of the enzymatic site. The folded structure, in turn, is stabilized through disulfide bonds and through a neat pattern of hydrogen bonds between pairs of - - C O - - N H - - groups which also may play an important role in electron transport to or from the enzymatic site2 The side chains (R groups) of proteins fall into two main categories-i.e., the nonpolar hydrocarbon groups contributed by alanine, valine, leueine, isoleucine, phenylalanine, and proline, and the various groups carrying active hydrogen or reactive sulfur atoms. The nonpolar class may play a role in the active site of enzymes through their affinity for similarly hydrophobic groups on the substrate. The chemical inertness of the hydrocarbon chain renders this class unsuitable to chemical study, 5R. M. Herriott, Advances in Protein Chem. 3, 169 (1947). 6H. S. Oleott and H. Fraenkel-Conrat, Chem. Revs. 41, 151 (1947). 7H. Fraenkel-Conrat, in "Amino Acids and Proteins" (Greenberg, ed.), p. 532. Charles C Thomas, Springfield, Ill. 1951. a F. W. Putnam, in "The Proteins" (Neurath and Bailey, eds.), Vol. I, Part B, p. 893. Academic Press, New York, 1953. 9K. Wirtz, Z. Naturforsch. 2b, 94 (1947); W. Schmitt, ibid. 2b, 98 (1947); T. A. Geiasmann, Biol. Revs. 24, 309 (1949).

250

TECHNIQUESFOR CHARACTERIZATION OF PROTEINS

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however, and probably justifies the belief t h a t their role is only secondary, their action radius being smaller than t h a t of the polar groups. The polar groups as listed below are in part dissociated under physiological conditions (the first four), but chemical modification is usually performed with the undissociated form. This probably accounts for the lack of good methods of a t t a c k on the completely dissociated guanidyl group. The various groups differ greatly in their chemical reactivity, decreasing in the approximate order listed (except for the low reactivity of the guanidyl and the high reactivity of free - - S H groups). Amino groups (lysine, N terminal amino acid) Imidazole groups (histidine) Guanidyl groups (arginine) Carboxyl groups (aspartic, glutamic acid, C terminal amino acid) Sulfhydryl-disulfide groups (cysteine, cystine) Phenol groups (tyrosine) Hydroxyl groups (serine, threonine) Indole groups (tryptophan) Amide groups (glutamine, asparagine, C terminal amide) Thio ether groups (methionine) (Phosphate ester groups in phosphoproteins; serine, threonine) The assumption t h a t the nature of the active site of any enzyme is a simple additive function of the properties of some of these polar and the nonpolar R groups m a y be an oversimplification. We now know of two specific instances in which reactions occur with proteins which cannot be duplicated with any simple mixture of amino acids or peptides, and in which a potentiating or synergistic effect of two or more of the typical R groups is believed to be operative. These are the inhibition of esterases b y D F P , to be discussed later, and the action of nitrogen trichloride on certain prolamines and no other proteins, causing the f o r m a t i o n - o f methionine sulfoximine presumably through cooperation of amide groups and methionine. 10 There exists also the definite possibility t h a t reactive groups of completely different nature from t h a t of any recognized protein building block m a y occur in certain enzymatic sites and m a y have escaped detection. Aldehyde or ketone groups have often been postulated, 11 and 10p. N. Campbell, T. S. Work, and E. Mellanby, Biochem. d. 48, 106 (1951); H. R. Bentley, E. E. McDermott, I. Moran, J. Pace, and J. K. Whitehead, Proc. Roy. Soc. B137, 402 (1950). 1~In one case the postulated occurrence of an aldehyde group in an enzyme [E. Werle and K. Heitzer, Biochem. Z. 299, 420 (1938)] has actually been confirmed later, though it was found to.be located in the coenzyme rather than in the protein (pyridoxal in histidine decarboxylase).

[11]

METHODS FOR I N V E S T I G A T I N G T H E ESSENTIAL GROUPS

251

hydroxylamino groups and others have been suggested. ~2 I t appears advisable to keep an open mind to this possibility, but also to shoulder the burden of proof before building extensive theories on such assumptions. The inactivation of an enzyme b y a so-called ketone reagent, for instance, is not sufficient proof for the ketonic nature of the enzyme until interaction of the reagent with all known types of protein groups has been thoroughly excluded.

Acylation of Amino Groups Discussion. M a n y different acyl residues have been introduced into proteins, b u t none appear to be preferable to the acetyl residue when water-soluble biologically active products are desired. Ketene has been often used for the acetylation of proteins1 ~ but acetic anhydride in neutral aqueous solution is easier to handle and appears as effective and less harmful to native proteins. 4,6-8 When it appears desirable to attach larger acyl groups to the amino groups, then phenylisocyanate is probably the reagent of choice. 13 In contrast to acetic anhydride, this reagent seems to attack also the masked - - S H groups of native proteins. TM Reaction with Acetic A n h y d r i d e . To the enzyme solution, preferably b u t not necessarily of high concentration (2 to 10%), is added an equal volume of saturated solution of sodium acetate; the solution (or suspension) is cooled in an ice bath and treated with a total a m o u n t of acetic anhydride approximately equal to the weight of enzyme used, but distributed over three to six additions in the course of 1 hour at 0 ° (e.g., 10 mg. of enzyme in 0.1 ml. of H:O in a small test tube, 0.1 ml. of sodium acetate, five times 2 ul. of acetic anhydride). The product is isolated b y dialysis. A n a l y t i c a l Characterization. This reaction is relatively specific for the amino groups. An easy approximation of the extent of acetylation of these groups is obtained by the ninhydrin test, 15 slightly simplified for use with proteins (see below). For critical evaluation the manometric Van Slyke m e t h o d is preferable. 1~ In such cases total acetyl introduced should also preferably be determined. 1~.17M a s k e d - - S H groups do not generally react,

1~S. Fiala and D. Burk, Arch. Biochem. 20, 172 (1949); H. Fraenkel-Conrat, ibid. 28, 452 (1950); H. Fraenkel-Conrat, N. S. Snell, and E. D. Ducay, Arch. Biochem. and Biophys. $9~ 97 (1952). la G. L. Miller and W. M. Stanley, J. Biol. Chem. 1411 905 (1941). 1~H. Fraenkel-Conrat, J..Biol. Chem. 1521 385 (1944). 1~S. Moore and W. H. Stein, J. Biol. Chem. 176, 367 (1948). 1~D. D. Van Slyke, J. Biol. Chem. 83~ 425 (1929). 17R. M. Herriott, J. Gen. Physiol. 19~ 283 (1935-1936).

252

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[11]

but free - - S H groups become acetylated. In proteins containing - - S H groups, their involvement should be ascertained analytically (see pp. 257ff.). Phenolic groups are not generally acetylated, but this should be ascretained by means of the Folin test or by ultraviolet spectrophotometry (see below). No other protein groups seem to be attacked. Procedure for Ninhydrin Reaction for Amino Groups in Proteins 15 (see also Vol. III [76]). The reagent is prepared as follows: 0.8 g. of stannous chloride (SnC12.2H20) is dissolved in 500 ml. of citrate buffer, pH 5 (21.0 g, of citric acid, 1 H~O; 200 ml. of N NaOH and water to 500 ml.). To this is added a solution of 20 g. of ninhydrin in 500 ml. of methyl Cellosolve. The solution is saturated with N2 gas and kept in a glass-stoppered bottle in the refrigerator. To test tubes containing 0.2 to 1 mg. of protein in 0.1 ml. of water or salt solution is added 1 ml. of reagent. The tubes are heated in a vigorously boiling water bath for 20 minutes, allowed to cool, rapidly mixed with 5 ml. of 50% aqueous isopropanol, and read within 15 minutes after removal from the bath in a Klett colorimeter with the green filter No. 54 against a blank treated with reagent in the same manner. Alanine (0.05 to 0.2 ~M.) is the standard. Proteins generally give about 80 % of the color expected from their amino-N content under these conditions. Various acetylated proteins show chromogenic values ranging from 10 to 60% of the original. Procedure for Folin-Herriott Test for Phenolic Groups in Proteins 1~,18 (see also Vol. I I I [76, 87]). To 1 to 5 mg. of protein dissolved in 8.5 ml. of water, dilute salt, acid or alkali, are added simultaneously 2.5 ml. of buffer (60 ml. of 0.5 M Na~HPO4 ~ 40 ml. of N NaOH) and 1.5 ml. of the threefold-diluted Folin phenol reagent. The solution is held for 1 hour at 40 °, cooled, and read in a colorimeter with a blue filter against a blank sample. Tyrosine or tryptophan may be used as standard. To regenerate labile phenol esters (Herriott's pH 11 procedure) water is added to only 5.5 ml., followed by 1.5 ml. of 0.1 N NaOH. After 15 minutes at room temperature, 1.5 ml. of 0.1 N HC1 is added, followed by buffer and reagent as usual. The severe limitations of the Folin tes~ have been amply discussed. ~7 As a comparative method, however, particularly to show the intactness or the alkali-labile acylation of phenolic groups, it continues to serve a useful purpose. Spectrophotometric Determination of Phenolic Groups. In alkaline solution, tyrosine, as well as the tyrosine residues in proteins, shows a characteristic maximum at a higher wavelength than that due to tryptophan (295 versus 280 mg). A method of determination of the two amino acids has been developed on this basis.19 The protein is dissolved in 0.1 N NaOH is R. M. Herriott and J. H. Northrop, J. Gen. Physiol. 18, 35 (1934-1935). 19 G. H. Beaven and E. R. Holiday, Advances in Protein Chem. 7, 320 (1952).

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METHODS FOR INVESTIGATING THE ESSENTIAL GROUPS

253

and its absorption read at the point of intersection of the two curves (294.4 m~), as well as at 280 m~. From the formulas M(Ty,) = (0.592,2944 -- 0.263,~8oo) X 10-3 M(T~yp) ---- (0.263,~8oo -- 0.170,2944) X 10-3 where K2944 and K2800 represent the extinction coefficient of the protein at the two wavelengths, the gram moles of tyrosine and tryptophan per gram of protein, M(tyo and M(tryp), can be calculated. For more accurate results nonspecific absorption is determined at 320 to 360 m~, extrapolated to 280 m~, and subtracted from the observed optical density. With proteins rich in disulfide bonds, readings must be taken immediately after addition of the alkali, since progressive spectral changes have been observed. When dealing with, or searching for, alkali-labile tyrosine derivatives, such as O-acetyl tyrosine, this method cannot be applied. With proteins of a high tyrosine-tryptophan ratio, however, a good indication of the extent of blocking of the phenolic groups should be derived from a comparison of the absorption at 275 m~ of neutral solutions of the original, the modified protein, and the latter after exposing it to alkali (e.g., pH 11) and bringing it back to neutrality. An alternate technique is to determine spectrophotometrically the pH needed for dissociation of the phenolic groups of the protein under study. This varies greatly for different proteins, s° At this pH, then, the technique of Beaven and Holiday can be applied. If the absorption (at 295 m~) increases with time, indicative of hydrolysis of phenol esters, then extrapolation to zero time will be necessary to evaluate the extent of acylation of the phenolic groups. (See also under Iodination.) Reaction with Phenylisocyanate.1. ~ To the protein buffered at pH 7.5 to 8.0 and held at 0 ° is added with efficient stirring a similar amount (1 ml./g, of protein) of phenylisocyanate in small aliquots distributed over 2 to 3 hours. When the smell of the reagent has disappeared, the diphenylurea is centrifuged off and the protein solution dialyzed and analyzed by the same methods as the acetylated product. Esterification of Carboxyl Groups Discussion. Proteins are readily esterified if they are suspended in alcohols containing about 0.02 N to 0.1 N HC1. ~l This reaction is selective for carboxyl groups. Denaturation can be avoided or minimized with many proteins, including serum albumin, if the reaction is performed at 20 j. L. Crammer and A. Neuberger, Biochem. J. 37, 302 (1943). 21 H. Fraenkel-Conrat and H. S. Olcott, J. Biol. Chem. 161, 259 (1945).

254

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[11]

lOW temperature, 6 but the danger of denaturation can never be completely excluded. T r e a t m e n t of proteins in aqueous solution with epoxides (e.g., propylene oxide) supplies a gentler means of esterifying carboxyl groups, but this reaction is not specific; amino, phenolic, and - - S H groups are also alkylated to a varying extent, depending on the p H of the medium. In acid solution, carboxyl groups react somewhat more readily than other groups, but the esterification never reaches completion. 22 M u s t a r d gas and related substances, including the nitrogen mustards, react in a m a n n e r similar to the epoxides, affecting primarily the - - S H and carboxyl groups below neutrality. 23,~4 Both diazoacetamide and diazoethylacetate seem to be specific reagents for the esterification of carboxyl groups. T h e y react under mild conditions, but a p p a r e n t l y with only a fraction of the carboxyl groups of serum albumin. 25 The esterification reactions are often reversible. M e t h y l esters are rather labile in dilute acid and alkali, and part of the original carboxyl groups can be regenerated by exposing esterified proteins to p H 2 or p H 10; 28 esters of higher alcohols form less readily and are more stable. T h e glycolic esters produced with the diazoacetyl reagents can also be split in dilute alkali. 25 Reaction with Methanol-HCl. ~I The d r y protein, preferably in the lyophilized state, is suspended in 100 parts of methanol cooled to - 5 to - 1 0 ° ; to this is added concentrated HC1 to a final concentration of 0.02 or 0.1 N, and the suspension is held at - 10 °, 0 o, or room t e m p e r a t u r e for one to several days, depending on the stability of the enzyme and the extent of esterification desired. M a n y proteins dissolve in the alcohol in the course of esterification. The reaction mixture is finally diluted with much ice water, dialyzed, and concentrated by pervaporation. A n a l y t i c a l Characterization. In view of the lability of m e t h y l esters, the only strictly q u a n t i t a t i v e m e t h o d of ascertaining the extent of methylation is methoxyl determination by the Zeisel method. ~7 Indications of the extent of esterification can be obtained through titration 22H. Fraenkel-Conrat, J. Biol. Chem. 154, 227 (1944). 2~R. M. Herriott, M. L. Anson, and J. H. Northrop, J. Gen. Physiol. 30, 185 (1946). ~4V. Desreux, E. Fredericq, and P. Fischer, Bull. soc. chim. biol. 28, 7 (1946). 2~We are indebted to Dr. P. E. Wilcox for letting us see a manuscript concerning this technique prior to publication; see also abstracts of papers, 12th Intern. Congr. Pure Appl. Chem., New York, pp. 60, 61 (1951). 36W. F. H. M. Mommaerts and H. Neurath, J. Biol. Chem. 185, 909 (1950). ~7E. P. Clark, J. Assoc. O~c. Agric. Chemists 15, 136 (1932).

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255

of the protein and its derivative from pH 2.5 to 6.0, ~3and through electrophoretic examination. Reaction with Epoxides. 22 To the enzyme solution (about 2 %) adjusted to pH 3.5 to 4.0 with acetic acid is added 0.1 vol. of propylene oxide. The reaction mixture is held at room temperature for 1 to 6 days, then dialyzed and assayed. Analytical Characterization, Determination of Total Acid Groups. 28 The extent of involvement of amino, phenolic, and sulfhydryl groups can be ascertained by methods described elsewhere in this chapter. The esterification of the carboxyl groups can be determined by analysis for total acid groups of the enzyme and the esterified product based on the capacity of the protein to bind a basic dye in alkaline solution. This method is not applicable to the more alkali-labile methyl esters. To a series of test tubes containing 2- to 3-rag. samples of protein (0.2 ml. of a 1 to 2% solution) are added 1.0 ml. of a buffer of pH 11.5 (250 ml. of 0.2 M Na2.HPO4, 200 ml. of 0.1 N NaOH, and water to 1000 ml.) and 1.0, 2.0, and 3.0 ml. of a 0.2% solution of Safranine 0. 29 The tubes are shaken for 20 hours at room temperature to ensure equilibration. The protein-dye complex is then centrifuged off, and the concentration of dye remaining in the supernatant is determined photometrically. To this end 0.5- or 1.0-ml. aliquots are diluted to 100 or 250 ml., and the absorption at 520 m~ is read in a spectrophotometer2 ° The concentration of dye in the stock solution is similarly determined; insoluble impurites and moisture can be corrected for if the dye concentration is calculated from the optical density, as compared to that of a purified and dry sample (0.111 for a solution of I ~,/ml. and a l-cm. light path). The difference between the amount of dye added and that found in the solution is the dye bound by the protein. This amount per milligram of protein should be independent of the amount of excess dye, as indicated by agreement of the results obtained with increasing amounts of dye. For greater accuracy, repeat analyses are performed, avoiding too great an excess (only two levels of dye from 75 to 300 % in excess of the amount bound). Multiplication by 28.2 will convert the amount of dye bound per milligram to total acid groups per 104 g. of protein. 2s H. F r a e n k e l - C o n r a t a n d M. Cooper, J. Biol. Chem. 154, 239 (1944). 29 If the Safranine is impure a n d a b r o w n precipitate settles out, this m a y be separated. Purification can be achieved b y dissolving the dye in 95 % ethanol containing a few drops of 6 N HC1, a n d precipitating with m u c h ether. 30 A colorimeter, e.g. Klett, can also be used, with a combination of Corning filters No. 3060, 3389, a n d 5543. Safranine has a great t e n d e n c y to adsorb on glass, a n d Corex cells were found unsuitable with the B e c k m a n n Model D U spectrophotometer. Plastic cells were found more convenient for this purpose.

256

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

[llJ

Reactions o f - - S H Groups Discussion. Several classes of reagents are available for the - - S H group, and several of the reagents show a high degree of specificity for this group. On the other hand, part or all of the - - S H groups of most native enzymes are to a varying extent masked and unreactive toward one, most, or all of the reagents. Thus, lack of effect of one or even several - - S H reagents on the enzymatic activity proves neither the absence nor the nonessentiality of - - S H groups. On the other hand, inactivation by one of these reagents should not be regarded as absolute proof of the presence and essentiality of such groups. Analytical methods are available for the determination of the reactive and of the total - - S H groups of a protein, and analytical proof for the blocking of - - S H groups by the reagents under study ought to be sought whenever possible. Free, fully reactive - - S H groups occur only in a few proteins. Enzyme activities are rarely if ever directly dependent on these groups. The essential - - S H groups--i.e., those located at or near the enzymatic site and believed to be involved in reactions with the substrate--are usually more or less masked; they rarely give the nitroprusside test and generally react with only a few of the - - S H reagents. Often the most effective class of reagents for these groups are the mercaptide-forming mercurials and arsenicals, such as p-chloromercuribenzoate. These reagents fulfill most of the requirements of an ideal protein reagent: They react stoichiometrically under gentle conditions, they show high specificity, and they can easily be split off and the original - - S H group regenerated. Another reagent which often attacks masked - - S H groups is iodine. Iodine reacts also with tyrosine and histidine residue (see p. 260), however, and thus its effect on enzymatic activity can only be interpreted as due to oxidation of - - S H groups, if the iodine used up is quantitatively accounted for by a corresponding decrease of - - S H groups. This has actually been found to be the case with only one protein, egg albumin. 81 With two other - - S H proteins, serum albumin and tobacco mosaic virus, al,3~ about twice the theoretical amount of iodine was used up in oxidative reactions for the abolishment of the - - S H group. In the case of the virus, the transformation of the - - S H to a stable sulfenyl-iodide group has recently been demonstrated. 3~a Certain alkylating agents, notably iodoacetic acid or its amide, usually show lesser reactivity toward masked - - S H groups but are com31 M. L. Anson, J. Gen. Physiol. 24, 399 (1940); M. L. Anson and W. M. Stanley, ibid. 24, 679 (1941). 32 W. L. Hughes, Jr., and R. Straessle, J. Am. Chem. Soc. 72, 452 (1950). 3~ H. Fraenkel-Conrat, J. Biol. Chem. 217, 373 (1955).

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paratively specific and react rapidly with free - - S H groups at room temperature and neutrality. Another reagent, only recently introduced, ~3 is N-ethylmaleimide (NEM), which adds to the - - S H group to give a stable thio ether. It appears to be a specific - - S H reagent when used near neutrality but shows little affinity for masked - - S H groups. In view of the great variations in the reactivity of SH groups toward different reagents, no general procedure can be advocated, and many pilot experiments are needed to decide which reactions are most applicable to a given problem. In all cases, it should be attempted to confirm all conclusions by the use of more than one type of reagent. Also, isolation of the modified enzyme is strongly advocated. Much confusion has been caused by the tendency of enzymologists to add reagents directly to solutions to be assayed; they then study the effect of reagents on the enzymatic reaction, rather than on the enzyme. Thus inhibitions and activations have been observed and interpreted in terms of the enzyme which had actually no relation to the chemical nature of the enzymatic site but represented various types of interaction or competition of the reagent with the substrate or coenzyme. Removal of excess reagent by dialysis is a simple procedure and should be carried out whenever possible; this will greatly facilitate the interpretation of enzymatic assay data. A suggested sequence of tests concerning the sulfhydryl nature of an unknown enzyme available in amounts of 5 to 50 rag. is as follows: (1) Nitroprusside test, native, and (2) nitroprusside test after denaturation. With limited material one should be prepared for (3) an immediate titration of the same sample with N E M if the nitroprusside test is positive. Another sample of enzyme should be reacted with p-chloromercuribenzoate by the ultraviolet spectrophotometric method as an indication of interaction24 Other samples can be treated with small amounts of iodine, iodosobenzoate, or iodoacetamide, and the change in N E M titration subsequently determined. Amperometric titration methods can also be employed to advantage. All samples that have been treated with the various reagents in the native state can be isolated by dialysis or some other convenient procedure and subjected to enzymatic assay, and to various analyses. Titration o f - - S H Groups with N-Ethylmaleimide. 3~ The reagents are (a) guanidine hydroehloride, saturated solution in 0.1 M acetate, pH 4.8; 33 T. Tsao and K. Bailey, Biochim. el Biophys. Acta 11, 102 (1953); E. Friedmann, D. H. Marrian, and I. Simon-Reuss, Brit. J. Pharmacol. 3, 335 (1948). 34 p. D. Boyer and H. L. Segal, in " T h e Mechanism of Enzyme Action" (McElroy and Glass, eds.), p. 520. Johns Hopkins Press, Baltimore, 1953; P. D. Boyer, J. Am. Chem. Soc. 76, 4331 (1954).

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METHODS FOR INVESTIGATING TttE ESSENTIAL GROUPS

259

ins, and readings must be taken at intervals until constant before more of the reagent is added. The n u m b e r of reactive SH groups can be estimated from the increment over the calculated absorption at 255 m# with 6200 as the molar extinction increment for the mercaptide formed at t h a t wavelength27 The same reaction can be performed at pH 7 (phosphate buffer), although the - - S H groups of native proteins seem to react more sluggishly at t h a t pH. (Under these conditions the molar extinction increment at 250 mt~ is about 7600.) These solutions m a y be used for enzymatic assay, preferably after thorough dialysis. M e r c u r y analyses and spectrophotometric analysis after dialysis will supply further proof for the extent of interaction. Dialysis against cysteine-HC1 solution reverses the reaction and removes most of the mercuribenzoate. The same spectrophotometric technique performed in 1% sodium dodecylsulfate will permit determination of the total (free + masked) - - S H groups of proteins. Reaction with Iodoacetamide, etc. Iodoacetamide is easily prepared by refluxing equimolar amounts of chloroacetamide with a slight excess of sodium iodide in alcoholic solution for 6 hours, filtering off the inorganic salt, evaporating part of the alcohol, and adding some water. The crude product separates on cooling and must be recrystallized from water and alcohol, without heating above about 60 °, to obtain a pure white product (melting point, 95°). 28 If iodoacetamide is found to inactivate an enzyme at neutrality and room temperature, then - - S H groups m a y be involved. The total extent of interaction can be ascertained from the acid liberated according to the equation R - - S H + ICH2CONH2--~ R - - S - - C H ~ - - C O N H 2 + HI. For this purpose an a u t o t i t r a t o r 39 can conveniently be employed, but the alkali needed to maintain a constant p H (pH 8) can also be determined malmally. 4° Analyses for amide-N in the isolated product can also serve as a basis for determining the extent of reaction with proteins containing m a n y reactive - - S H g r o u p s ) ° The involvement of the - - S H groups is demonstrated by means of N E M titrations in guanidine (methods 2 and 3). Iodoacetic acid can be used in the same manner if a white (iodinefree) preparation is available. 37According to Boyer34the molar absorption increment varies somewhat for different mercaptides and proteins, so that it must be determined by the use of excess thiol, when exact absolute values are needed. ~s j. yon Braun, Bet. 41, 2144 (1908). ~ C. F. Jacobsen and J. Leonis, Compt. rend. tray. lab. Carlsberg, S~r. chim. 27, 333 (1951). 40H. Fraenkel-Conrat, A. Mohammad, E. D. Ducay, and D. K. Mecham, J. Am. Chem. Soc. 73, 625 (1951).

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TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

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Iodine, when applied as an - - S H reagent, is added in small amounts (no more than three times the amount needed for the oxidation of all - - S H groups) and preferably in acid solution (pH 3.2 to 6.8). The concentration of potassium iodide may play an important but variable role. 3~ The extent of oxidation of - - S H groups can be ascertained by N E M titrations in guanidine hydrochloride (methods 2 and 3). o-Iodosobenzene, when used near pH 7, 36,41 is probably the most specific of the oxidizing agents which, in general, have lost their formerly predominating role as - - S H reagents. One of the objections to these reagents, the need to find a pair of reactive - - S H groups for oxidation to the disulfide form, holds also for o-iodosobenzoate. 4~ Thus complete reaction can never be assumed. For details of this procedure, as well as of many other useful procedures for - - S H groups, the reader is referred to a recent review2 8 Iodination

Discussion. Most proteins rapidly decolorize iodine in neutral solution. When SH groups are absent, or when the consumption of iodine exceeds their content and iodine is actually incorporated into the protein, then iodination of phenolic groups is usually assumed. In most proteins this reaction is comparatively specific, but in some proteins the histidine residues react more readily than the phenolic groups. Thus, in the case of lysozyme, iodination caused loss of activity which was reversible in the early stages of the reaction and which was found to involve the single histidine residue of this protein. 48 Since oxidative side reactions can never be completely excluded and good analytical techniques for allocation of the iodine atoms are lacking, the effect of iodination on an enzyme can not readily be interpreted in terms of essential groups. Reaction. Varying amounts of an iodine solution (0.01 M Is in 0.5 M KI) are added to the enzyme solutions buffered with acetate (pH 5 to 6), ~4 phosphate (pH 7 to 8), 48 or carbonate-bicarbonate (pH 9 to 9.5) ~2 and held at 0 ° or room temperature. The rate of disappearance of the yellow color is an indication of the reactivity of the groups of a given protein; this varies greatly for different proteins. The most alkaline pH at which an enzyme is stable should be used to minimize oxidative reactions. ~ The 4~L. Hellermann, F. P. Chinard, and P. A. Ramsdell, J. Am. Chem. Soc. 63, 2551 (1941). 43 The possibility of oxidation of single masked - - S H groups directly to - - S - - O H groups appears not to have been considered, although some of the analytical data favor this concept (see ref. 32a). 4a H. Fraenkel-Conrat, Arch. Biochem. 27, 109 (1950). 44 R. M. Herriott, J. Gen. Physiol. 20, 335 (1936-37); 31, 19 (1947).

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extent of modification can be controlled b y the a m o u n t of iodine added (usually from 4 to 40 equivalents per mole of protein). Any residual iodine can be titrated back with thiosulfate after a suitable time interval. T h e enzyme is then dialyzed (or reisolated) and assayed. Analytical Characterization. Oxidation of - - S H groups, if any are present, must first be ascertained (see p. 258), or excluded b y blocking them with mercuribenzoate or iodoacetamide. Apart from the reaction with - - S H groups, which only in exceptional cases is one of substitution, TM most of the iodine used up rapidly in dilute solution generally acts in substitution reactions, further oxidation (of disulfide bonds, indole residues, etc.) occurring only under more extreme conditions, particularly with a great excess of iodine22 T h e total extent of substitution is established b y iodine analysis of the thoroughly dialyzed product; the extent of oxidation can be estimated by comparing the a m o u n t of iodine used up (added minus b a c k t i t r a t e d iodine) to t h a t found in the product; this ratio equals 2 for substitution, ~o for oxidation. The use of I TM is v e r y advantageous for analytical purposes, particularly if a well-type scintillation counter is available. Naturally, the dilution of the I TM used by the K I 1~7 serving as solvent must be taken into account. The main difficulty lies in ascertaining the relative extent of substitution on imidazole and mono- and disubstitution on phenol groups. Methods have been described for the determination of monoiodo- and diiodotyrosine, 45 b u t the lability of these and even more so of the iodinated histidines under conditions of hydrolysis prevents a definite quantitative allocation of the iodine atoms in the protein. The shift of the ultraviolet absorption maximum of both the dissociated and undissociated form of tyrosine on mono- and diiodination to higher wavelengths and absorptions 44 is observed also in proteins and can be used as an indication of the extent of involvement of these residues. ~2

Coupling with Diazo Compounds Discussion. The reaction of proteins with diazobenzene sulfonic acid or other diazobenzene derivatives occurs with great ease near neutrality. 4,4° The reaction, like iodination, involves phenolic and imidazole groups, b u t in this case the imidiazole groups of most proteins react at least as fast as do the phenolic groups. Thus this reaction, like iodination, is easy to perform, b u t generally difficult to interpret. Reaction with Diazobenzene Sulfonic Acid. Diazobenzene sulfonic acid 4~The Millon reaction serves to differentiate tyrosine from diiodotyrosin, which gives no color, and from monoiodotyrosine, which gives a color with a slightly different maximum [J. Roche, R. Michel, and M. Lafon, Compt. re~d. 224, 233 (1947)].

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METHODS FOR INVESTIGATING THE ESSENTIAL GROUPS

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this reagent can be bound through methylene groups to the amino groups of the protein and cross-linking of the methylol with other protein groups can be minimized. Similarly, addition of amines or amino acids to the reaction mixture directs most of the reaction of formaldehyde toward the amide and guanidyl groups of the protein. 4s Reaction and Analyses. The reactions are performed at room temperature and neutrality, usually with 1% formaldehyde and about 10 % concentration of the small-molecular additives for 24 to 72 hours. For analytical evaluation the following methods are employed: the ninhydrin or Van Slyke reactions for amino groups; NEM titrations for - - S H groups; amide N by autoclaving with 1.2 N sulfuric acid for 2 hours, distilling in a micro-Kjeldahl apparatus with an antifoam agent after addition of buffer (pH 7.8 to 9), and titrating the ammonia as in the Kjeldahl procedure. Formaldehyde is released and steam-distilled by distillation from 1 N H2SOt until the acid begins to fume. The distillate is collected in water, and aliquots analyzed by means of chromotropic acid. 4~ Reduction of Disulfide Bonds Some enzymes containing - - S H groups essential for their activity are autoxidizable when removed from their natural, presumably reducing, medium. They are characterized by being activated by reducing agents, and they are preferably maintained in a reducing medium throughout their isolation. 5° Suitable reducing media are 0.01 to 0.1 M cysteine, mercaptoethanol, thioglycolate, or cyanide at pH 5 to 7. The same reagents may be used to reduce the - - S - - S - - bonds originally occurring in most native proteins. These are usually, at least in part, masked and of a low order of reactivity so that quite high concentrations of the mercaptans are needed for extensive reduction (e.g., 0.1 to 1 M at pH 7.5), and complete reduction is possible in most proteins only in solution in denaturing and dispersion agents (e.g., saturated urea4°), if at all. Since the disulfide bonds play an important structural role, it is not surprising that their reduction usually causes inactivation. Because of the high reactivity of the resulting free - - S H groups, reduced proteins tend to undergo rearrangements and form aggregates leading to precipitation, gelation, etc., even if reoxidation is prevented. 5°~ It is, therefore, advisable to " f i x " the reduced product by adding a great 49D. A. MacFadyen, J. Biol. Chem. 168, 107 (1945). 5oj. R. Kimmel and E. L. Smith, J. Biol. Chem. 207, 515 (1954); A. A. Green and G. T. Cori, ibid. 151, 21 (1943). ~0~C. Huggins, D. F. Tapley, and E. V. Jensen, Nature 167, 592 (1951).

264

TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

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excess of an SH reagent, such as iodoacetamide, to the reaction mixture, maintaining the pH near 8 until the reaction is complete and a stable derivative has been obtained. 4° No protein groups except the disulfide are known to be affected by mercaptans. Oxidation

Many reagents have been used for the oxidative modification of proteins, but most of them show a low degree of specificity. They generally attack sulfhydryl and disulfide groups, thio ether, indole, phenol, imidazole, and other groups. Hydrogen peroxide, under certain conditions, shows a specific affinity for the sulfur amino acid residues. A manometric technique of photooxidation with methylene blue as catalyst has been described ~1 which causes destruction of most of the histidine prior to any appreciable attack on the indole, and later on the phenol groups. This technique has supplied corroborative evidence concerning the essential histidine group of lysozyme and the mechanism of action of D F P on chymotrypsin. ~1 For the details of this technique the reader is referred to the original literature. A new technique for the selective oxidation of protein is by means of peroxidase and hydrogen peroxide. 5~ Tryptophan appears most susceptible under these conditions. Interesting results were again obtained with chymotrypsin. Sulfation

No gentle and specific reagents are known to attack aliphatic hydroxyl groups (serine, threonine). These groups can quantitatively and rapidly be transformed to sulfate esters by means of concentrated sulfuric acid at - 1 0 °. No peptide bonds are ~plit, and sulfonation of some of the phenolic groups is the only side reaction. 5~ Insulin proved active after sulfation, 53 and the derivative has been used in studies of its mode of fixation in the tissues. ~4 Yet this technique can definitely be denaturing and is not advocated for most enzymes. ~5 ~ L. Weil and A. R. Buchert, Arch. Biochem. and Biophys. 34, 1 (1951); L. Weil, A. R. Buchert, and J. Maker, ibid. 40, 245 (1952); L. Weil, S. James, and A. R. Buchert, ibid. 46, 266 (1953). 51~ H. N. Wood and A. K. Balls, J. Biol. Chem. 213, 297 (1955). 5~ H. C. Reitz, R. E. Ferrel, H. Fraenkel-Conrat, and H. S. Olcott, J. Am. Chem. Soc. 68, 1024 (1946). 53 M. B. Glendening, D. M. Greenberg, and H. Fraenkel-Conrat, J. Biol. Chem. 167, 125 (1947). ~ W. C. Stadie, N. Haugaard, and M. Vaughan, J. Biol. Chem. 199, 729 (1952). 65 Methods to produce the acyl shift have been studied in recent years [D. F. Elliott, Biochem. J. 50, 542 (1952)]. Extensive transformation of the peptide linkages

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METHODS FOR I N V E S T I G A T I N G T H E E S S E N T I A L GROUPS

265

Deamination Nitrous acid has long been in use as a protein reagent. For the analytical purpose of determining the amino-N it is still unsurpassed. 18 B u t no conditions have been found under which its action can be confined to the amino groups, and under gentle conditions (pH 4, 0 °) designed to minimize side reactions (diazotization of phenolic groups, oxidation of - - S H groups, etc.) the deamination is far from complete. ~8

Guanidination A technique has been described for the transformation of amino to guanidine groups 57 which m a y prove useful in characterizing enzymatic mechanisms. T h e protein is allowed to interact with 0.5 M O-methylisourea at p H 8.5 to 10.5 and 0 ° for several days. Depending on the pH, a variable fraction of the amino groups will be guanidinated, as indicated b y a drop in amino-N.

Enzymatic Attack on Enzymes D i s c u s s i o n . N a t i v e proteins are attacked b y endopeptidases at greatly varying rates and to different extents. T h e y are resistant to most other enzymes, with a few notable exceptions. Among these are carboxypeptidase which splits amino acids from the C terminal end, certain phosphatases which split phosphate from serine residues, ~s and tyrosinase which m a y attack the tyrosine residues in proteins. 59 A s t u d y of the effect of some of these enzymes, including limited proteolytic digestion on enzymatic activity, appears well worth attempting. Although the enzymatic attack is not v e r y likely to supply a direct indication of the nature of the active site, it m a y help to focus our attention on it b y trimming away unessential parts of the molecule. In view of the great variations in susceptibility to the proteolytic enzymes, no general techniques can be proposed. E n z y m a t i c activity (ATPase) has been retained after brief digestion with trypsin or chymo-

involving hydroxyamino acids to ester linkages with the --OH group is being achieved. This reaction is catalyzed by acids and occurs under prolonged attack of concentrated sulfuric acid. The peptide bond is re-formed above pH 7. It appears possible that such acyl-shifted linkages occur naturally in some proteins. 56j. S. L. Philpot and P. A. Small, Biochem. J. 32, 542 (1938). 5vW. L. Hughes, Jr., H. A. Saroff, and A. L. Carney, J. Am. Chem. Soc. 71, 2476 (1949). ~8D. L. Harris, J. Biol. Chem. 166, 541 (1946); G. E. Perlmann, Nature 166, 870 (1950) 59I. W. Sizer, J. Biol. Chem. 169, 303 (1947).

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TECHNIQUES FOR CHARACTERIZATION OF PROTEINS

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trypsin near the pH optima of these enzymes.S° Pepsin may be used with advantage at a higher than optimal pH (e.g., pH 4). 6~ The extent of digestion after various time intervals can be ascertained by means of the ninhydrin reaction (see p. 252). The termination of the reaction represents a definite problem. Trypsin can apparently be specifically inhibited even in the presence of substrate by addition of the soybean trypsin inhibitor. 60 In other cases the substrate enzyme may be removable by fractionation methods (e.g., isoelectric precipitation, ultracentrifugation), or further digestion may be prevented by change in pH and/or temperature. Carboxypeptidase has been used extensively for the prime purpose of elucidating the C terminal and adjacent amino acids. In general, the biologically active proteins investigated have retained their activity after loss of their C terminal amino acid. 62,83 Digestion with Carboxypeptidase (see Vol. II [8]). Preliminary experiments may be performed with commercial crystalline carboxypeptidase, although it is recognized that traces of endopeptidases are often present in such preparations. The carboxypeptidase crystal suspension (1 mg. of enzyme) is dissolved with a minimum of alkali (about 0.015 ml. of 0.1 N NaOH), buffered with 0.04 ml. of 1% bicarbonate, and diluted to 0.35 ml. To the substrate, adjusted to pH 7 to 8, is added one-fiftieth of its weight of carboxypeptidase. Amino-N (ninhydrin) is determined prior to addition of the peptidase and again after 2 hours at room temperature; if no appreciable increase (of the order of 0.2 to 1 equivalents per mole of substrate) has occurred, the digest may be incubated at 40 ° and again analyzed at intervals. Aliquots are also taken at various stages for assay of the enzymatic activity of the substrate. Depending on the rate of digestion, more or less (1/~0 to 1/~o0) carboxypeptidase may be preferable. The nature and the approximate amount of amino acids liberated can often be very simply ascertained by chromatographying aliquots (corresponding to about 0.05 ~M. of amino-N increase) of the digest directly, usually first in butanol-acetic-water. If the particular protein causes streaking, then other techniques must be employed 63 the detailed description of which are outside of the scope of this chapter. Before reliance can be placed on any results obtained with carboxys0 J. Gergeley, J. Biol. Chem. 200, 543 (1953); 19th Intern. Physiol. Congr., Montreal, p. 389 (1953); E. Mih~flyi and A. G. Szent-Gy6rgyi, J. Biol. Chem. 201, 189, 197, 211 (1953). el M. L. Petermann and A. M. Pappenheimer, Jr., J. Phys. Chem. 45, 1 (1941). 62 j . I. Harris and C. H. Li, J. Am. Chem. Soc. 74, 2945 (1952); J. I. Harris and C. A. Knight, Nature 170, 613 (1952). 63 H. Fraenkcl-Conrat, J. I. Harris, and A. L. Levy, in " M e t h o d s of Biochemical Analysis" (Glick, ed.), Vol. II, p. 360. Interscience, New York (1955).

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peptidase, contaminating pancreatic endopeptidases (mainly chymotrypsin) must be inactivated, which is usually done by adding the very toxic inhibitor diisopropylfluorophosphate (DFP)64 (see next section) to the enzyme solution (molar ratio about 50:1, or 0.02 ml. of 2%, i.e., 0.1 M DFP per milligram of enzyme) at pH 8, and holding the mixture for at least i hour at room temperature prior to addition to the substrate. Action of Diisopropylfluorophosphate (DFP) on Esterases ~5

The protein reaction which pinpoints most clearly the enzymatically active site is the stoichiometric interaction of many esterases, including trypsin and chymotrypsin (but excluding papain, carboxypeptidase, and phosphate ester-splitting enzymes) with DFP and related polyalkyl phosphates. This reaction has not been completely elucidated, but it appears certain that a particular spatial arrangement of several amino acid residues, probably involving a histidine and a serine residue, is needed for the substitution of one H atom by the dialkyl phosphate group. ~6 It also appears very probable that this same site is functionally involved in the fixation of the substrate ester which, in contrast to the dialkyl phosphate ester, can be split and released. The high affinity and specificity of DFP for the reactive group which favors stoichiometric reaction with esterases in quite dilute solution, and yet prevents any interaction with all inert proteins including the zymogens (chymotrypsinogen) and the denatured enzymes, makes this the ideal reagent for the study of this particular enzymatic site. All enzymes should be investigated for their susceptibility to DFP, and an active search for new specific inhibitors of other enzymatic sites should be vigorously pursued. The technique of the reaction of esterases with DFP has been described in the preceding section. If inactivation is observed, then phosphorus and other analyses are required as evidence for the mode and extent of reaction. For these the reader is referred to the original literature. ~5 Conclusions

The specificities of each of the reactions which have been discussed are summarized briefly in the table. The danger of causing denaturation with the various reagents is also estimated in the table, although gen64All direct contact with DFP and its fumes should be avoided. 85E. F. Jansen, M. D. F. Nutting, R. Jang, and A. K. Balls, J. Biol. Chem. 179~ 189 (1949); E. F. Jansen and A. K. Balls, ibid. 194~ 721 (1952). ~6 T. Wagner-Jauregg and B. E. Hackley, Jr., J. Am. Chem. Soc. 75~ 2125 (I953); R. E. Plapinger and T. Wagner-Jauregg, ibid. 76, 5757 (1953); N. K. Schaffer, S. C. May, Jr., and W. H. Summerson, J. Biol. Chem. 202, 67 (1953); 206, 201 (1954); W. N. Aldridge and A. N. Davison, Biochem. J. 55, 763 (1953).

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eralizations in this regard are very hazardous, owing to the wide range in the stability of enzymes. A considerable number of reactions can be performed in aqueous solution near neutrality and at room temperature, and these are regarded as nondenaturing conditions for most enzymes. Yet, some of the more specific are not among the safest reactions. The investigator will have to choose those methods which are within the stability range of his enzyme and run suitable controls to ascertain the effect of the reaction conditions per se. Considerable information has been accumulated for many enzymes, concerning the groups which are termed essential and unessential for their activity. A review of these studies appears outside of the scope of this book, and the reader is referred to other sources. ~-7,67 At a recent symposium on the Mechanism of Enzyme Action the newer developments i~1 our understanding of the nature and the mode of action of certain enzymatic sites were described. I t now appears probable that primary bonds may be formed between certain protein groups and the substrate. Thus, in the case of glyceraldehyde-3-phosphate dehydrogenase an - - S H group is believed to react with the aldedydic substrate to yield a hemimercaptal which is oxidized to the thiol ester at the expense of the bound coenzyme, DPN. The free D P N enzyme is regenerated by another molecule of coenzyme and by an acceptor for the newly formed acyl group. 34 Although the complete structure of an enzymatic site has as yet not been unequivocally established, working hypotheses are under active study in many laboratories. Data of the type described in this chapter will be needed as a basis, and as a test, of any such hypotheses. ~7 R. R. Porter, in " T h e Proteins" (Neurath and Bailey, eds.), Vol. I, Part B, p. 973. Academic Press, New York, 1953.

[19.]

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[12] T e c h n i q u e s f o r t h e A s s a y of t h e R e s p i r a t o r y E n z y m e s

By BRITTON CHANCE Introduction The classic method for the identification and characterization of respiratory enzymes is based on the appearance of the sharp absorption bands of their reduced forms when the respiring cells become anaerobic owing to their own respiration. This oxidation-reduction reaction served as the basis for the historic studies of MacMunn ~ and of Keilin 2 on cytochromes. As more components of the respiratory mechanism have become identified, attention has been directed toward quantitative estimates of their concentration. For this reason there has been considerable development, pioneered by Keilin, 2,3 of specialized chemical reactions that reveal the characteristic absorption bands of only one component of the respiratory system. Such reactions have been especially useful in conjunction with spectrophotometric recording of the absorption bands of the pigments and have served as the basis of a better differentiation of the respiratory components and their quantitative estimation. 4-6 Various chemical reactions that we have found useful in our spectrophotometric studies of members of the respiratory chain are briefly outlined.

Specific Chemical Reactions Carbon Monoxide. Carbon monoxide is an example of the ideal chemical reactant, since it combines specifically with the iron atom of the mammalian terminal oxidase, cytochrome a3. m,9,~ In the visible region of the spectrum no very distinctive effects are obtained when the reduced form of cytochrome a3 is treated with carbon monoxide, but the differential recording method in the region of the Sorer band brings out very 1 C. A. MacMunn, J. Physiol. 6, 22 (1885). D. Keilin, Proc. Roy. Soc. B98, 312 (1925). 3 D. Keilin and E. C. Slater, Brit. Med. Bull. 9, 89 (1953). 4 B. Chance, in " T h e Mechanism of Enzyme Action" (McElroy and Glass, eds.), p. 399. Johns Hopkins Press, Baltimore, 1953. b B. Chance, Harvey Lectures 49, 145 (1954-55). B. Chance and G. R. Williams, J. Biol. Chem. 217, 395 (1955). 7 O. Warburg, Biochem. Z. 177, 471 (1926). s D. Keilin and E. F. Hartree, Proc. Roy. Soc. B127, 167 (1939). 9 B. Chance, J. Biol. Chem. 202, 383 (1953). 9, L. N. Castor and B. Chance, J. Biol. Chem. 217, 453 (1955).

274

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distinctly the absorption band due to the carbon monoxide compound of cytochrome a3. 9 In this method, two samples of the material containing the respiratory chain are used, and both are made anaerobic by their own respiration. The difference of absorbancy between the two samples is then recorded, with the split-beam method, l°,H over a convenient span of wavelengths, usually 400 to 480 m~. This trace, of course, should show no distinctive absorption band, since the concentration of pigment and the optical path lengths in the two samples have been adjusted to be identical. One of the suspensions is now bubbled with a fine stream of CO gas for about 1 minute. The spectrum representing the difference of absorbancy between the two samples is again, recorded, and if cytochrome a3 is present in sufficient concentrations ( ~ 10-7 M), the sharp absorption band having its peak at 429.5 m~ and a trough at 445 m~ will be clearly defined (Fig. 1). This is a completely satisfactory identification of cytochrome a3, and the quantitative amount present may be estimated by dividing the total optical density change (the increase at 430 m~ minus the decrease at 444 m~) by the change of extinction coefficient, 91 cm. -1 mM.-l. 12 The pigments of mammalian tissues that interfere with this assay are hemoglobin, myoglobin, and a pigment found in microsomes. 18Various protein hemochromogens will not interfere because they are not reduced by substrate under anaerobic conditions and require a chemical reducing agent such as dithionite. Several methods are available for reducing the contamination by blood pigments. First, erythrocytes are usually completely removed from mitochondria in the process of differential centrifugation necessary for preparation of mitochondria. In cases where hemoglobin or myoglobin contamination is unavoidable, oxidation of these pigments by nitrite, followed by dialysis to remove the nitrite, has often been used, for example, in the case of leucocytes 14 and of heart muscle preparation. 1~ The ferric forms of the blood pigments are not reduced to ferrous by anaerobiosis and hence do not contribute to the carbon monoxide spectrum. The ferrous forms of hemoglobin or myoglobin combine with carbon monoxide and give rise to an absorption peak at 419 m~ and a trough at 432 m~. The effect of hemoglobin upon the assay of cytochrome aa is illustrated by the traces of Fig. 2. It is seen that the trough at 444 m~ is very little affected while the peak at 430 m~ of the CO compound of cytochrome a3 is shifted to shorter wavelengths 10B. Chance, Science 121, 621 (1955). 11C. C. Yang and V. Legallais, Rev. Sci. Instr. 25, 801 (1954). 12B. Chance, J . Biol. Chem. 202, 397 (1953). ~aM. Klingenberg,unpublished data. 14B. Chance and L. N. Castor, Science 116, 200 (1952). 15B. Chance, J. Biol. Chem. 197~557 (1952).

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?~%u) FIo. 1. Illustrating the measurement of the concentration of cytochrome a3 by the formation of its carbon monoxide compound; a reproduction of original experimental records of spectra representing the difference of absorption between the oxidized and reduced forms of the cytochromes of a Keilin and Hartree heart muscle preparation (trace a-b) and between the reduced forms and the carbon monoxide compound of cytochrome a3 (trace d-c). Trace a represents a base line with the two cuvettes of the split-beam apparatus filled with a suspension of Keilin and Hartree's heart muscle particles. On the addition of succinate to one of the cuvettes, and after anaerobiosis, trace b is recorded. Trace c is recorded after addition of succinate to the aerobic cuvette and after both cuvettes have become anaerobic. Trace d is the one referred to in the text and is obtained after bubbling carbon monoxide gas through one of the anaerobic samples. The peak at 429.5 m# and the trough at 445 m# identify the carbon monoxide compound of cytochrome a3. The optical density change due to this compound is read off the scale of the graph. Trace e is obtained after carbon monoxide treatment of both samples. The small displacement of trace d with respect to the others was accomplished electrically in order to avoid confusion with the remaining traces. This method of chemical-spectral analysis differs from that of Lundeg~rdh ~a who subtracts the spectrum of the known cytochromes from the total spectrum of all components. (Keilin and Hartree heart muscle preparation, pH 7.2, 0.15 M phosphate buffer, 13 raM. of succinate added. (Courtesy of The Journal of Biological Chemistry.) b y a low c o n c e n t r a t i o n of h e m o g l o b i n - C O c o m p o u n d a n d t h e 430 m # p e a k m a y be o b l i t e r a t e d b y t h e H b - C O peak a t h i g h e r c o n c e n t r a t i o n s . N i t r i t e t r e a t m e n t s h o u l d be used u n d e r these c o n d i t i o n s . 14,15 T h e m i c r o s o m a l p i g m e n t whose c a r b o n m o n o x i d e c o m p o u n d a b s o r b s a t 450 m # has n o t b e e n f o u n d to be a s s o c i a t e d w i t h c y t o c h r o m e a3 a n d i n t e r f e r e n c e of t h e two c o m p o n e n t s is r e n d e r e d u n l i k e l y b y the r a t h e r wide s e p a r a t i o n of t h e i r a b s o r p t i o n b a n d s . I n a d d i t i o n , t h e 450-m# CO c o m p o u n d f o r m s r a t h e r slowly 13 a n d m a y t h e r e b y be d i s t i n g u i s h e d f r o m t h a t of c y t o c h r o m e a3, w h i c h f o r m s rapidly.16 ~eB. Chance, J. Biol. Chem. 202, 407 (1953). 1~ H. Lundeg~rdh, Biochim. et Biophys. Acta 20, 469 (1956).

276

TECHNIQUES FOR METABOLIC STUDIES

[12]

In bacteria, three other types of carbon monoxide compounds have been observed2 The first is cytochrome a~ the terminal oxidase of Acetobacter pasteurianum. This terminal oxidase is similar t o t h a t of the mammalian system in m a n y ways and, in fact, was mistaken for it by Warburg in his pioneer studies of the photochemical dissociation of their carbon monoxide compounds.~7 The peak of the absorption band of the carbon monoxide compound cytochrome a~ lies at 427 m~, and the corresponding trough is observed at 442 m~. ÷0.1~ I¢EJ v

e-, / s

'l I

i°

\

-o.i

I

•

¢.J

o

i

380

i

400

420

i

|

i

440

460

480

~(rnjj) Fro. 2. Illustrating the effects of hemoglobin contamination upon the estimation of cytochrome aa of a heart muscle preparation in terms of its carbon monoxide compound. A small contamination is apparent in the solid trace and a larger value in the dashed trace (cf. trace d of Fig. 1). T h e second cytochrome is called the " C O - b i n d i n g p i g m e n t " because of its characteristic reaction with carbon monoxide2 In this case, the peak of the absorption band of the CO compound in the Soret region lies at 416 m~, with a corresponding trough at 432 m~ (Fig. 3). This compound is readily observed in m a n y types of bacteria, particularly M icr ococcus pyogenes var. albus and A cetobacter suboxydans. 18 T h e third carbon monoxide compound is t h a t of cytochrome as. This one has an indistinct Soret band t h a t is difficult to distinguish in the living cell but has a readily observable 647-m~ band with a corresponding trough at 630 m~.18 In m a n y cases, the terminal oxidases of bacteria are mixed, and various combinations of the spectra due to the carbon monoxide compounds 1~O. Warburg, "Schwermetalle," Freiburg, 1949. is L. Smith, Bacteriol. Revs. 18, 106 (1954).

[12]

TECHNIQUES FOR &SSAY O ? REBPIR&TORY ENZYMES

277

of cytochromes al, a2, and the CO-binding p i g m e n t h a v e been observed. E x a m p l e s of the complicated form t h a t the difference s p e c t r u m result in are given elsewhere. 9 +0.04.

Reduced , . + 0.02.

E

u

Reduced + CO

C

E u

_c

O,

C

a O

~_ - 0 . 0 2 . (3. o 380

420 ),,(re,u)

460

Fie.-,. 3. Identification of a terminal respiratory enzyme, the "CO-binding pigment," by means of its carbon monoxide compound. The spectrum represents the difference between the carbon monoxide compound of the "CO-binding pigment" and the reduced forms of the cytochromes of Micrococcus pyogenes var. albus. In addition, the spectrum representing the difference between the steady-state oxidized and reduced forms of the respiratory enzymes of this bacteria is presented. (Courtesy of Journal of Biological Chemistry.) Carbon monoxide is the m o s t effective reagent for identification of terminal oxidases; it is highly specific, and the carbon monoxide compounds h a v e distinctive absorption b a n d s in the Sorer region. Other studies show t h a t these CO c o m p o u n d s are inhibitors of cellular respiration because of the correspondence of the photodissociation and photochemical action s p e c t r u m for the relief of carbon monoxide inhibition of respiration. 9~ Cyanide. Cyanide is not nearly so useful as carbon monoxide because of the lack of spectroscopically distinct c o m p o u n d s of the terminal o×idases. 8,18 I t s effects on the peroxidase of y e a s t cells and on the catalase of bacteria are m u c h more pronounced t h a n on the terminal oxidases.

278

TECHNIQUES FOR METABOLIC STUDIES

[12]

In addition, its combination with oxidized hemoglobin and myoglobin will cause spectroscopic interference. Other r e a g e n t s - - a z i d e , ethyl hydrogen peroxide, and so f o r t h - - g i v e less distinctive spectroscopic effects 8 and do not compare with carbon monoxide as a reagent for q u a n t i t a t i v e analysis of the terminal oxidases. Antimycin A, Hydroxyquinoline-N-Oxirne. When the m a m m a l i a n respiratory chain is a p p r o p r i a t e l y t r e a t e d with one of these substances, the principal b a n d which a p p e a r s is t h a t of reduced c y t o c h r o m e b with

A c ~ ~cm) *.080"

J.

E

+.060.

'+.015

~E u

u

-

+.040'

•+.010

~

+.020.

•+.0O5 u~

j

o

' L_.-------

c

~

g

,_005 g~

-.0~0 °

o

._u -o040o =060

°

'E

,-.o10

8

o

4bo

4go

5bo

"A(m)J)

5io

660

-.ol5

FIG. 4. Identification of cytochrome b by means of antimycin A treatment of respiring cells; the spectrum represents the difference between antimyein A-treated yeast cells and aerobic yeast cells. The absorption peaks shown are those of yeast cytochrome b together with a small quantity of flavoprotein. A similar spectrum for liver mitochondria is given in Fig. 9. (Courtesy of Johns Hopkins Press.) its Soret b a n d at 430 mtL and its a - b a n d at 562 mt~. 8,19-2j I n order to obtain the m o s t distinctive results, two samples of the respiratory s y s t e m are oxygenated and t r e a t e d with an a p p r o p r i a t e s u b s t r a t e so t h a t electron transfer proceeds at a m o d e r a t e rate. A base line is then run with the s p e c t r o p h o t o m e t e r to indicate equality of a b s o r b a n c y of the two specimens. One is then treated with the a p p r o p r i a t e a m o u n t of the inhibitor, and the difference in a b s o r h a n c y between the two samples is again plotted. This will reveal a sharp p e a k at 430 m~ with a corresponding trough at a b o u t 412 mtL (Fig. 4). I n the region of the ~-band, the peak and trough lie at 562 and 575 mt~. Depending on the intensity of respiration, other spectroscopic changes will a c c o m p a n y t h a t of c y t o c h r o m e b; for example, flavoprotein and pyridine nucleotide will become reduced ~9B. Chance, Nature 169, 215 (1952). ~0E. C. Slater, Biochern. J. 45, 14 (1949). 31 V. R. Potter and A. E. Rief, Federation Proc. 10, 234 (1951).

[12]

TECHNIQUES F O R ASSAY OF RESPIRATORY ENZYMES

279

due to the action of the inhibitor. Cytochromes c, cl, and a will become more oxidized, an illustration of the "pseudo-reversibility" of the respiratory chain. The effect on cytochrome b, however, so dominates the spectroscopic results that no confusion will result. TM Antimycin A and hydroxyquinoline-NIoxime are the most useful inhibitors. They act rapidly and at a low concentration with the intact respiratory chain. Usual values for maximal spectroscopic change are of the same order as the amount of cytochrome b involved. In Keilin and Hartree's succinic oxidase preparations, the end point of the titration of cytochrome b with antimycin A has been found to be half that of the cytochrome b content.19 Intact mitochondria respond very sensitively to these inhibitors, as do yeast and ascites tumor cells, although the rate of penetration of the inhibitor may be quite slow. In intact muscle, for example, frog's sartorius muscle, rather high concentrations and long incubation periods have been found to be necessary in order to obtain proper penetration of the antimycin A. 22 Hydroxyquinoline-N-oxime also interrupts the respiratory chain of certain bacteria but causes oxidation of reduced cytochrome bl in E. coli and Pr. vulgaris. 23 Chin has reported that urethan acts on the respiratory chain of Acetobacter peroxydans, although his report has not been confirmed. 24 Taken all in all, these inhibitors of the oxidation of reduced cytochrome b give very distinctive spectroscopic effects which can easily be observed spectrophotometrically at both 562 and 430 m~. The molarity of cytochrome b may be estimated by the optical density change measured at 562 and 575 m~. This optical density change may be divided by 22 to give the millimolar concentration of cytochrome b (see discussion below and ref. 19) (optical path length, 1 cm.). The concentration of cytochrome b may also be estimated from the optical density change measured at 430 and 410 m~, the peak and trough of the changes at the region of the Soret band, but in this case there is some interference due to cytochrome c, which, as mentioned above, becomes oxidized when cytochrome b becomes reduced. If, however, cytochrome c is known independently already to be oxidized before the addition of the inhibitor, then estimation of cytochrome b in the region of the Soret band ~a A condition which would render the estimation of cytochrome b difficult b y this m e t h o d is the presence, in cells containing endogenous substrate, of a strong dehydrogenase a n d a weak oxidase system which would cause the cytochrome b to be already reduced before t h e addition of the inhibitor. This difficulty is readily avoided if the substrate is r e m o v e d from the system b y vigorous aeration for a reasonable interval (18 hours is sufficient for b a k e r ' s yeast). 22 A. M. Weber, u n p u b l i s h e d data. 23 j . W. Lightbown a n d F. L. Jackson, Biochem. J . 63, 130 (1956). 24 C. H. Chin, Dissertation, Cambridge University, 1952.

280

TECHNIQUES FOR METABOLIC STUDIES

[12]

is satisfactory and an extinction coefficient of about 180 cm. -1 mM. -~ may be used for liver mitochondria (see below). Except for one recent report in which optical constants were not given, 25 cytochrome b, as distinguished from cytochrome bs, has not been highly purified, and the molecular extinction coefficients have been derived by analogy with other purified hemoproteins. We have first used the characteristics of cytochrome b obtained from bacteria by Pappenheimer et al. ~'6 In a personal communication he has indicated that the optical density difference between the oxidized and reduced forms of cytochrome b at 560 m~ is about the same as that obtained at 554 m~ for the pyridine hemochromogen, corresponding to a value of 22 cm. -1 raM. -~. This value is in good agreement with the change of molecular extinction coefficient that has been calculated from the original data of Bach et al. ~7 on cytochrome b2, which they state to be essentially the same as that of cytochrome c (19.1 cm. -~ mM.-1). Later studies of cytochrome b2 give a value of 23 cm. -~ mh/[.-1. 28-3° Recent studies of the cytochrome b5 system give a similar value.3° Strittmatter and Velick3~ purified cytochrome bs, and we read from their graphs changes of extinction coefficients of 22 cm. -~ mM. -~ for the a-band and 160 cm. -1 mM. -~ for the -~ band. We therefore take 22 cm. -1 mM. -~ to be correct for the a-band of cytochrome b, as measured at 562 and 575 m~. In difference spectra of cytoehrome b, as measured in various materials, the ratio of the optical density changes at 430 and 410 m~ to those at 562 and 575 m~ varies from 8:1 in liver mitochondria to 10:1 in heart muscle succinic oxidase preparations. A m y t a l . Amytal inhibits electron transport near the lowest member of the respiratory chain, 32,33and therefore the changes of the cytochromes are in the direction of oxidation; such changes are large enough to be noticeable only if adequate levels of substrate are already present. Thus, on addition of Amytal to a respiratory chain supplied with substrate and oxygen, the spectra shifts observed are oxidations of eytoehromes a, a3, 2~I. Sekuzu and K. Okunuki, J. Biochem. (Japan) 48, 107 (1956). 2s A. M. Pappenheimer, Jr., and E. D. ttendee, J. Biol. Chem. 171, 701 (1947). 27 S. J. Bach, M. Dixon, and L. G. Zerfas, Biochem. J. 49~ 229 (1946). 28 C. A. Appleby and R. K. Morton, Nature 175~ 749 (1954). 29 E. Boeri, E. Cutolo, M. Luzzati, and L. Tosi, Arch. Biochem. and Biophys. 56, 487

(1955). 30 B. Chance, M. Klingenberg, and E. Boeri, Federation Proc. 15, 231 (1956). 81 p. Strittmatter and S. Velick, J. Biol. Chem. 221, 258 (1956). a2L. Ernster, O. Jalling, H. LSw, and O. Lindberg, Exptl. Cell Research Suppl. 3, 124 (1955). 3~B. Chance, in "Enzymes: Units of Biological Structure and Function" (Gaebler, ed.), p. 447. Academic Press, New York, 1956.

[12]

TECHNIQUES FOR ASSAY OF RESPIRATORY ENZYMES

281

b, c, c~, and, in the case of liver mitochondria, flavoprotein. The distinctive change that is observed is the reduction of pyridine nucleotides (Fig. 5). Thus, Amytal is a specific reagent for the presence of pyridine nucleotide in the respiratory chain. Such a specific reagent is necessary DPNH

/

* 0.03-

/

/

/'\

'\ ' \

/

\ '

\ \

÷0.01

I

'

\

/

tO02

\~

Amytol to State 5 \ (AOH as subs~rale

E

k C

0

\

¼_

/

E

,\

/

//

-OOI-

--0102

© -0,03

3~0

3~0

4bo

:k (m~u)

4~0

sbo

FIG. 5. Identification of reduced pyridine nucleotide in rat liver mitochondria by means of Amytal treatment; the spectrum represents tim difference between Amytaltreated mitochondria (2 mM. Amytal) and aerobic mitochondria in state 3 (supplied with phosphate acceptor, phosphate, substrate, and oxygen). The trough at 430 m# is due to the oxidation of reduced cytochrome b, and the peak at 340 m# is due to the reduction of pyridine nucleotide (experiment 522-A).

in cases where only small amounts of pyridine nucleotide are present: the transition from aerobiosis to anaerobiosis may be unsuitable for revealing pyridine nucleotide, since this change causes reduction of cytochromes that have their ~-bands in the region of 320 m#. An example of

282

TECHNIQUES FOR METABOLIC STUDIES

[12]

this is afforded by the work of Holton, ~4 whose data show no distinctive band at 340 m~ in the aerobic-anaerobic difference spectra of the a-ketoglutarate oxidase system of the heart muscle sarcosomes. Amytal treatment, on the other hand, gives an optical density change attributable to reduced pyridine nucleotide (PNH), and this change is readily measurable with a sensitive spectrophotometer25 The concentration of PNH has been estimated by dividing the optical density change measured at 340 and 374 m~ by 6.38 Pyridine Nucleotide-Linked Substrates. Oxidizing and reducing substrates for PN-linked dehydrogenases are nearly ideal reagents for the assay of a single component of the respiratory chain. If, for example, a PN-linked substrate were added to material made anaerobic with a nonPN-linked substrate, one would record only the absorption due to PNH. Unfortunately, in mitochondria, all substrates, including succinate, cause reduction of pyridine nucleotide. ~3 On the other hand, the reversibility of the dehydrogenase system does provide an excellent opportunity to obtain a highly specific interaction of a substrate with a respiratory chain. Mitochondria are made anaerobic by the addition of phosphate acceptor plus a low concentration of the DPN-linked substrate, ~-hydroxybutyric acid. Addition in excess of the reaction product, acetoacetic acid, plus ADP, will cause oxidation of pyridine nucleotide without affecting any other component. No measurable amount of flavoprotein oxidation has been observed under these conditions. Similar reactions can be obtained by reduction of pyridine nucleotide with a small concentration of glutamate, followed by oxidation with ammonium chloride. Reactions such as these are applicable to intact yeast cells which contain an active alcohol dehydrogenase system. A low concentration of ethanol is used to reduce the pyridine nucleotide of the anaerobic cells. An excess of acetaldehyde causes oxidation of the pyridine nucleotide without affecting other components 5 (Fig. 6). Intracellular pyridine nucleotides can be oxidized by a-glycerol phosphate dehydrogenase activity. 4 DPN is reduced by addition of ethanol to anaerobic yeast. The cells are then heated with iodoacetate to inhibit glyceraldehyde-3phosphate dehydrogenase activity. The D P N H is reoxidized on addition of glucose from which dihydroxyacetone phosphate is formed and rapidly reacts with D P N H to give DPN and a-glycerol phosphate. Penetration of hydrogen ions into the yeast cells also causes an oxidation of reduced pyridine nucleotides because of the pH-sensitive equilib34 F. A. Holton, Biochem. J. 61~ 46 (1955). 35 B. Chance and M. Baltscheffsky, Biochem J. in press. 38 P a b s t Laboratories Circular OR-7 (1955).

[19.]

TECHNIQUES FOR ASSAY OF RESPIRATORY ENZYMES

283

rium of the reduced and oxidized forms. Thus, ethanol-reduced DPN can be largely reoxidized by addition of organic acids or hydrazoic acid. ~ It is also possible to distinguish between D P N H and T P N H by these methods; for example, if glucose is added to the starved yeast cells in

I 500

520

I

I

I

+0.10

I

,~(m~ 540 560 I

I

380 I

400_

I

i

I -I- 0.05 E v ID

E '/I -

--

,~nuerobi~

~

~ e a s t

(ethano~

eect)

j,

~ ~-

0

I

\

a°

Addition of 15ran /

\~-'acetoIdehyde-y

\

I/

:-

~-'5

-o.o

\__J

0.10 FIG. 6. Identification of intracellular pyridine nueleotide in yeast cells by a shift of the oxidation-reduction equilibrium by means of acetaldehyde; the spectrum represents the difference between acetaldehyde-treated yeast cells and yeast ceils treated with alcohol alone. The broad trough indicates the oxidation of intracellular DPNH.

which the terminal oxidase is inhibited by addition of cyanide or by anaerobiosis, all the pyridine nucleotides become reduced. On the other hand, if the yeast cells are pretreated with iodoacetic acid in order to inactivate glyceraldehyde-3-phosphate dehydrogenase, the addition of glucose activates zwischenferment and causes reduction of T P N only.

284

TECHNIQUES FOR METABOLIC STUDIES

[12]

With a particular yeast, only 10% of the total pyridine nucleotide was so reducible, a result that is in accord with chemical assays. 37 This result also indicates that the intracellular transhydrogenase activity of these yeast cells can be neglected; otherwise the reaction T P N H + D P N ~-* T P N -}- D P N H would have proceeded. 3s Apparently the reaction between D P N and flavoprotein is not sufficiently reversible to cause appreciable oxidation of flavoprotein on the addition of these DPN-linked substrates. This has the advantage in that there is no interference from flavoprotein, and the disadvantage that these methods cannot be used to assay independently for flavoprotein. In order to obtain a quantitative measure of the D P N H absorption, the absorbancy changes are measured at 340 and 374 mp for the following reasons: The choice of 340 m~ is an appropriate one for mitochondria and sarcosomes, since we find there that the pyridine nucleotide that is involved in the oxidative phosphorylation reaction is not bound in such a way as to shift its absorption maximum from 340 m~ (Fig. 7). It has been shown, however, in yeast and in certain bacteria that the absorption maximum occurs at a wavelength of about 330 m~, and this result has been verified by Lundeg~rdh. TM This shift has been attributed to binding of D P N H to alcohol or lactic dehydrogenase. 4,j9,~9,4° The appropriate molecular extinction coefficient in this case is 5.8 cm. -1 miV[.-1. 89 In metabolizing cells the possibility of pyruvate accumulation often exists and its absorption band may interfere with the assay of pyridine nucleotide. The difficulty is readily avoided by (a) reducing the oxygen concentration in the cell suspension to a low value before adding a substrate, such as glucose which leads to pyruvate formation, and then measuring the pyridine nucleotide as soon as anaerobiosis has occurred; (b) establishing anaerobiosis with ethanol, then adding glucose; or (c) oxidizing the reduced pyridine nucleotide by acetaldehyde, etc. The choice of 374 mu as the reference wavelength for the measurement of reduced pyridine nucleotide is based on the following considerations: First, a wavelength shorter than 340 m~ is undesirable because the opacity of the sample increases rapidly with decreasing wavelengths. Second, in cells which contain glycolytic as well as respiratory chains, a considerable amount of glyceraldehyde-3-phosphate-dehydrogenase-DPN compound tl may be present. This compound has an absorption peak at 365 m~ that 87H. yon Euler, F. Schlenk, H. Heiwinkel, and B. H6gberg, Z. physiol. Chem. 256, 208 (1938). 38S. P. Colowick, N. O. Kaplan, and M. M. Ciotti, Federation Proc. 10, 174 (1951). 39H. Theorell and R. K. Bonnichsen, Acta Chem. Scand. 5, 1105 (1951). 40B. Chance and J. B. Neilands, J. Biol. Chem. 199, 383 (1952). 41E. F. Racker and I. Krimsky, J. Biol. Chem. 198, 731 (1952).

[12]

TECHNIQUES FOR ASSAY OF RESPIRATORY ENZYMES

285

disappears on the reduction of pyridine nucleotides or on addition of iodoacetate 4z (Fig. 8 4). On the other hand, this absorption band is so broad that it has about equal values of extinction at 340 and 374 m~. 4 Thus, measurements made differentially between these two wavelengths

-I- 0.01

I

-

E U v

C

E

o~

c

u~

-0.01

0

_~

-0.02

o

-0,03-

350

s6o (re,u)

FzG. 7. Identification of reduced pyridine nucleotide in mitochondria by treatment with adenosine diphosphate. The spectrum represents the difference between mitochondria treated with ADP and those to which only substrate and phosphate were added. The oxidation of reduced pyridine nucleotide is indicated by the trough at 340 m~. The oxidation of reduced cytochrome b is indicated by the trough at 430 m~ (experiment 522-A).

would not be affected by the glyceraldehyde-3-phosphate-dehydrogenaseDPN compound. Wavelengths longer than 374 m~ are undesirable, since the Soret bands of the cytochromes interfere, and, in addition, the difference between 340 m~ and the reference wavelengths should be kept at a minimum. As will be shown below, flavoprotein interference at 374 m~ is not significant. Judging from the published data on most purified flavoproteins, a major absorption band in the vicinity of 360 mu (for a recent summary see reference 42) should be observed in the intact cell and should interfere with the measurement of pyridine nucleotide when the reference wavelength is 374 m~. An exception is the flavoprotein component of purified 4~ H. R. Mahler, Advances in Enzymol. 17, 233 (1956).

286

TECHNIQUES FOR METABOLIC STUDIES

[12]

succinic dehydrogenase which does not show a 360 m~ absorption band. 42, We have, however, obtained no evidence for such an absorption band in i n t a c t cells or in mitochondria. A r a t h e r crucial test for the presence of such an absorption band is afforded b y studies of guinea pig liver mitochondria in which the absorption of the oxidized form of flavoprotein at 465 m~ is nearly as great as the absorption of reduced cytochrome a~ at 445 m#. 6 Nevertheless, no definite evidence of an absorption band at 360 m# has been obtained in the oxidized minus reduced difference spectra IE ¢J v

>-,

ooo:t\

Qa

r~ 0

300

350

400

450

500

(mjJ)

Q.

0

FIO. 8. Identification of glyceraldehyde-3-dehydrogenase by means of the effect of iodoacetate on the spectrum of its DPN compound. The spectrum represents the difference between yeast cells treated with iodoacetate and aerobic yeast cells. The trough in the neighborhood of 360 m~ indicates the disappearance of the absorption band of the glyceraldehydephosphate dehydrogenase-DPN compound on the addition of iodoacetic acid. (Courtesy of Johns Hopkins Press.) or n the r a t h e r more critical test obtained b y adding antimycin A to mitochondria in the aerobic quiescent state (state 4). In this state the pyridine nucleotide is already over 99 % reduced and the flavoprotein is considerably oxidized. 43 T h e addition of the inhibitor causes reduction of the flavoprotein (and cytochrome b as well; see above) and would presumably reveal the second flavoprotein absorption band if it were of appreciable magnitude. Anaerobiosis. B y definition, those pigments whose oxidation state changes from oxidized to reduced under anaerobic conditions are respirat o r y pigments. Absorption bands due to cytochromes a3, a, c, cl, and b, and reduced pyridine nucleotide appear under these conditions, whereas the absorption band due to oxidized flavoprotein disappears (Fig. 9). Thus, m a n y absorption bands appear nearly simultaneously in anaerobiosis, but the several' components m a y be separately assayed b y measur42=T. P. Singer, E. B. Keaney, and V. Massey, "Enzymes: Units of Biological Structure and Function" (Gaebler, ed.), p. 417. Academic Press, New York, 1956. 43B. Chance and G. R. Williams, J. Biol. Chem. 217, 409 (1955).

[12]

287

TECHNIQUES FOR ASSAY OF RESPIRATORY ENZYMES

ing their absorption at appropriate pairs of wavelengths. This procedure is necessary for four of the components that are not individually affected by the chemical treatment described. These components are cytochromes a, c, and c~, and flavoprotein.

<- Reduced , ~ Pyridine i

~Nucleotide t

i

-~ +.08

,

i

E I

E

0

E

oe- +.04

+.01

E ¢1) E 0 eB

u)

+005

,i

a)

0

-0

¢1.

e.

c~ 0

0

;i°~i n

-.04-

340

4()0

460 550 A(m~)

0

6()0

FIG. 9. Identification of cytochromes a, c, and a3, and flavoprotein and reduced pyridine nucleotide by means of anaerobiosis. The spectrum represents the difference between anaerobic mitochondria and aerobic mitochondria. The aerobic mitochondria are, in this case, in the phosphorylating state 3 (phosphate, phosphate acceptor, substrate, and oxygen are present). The dashed spectrum represents the difference between mitochondria treated with antimycin A and those in the aerobic state 2 (phosphate, phosphate acceptor, oxygen, but no substrate present). (Courtesy of Advances in Enzymology.)

Cytochrome a, for example, is measured almost independently of cytochrome as if the wavelengths of 605 and 630 m~ are chosen. A change of extinction coefficient of 16 cm. -1 mM. -1 has been assumed for this component on the basis of an analogy between the oxidized and reduced spectrum of cytochrome a and the corresponding spectrum for the hemoprotein verdoperoxidase 19 Cytochrome a~ is difficult to measure at 600 m~ and may be measured by the difference of absorbancy at 445 and 465 m~. This separation of wavelengths gives very nearly the full value

288

TECHNIQUES FOR METABOLIC STUDIES

[12]

for the cytochrome a3 band. The value of change of extinction coefficient for the wavelengths 445 and 465 m~ is 91 cm. -1 mlV[.-1. ~6 The trough at 465 m~ may interfere, especially in mitochondria prepared from guinea pig liver. This interference is reduced by choosing 455 m~ for the reference wavelength, but in this case the change of extinction coefficient is reduced to about 60 cm. -1 mlV[.-1 (see also p. 274). As mentioned previously, cytochrome b may be measured under anaerobic conditions at the same wavelengths as were recommended where antimycin A treatment was used. The contribution due to cytochrome c at the pair of wavelengths suggested (562 and 575 m~) is rather small, and, in most cases where the cytochrome b content is approximately equal to cytochrome c, little difficulty will be encountered. Cytochrome c is usually measured by the difference of absorbancy measured at 550 and 540 m~. The change in molecular extinction coefficient for the purified cytochrome c is 19.1 cm. -1 mM.-l. 44 Under these conditions, there may be appreciable contribution due to cytochrome cl, and in liver mitochondria it was estimated that one-third of the absorption attributed to cytochrome c is due to cytochrome cl. 43 The quantity evaluated at 550 m~ is therefore termed c -~ cl, and the contribution due to cl is substracted after its estimation by the extraction procedure described on p. 289. Cytochrome cl is measured by the difference of absorbancy at 553 and 540 m~. There is a considerable contribution of cytochrome c to this absorption, and it is preferable to use liquid air temperatures or to follow the procedure described below for the removal of cytochrome c and for the measurement of eytochrome c~ in the remaining material. Pending the purification of cytochrome c~ we have arbitrarily used the same molecular extinction coefficient as for cytochrome c. Flavoprotein in intact mitochondria may be measured by the difference of the absorbancies at 465 and 500 m~. After considerable experience with this type of measurement in the case of flavohemoprotein cytochrome b~, there is some indication that a wavelength of 510 m~ is preferable to 500 m~, although it is quite probable that the details of the absorption band of flavoprotein in the respiratory chain will differ somewhat from that in the flavohemoprotein. In this case, a value of 11 cm. -I miV[.-1 has been used for the change of molecular extinction coefficient.45 Pyridine nucleotide is assayed on the basis of the oxidized minus reduced spectrum, providing it is present in a concentration large compared to th at of the cytochrome. This is the case with whole cells and with intact liver mitochondria, and under these circumstances the wavelengths of 340 and 374 m~ are used, together with a change of extinction 44 H. Theorell, Biochem. Z. 286, 207 (1936). 45 D. Keflin and E. F. Hartree, Biochem. J. 42, 221 (1948).

[12]

TECHNIQUES FOR ASSAY OF RESPIRATORY ENZYMES

289

coefficient of 6 cm. -1 m M . -1. I n cases where the pyridine nucleotide cont e n t is smaller, the use of A m y t a l or specific D P N - l i n k e d substrates is r e c o m m e n d e d (see p. 282). Usually no a t t e m p t has been m a d e to evaluate the concentrations of cytochromes b and c in the region of the Soret b a n d f r o m the oxidized minus reduced spectrum, the a - b a n d s of these pigments being m u c h more clearly distinguished t h a n the -y-bands. I t is of interest to note t h a t the Sorer b a n d s of intact frog muscle are more clearly separated t h a n those of liver mitochondria. ( C o m p a r e Figs. 16 and 18.)

E +.008o

(]D(

Reduced~

c

~ "t-.004

"

0

-5

~- -.oo4

Z(m.~) FIG. 10. Identification of cytochrome el in water-and-saline-washed mitochondria. The spectrum represents the difference between suecinate-reduced and oxidized particles derived from rat liver mitoehondria by water and saline washing. The absorption band of cytochrome cl can be clearly distinguished, since most of the cytochrome c is removed by this treatment. (Courtesy of Journal of Biological Chemistry.)

Saline Washing of Lysed Mitochondria. I n a n u m b e r of cases where an accurate estimate of the content of c y t o c h r o m e cl is required, it is desirable to remove c y t o c h r o m e c. This c o m p o n e n t can readily be rem o v e d f r o m liver mitochondria b y t r e a t i n g t h e m with w a t e r in order to disrupt the m e m b r a n e , and t h e n with p h o s p h a t e in order to extract the c y t o c h r o m e c. This extraction can be m a d e nearly complete, and a p p a r ently none, or v e r y little, of the c y t o c h r o m e cl is r e m o v e d 48 (Fig. 10). A trace of c y t o c h r o m e c remains to support a feeble electron t r a n s p o r t and causes anaerobiosis in a reasonable period. T h e oxidized minus reduced s p e c t r u m can then be used for an assay of cytochrome cl as was described above. Reduction with Dithionite. As mentioned above, the respiratory enzymes are defined b y their reduction in anaerobiosis. T h e r e are, however, pigments accessory to the respiratory chain which do not transfer elec-

290

TECHNIQUES FOR METABOLIC STUDIES

[12]

trons but which may be necessary for phosphorylation or other processes. These pigments may be evaluated by the absorbancy changes observed on addition of dithionite to the anaerobic system. If a split-beam spectraphotometer is used, both samples are made anaerobic, a base line is run, and then dithionite is added to one of the samples. The resulting difference spectrum for liver mitochondria that are free of microsomes shows a Soret band that is attributable to the pigment called "mitochrome ''46 I E U

to anaerobic(A) •/Na2S204 ~ / N o 2 $204 !o

v

E L. c'-

~

•'-

O.

0

t--

0 i

.I

0

4io

4- o ~,( m.u )

FIG. 11. Identification of mitochrome and related pigments by dithionite treatment of the anaerobic material. Curve A, a spectrum representing the difference between anaerobic mitochondria treated with sodium dithionite and anaerobic mitochondria reduced by substrate. The peak at 427 m~ may be attributed to the pigment called "mitochrome," although the similarity of the spectra of traces A and B indicates that this material combines only to a small extent with carbon monoxide. The large trough at 470 m~ is due to the reduction of flavoprotein. (Courtesy of Journal of

Biological Chemistry.)

(Fig. 11). The optical density change at 427 and 410 m~ has been used for evaluating the concentration of this pigment, and a molecular extinction coefficient typical of hemoproteins (200 cm.-1 raM. -1) is assumed to be valid for mitochrome. Separate estimation of cytochrome b5 and mitochrome is discussed on p. 295. In preparations which contain the ferric forms of the blood pigment, peroxidases, or denatured hemochromogens, this test has little meaning owing to the overlapping of the Soret bands of these pigments. Some discrimination is possible, since in the intact mitochondria mitochrome does not combine with carbon monoxide, whereas the reduced forms of most hemoproteins do. ~s D. B. Polls and H. W. Schmukler, Abstr. 128th Meeting Am. Chem. Sac. p. 196. Minneapolis, September, 1955.

[12]

T E C H N I Q U E S FOR ASSAY OF R E S P I R A T O R Y ENZYMES

291

In heart muscle preparations, Ball 47 has reported that succinate reduction of the anaerobic material does not cause complete reduction of cytochrome b and that dithionite addition is necessary for complete reduction. Although we have confirmed the increased absorption at about 560 m~, the pigment reduced has an absorption band that differs distinctly from that of cytochrome b and is not identical to that observed on hydrosulfite addition to anaerobic liver mitochondria. Thus, the pigment observed by Ball may be due to denaturation of one of the hemoproteins of heart muscle, and the practically complete reduction of cytochrome b by excess succinate can be relied u p o n . TM Low-Temperature Spectroscopy. An excellent method for the estimation of cytochromes c, c,, and b is to freeze the particulate suspension in a glycerol-buffer glass so that intensification and sharpening of the absorption bands occurs as has been described by Keilin and Hartree. 48,4° Under these conditions, cytochromes c and cl can be distinguished as was shown by Estabrook 5°,~1 and by Keilin and Hartree. 5~Three of these components can be estimated separately (Fig. 12). The low-temperature attachment for the split-beam recording spectrophotometer is described later (see Figs. 31 and 32) ; the technique for using it is as follows: The material in dilute buffer is diluted with an equal volume of glycerol and placed in a 1or 2-mm.-path cuvette, depending on the pigment concentration. The reference cuvette may contain the same material if a difference spectrum is desired, or it may contain 50% glycerol-water for recording absolute spectra. The procedure for obtaining an intensification of the absorption bands closely follows that of Keilin and Hartree. 4° The cuvettes are placed in the holder and plunged beneath the surface of the liquid air. Although sharpening of the bands has occurred, there has been no intensification. The frozen samples are then withdrawn and allowed to stand in the air until devitrification occurs. This is observed visually as a "sunset glow" that spreads through the cuvettes as the solid reaches a temperature of - 5 5 °. The samples are then cooled and placed in the optical path so that their difference spectra can be recorded. The absorption peaks of the cytochromes are now very sharp and are clearly separated. At liquid air temperatures the principal peaks lie at 560 mu for ba, 554 mu for c~, and 549 mu for c,,, and 546 for c,~. Thus, the method is most useful for the 47 E. G. Ball, Biochem. Z. 296, 262 (1938). 48 D. Keilin and E. F. Hartree, Nature 164, 254 (1949). 49 E. F. Hartree, in "Modern Methods of Plant Analysis" (Paech and Tracey, eds.), Vol. 4, p. 197. Springer-Verlag, Berlin, 1955. 5o R. Estubrook, Federation Proc. 14, 45 (1955). 51 p~. Estabrook, J. Biol. Chem. 223, 781 (1956). 5~ D. Keilin and E. F. Hartree, Nature 176, 200 (1955).

292

TECHNIQUES FOR METABOLIC STUDIES

[12]

detection of these cytochromes, especially c y t o c h r o m e c in the presence of excess c y t o c h r o m e cl. I n a particular case, c y t o c h r o m e c has been detected in the presence of at least a fivefold excess of cl. T h e use of lowt e m p e r a t u r e spectra for q u a n t i t a t i v e estimation of cytochromes c, c~, and b is more difficult because the intensification of the absorption bands varies with the n a t u r e of the particles and with the concentration of the cytochromes. cc~

b

Optical Density Increment 0.02

T

I

490

'

I

510

'

I

550 (mp)

'

I

550

'57

FIo. 12. Identification of cytochrome cz by low-temperature spectroscopy; an apparent absolute spectrum of cytochromes c, cl, and b of a Keilin and Hartree heart muscle preparation as measured at temperatures of liquid air. The sharpening of the absorption bands allows a clear separation of the absorption bands of cytochrome c and ci. (Courtesy of Dr. Ronald W. Estabrook.) T h e following procedure is therefore r e c o m m e n d e d : (1) Measure the sequence of optical density changes, c : c1: b :a, at liquid air temperatures, using the respective pairs of wavelengths, 549-540 m~, 554-540 m#, 560575 m#, and 601-630 m/~. (2) Divide this sequence b y the r o o m - t e m p e r a ture molecular extinction coefficients which are t a k e n to be 19 : 19:20 : 16, respectively. This gives the sequence of concentrations relative to cytochrome a, (3) C o n v e r t the sequence to an absolute basis from a deter-

[12]

TECHNIQUES FOR ASSAY OF RESPIRATORY ENZYMES

293

mination of the concentration of any one of the components, preferably cytochrome b (Ae56~_575 = 22 cm.-1 mM.-1), from the room temperature oxidized-reduced spectrum and the protein content, and convert the other values in the sequence of relative concentrations to absolute values. This procedure assumes that the intensification of the absorption bands of the four components at low-temperature is the same (Estabrook, unpublished data). _.--.

IE~+.050. ..

.| a

0

~

q

0

-.OK)

i

-.O~

Methyl hydrogen peroxide oclcled to oerobic storved yeost cells

soo

66o

FIG. 13. Identification of peroxidase of yeast cells by the addition of a substrate, methyl hydrogen peroxide. The spectrum represents the difference between aerobic yeast cells, treated with methyl hydrogen peroxide, and aerobic yeast cells. The compound formed is peroxidase complex II. (Courtesy of Johns Hopkins Press.)

The Estimation of Catalases and Peroxidases in Intact Cells or Mitochondria. The intact yeast cell contains a considerable amount of peroxidase, and this enzyme can be estimated by adding methyl hydrogen peroxide to the starved yeast cells in the aerobic state. 4 On addition of roughly 0.1 mlV[. methyl hydrogen peroxide, the absorption band of complex II is observed with a peak at 426 m~ and a trough at 400 m~ (Fig. 13). The compound is stable for a minute or so, provided that endogenous substrate has been reduced to a low concentration by the starvation process. The optical density change measured at these two wavelengths is divided by about 100 cm. -1 mS~. -1 to give the concentration of the enzyme. The aerobic starved state (state 2) was chosen for this estimation for two ~easons: (1) to eliminate endogenous donor for complex II and thereby prolong its lifetime and increase its steady-state concentration, and (2) to ensure that the cytochromes would be in the oxidized state so that the oxidation of their reduced forms by complex II

294

TECHNIQUES FOR METABOLIC STUDIES

[12]

of peroxidase would not interfere with a spectroscopic estimation of this compound. When peroxide is added to anaerobic cells, it is preferable to add the alkyl hydrogen peroxide instead of the hydrogen peroxide itself because of the possible decomposition of the latter into oxygen and water. This reaction does not occur with the methyl hydrogen peroxide. In addition, methyl hydrogen peroxide is advantageous for studies with catalase because all four of the catalase hemes are bound to the alkyl hydrogen peroxide, whereas only one-fourth of the heroes are bound, on the average, with hydrogen peroxide as the substrate. 53 Rather different considerations enter into the estimation of catalase in bacterial cells such as Micrococcus lysodeikticus and Bacillus subtilis. In this case, respiration leads to peroxide generation, and the enzyme is already partially saturated with substrate. 54 In order to have the free enzyme ready to combine with added peroxide, the cells are put in the anaerobic state in which no hydrogen peroxide is generated. Under these conditions, addition of a low concentration of methyl hydrogen peroxide will give the characteristic trough due to the partial disappearance of the 405-m~ band of catalase (Fig. 14). The change of the molecular extinction coefficient to this wavelength, corresponding to the formation of complex I of catalase methyl hydrogen peroxide, is 200 cm. -I mS/[. -~. The reason that this type of experiment is successful with catalase, but not with peroxidase, is that the catalase complex I does not react with the respiratory chain, whereas the peroxidase complex II does. Thus, no oxidation of cytochrome occurs on addition of methyl hydrogen peroxide to anaerobic cells containing only catalase. This observation has also been verified with liver mitochondria. In liver mitochondria special problems in the estimation of catalase arise owing to the presence of a hydrogen donor for catalase complex I in the mitochondria. An easy method for avoiding the difficulties due to the hydrogen donor is to allow the mitochondria to stand overnight at 4 °, or at room temperature for a few hours, so that the mitochondria will lose their endogenous substrate and their capability for oxidative phosphorylation. Addition of methyl hydrogen peroxide to the aerobic mitochondria in these conditions gives the characteristic change of absorption due to the formation of catalase complex I. No evidence has been obtained for the existence of measurable amounts of peroxidase complex II in liver mitochondria although a liver peroxidase has been described. 54" Cytochrome bs. A solution of D P N H has been found extremely useful 5s B. Chance, Acta Chem. Scand. It 236 (1947). 5~ B. Chance~ Science 120~ 767 (1954). a~ M. J. Hunter, Vol. II [141], p. 791.

[12]

TECHNIQUES

FOR

ASSAY

OF

RESPIRATORY

ENZYMES

295

in testing for the presence of c t y o c h r o m e b5 in crude h o m o g e n a t e s or in partially purified mitochondria. 43 T h e utility of D P N H for this purpose rests on the fact t h a t intact m i t o c h o n d r i a are i m p e r m e a b l e to a solution of D P N H and therefore the intramitochondrial respiratory chain will not be activated. On the other hand, microsomes in the suspension or a t t a c h e d to the m i t o c h o n d r i a are accessible to a solution of D P N H . In addition, cytochrome b5 is not associated with an oxidase of appreciable activity. Consequently, addition of a solution of D P N H causes

Aerobic cells plus I.

-to.o~

~ R e .it. . ~ , _

~

~,duced C~/e°C0~~ie~

C

E ~e"

C

o

0

-0.020

0

0

570

590

410

450 (rap)

450

Fro. 14. An estimation of the catalase content of intact bacterial cells. The spectrum, obtained on addition of a hydrogen donor for catalase complex I (HCOOH), represents the difference between cells containing catalase complex I and cells substantially free of catalase complex I. For comparison, the spectrum representing the difference between the anaerobic bacterial cells and the aerobic bacterial ceils is included. The bacteria used are Micrococcus lysodeikticus. (Courtesy of Johns Hopkins Press.) a nearly complete reduction of cytochrome b5 and does not appreciably affect the other respiratory pigments. I n this case, the optical density change measured at 426 and 405 m~ is divided b y 160 cm. -1 raM. -1 in order to give the concentration of cytochrome b~. I t should be pointed out t h a t in the dithionite t r e a t m e n t described a b o v e c y t o c h r o m e bs, as well as m i t o c h r o m e and other pigments, will be reduced. If one wishes to differentiate between c y t o c h r o m e b5 and mitochrome and other pigments, it is preferable to m a k e the particle s y s t e m anaerobic with a natural substrate, and then to add D P N H in order to reduce c y t o c h r o m e bs, and then finally to add dithionite to reduce mitochrome and other pigments. An example of this is indicated in Fig. 15.

[12]

TECHNIQUES

FOR ASSAY OF RESPIRATORY

297

ENZYMES

contributes at this wavelength, but its magnitude is considerably less t h a n t h a t of cytochrome a3, as is indicated by the extent to which this band disappears on the addition of carbon monoxide. The Soret bands of cytochromes b and c are seen in the region of 420 to 430 m~. The fact t h a t t h e y tend to merge makes the detection of the Soret band of cytochrome c rather difficult. These bands merge because the trough of the cytochrome b band diminishes the peak of the cytochrome c band. In

-~20] I

Reduced pyridine nucfeotides

":+.,oi /

c

~

I//

' +O8 C~

\*-.

h Anaerobic

b~. Pl~xAnoerobicceils

~Anaerobicb,cells w~'= o(+o f ~1~ ,~,

0

o -,10 Storved Aerobic cells

5O0

350

400

:~(my)

.-..

'+,06 IE u ' ~.04

,~02 E (D -0

--

'-.02

"

•; 0 4

_

,-.06 ~. 0 65O

FIG. 16. The absorption bands of the respiratory components of baker's yeast cells. The spectrum represents the difference between ethanol-reduced anaerobic yeast cells and aerobic starved yeast cells. The absorption bands due to cytochromes, flavoprotein, and reduced pyridine nucleotide are labeled in the figure. Note that the yeast suspension is 2.9 times as dilute in the region of 300 to 500 m~ as in the region of 520 to 660 mg and that the left-hand scale of ordinates applies only to the region 300370 mg, (Courtesy of Johns Hopkins Press.) addition, the relatively smaller size of the ~-band of cytochrome c, compared to t h a t of cytochrome b, makes the former band less distinct. If hemoglobin is present in the cell, the reduced form will absorb in this wavelength region and will accentuate the absorption t h a t would otherwise be a t t r i b u t e d to cytochrome b. This error is easily avoided if antimycin A t r e a t m e n t is used to estimate the a m o u n t of cytochrome b. T h e deep trough at about 405 m~ has not been used for the identification of cytochrome; it marks the wavelength region where the bands of the oxidized forms of cytochromes b and c have disappeared. The D P N H band rises smoothly from 380 m~ to a peak at slightly less t h a n 340 m~. There is no shoulder on this band, owing to a second flavoprotein band, as has been discussed in detail above. As far as we have been able to tell, there is no distinctive absorption band in the difference spectrum below 310 m#, although more sensitive techniques m a y reveal such effects. T h e ~-bands of the cytochromes which appear in the region of 310 to 320 m~ are largely obscured b y the D P N H absorption.

298

TECHNIQUES FOR METABOLIC STUDIES

[12]

Yeast Mitochondria. Mitochondria prepared from yeast according to the method of Nossal et al. 55 show the respiratory pigments of Fig. 17 in the spectrum representing the difference of optical density between the aerobic and anaerobic states. Cytochromes a and b appear to be present in the same relative concentrations observed in whole cell, but the a m o u n t of

f.02

5 fold dilution

Undiluted .~Reduced

i

E

-I-.01

0

Reduced

m

¢

O C

E -.01

~-.02 O O

c~ -.03 380

420

460

5b0500' (m,u)

540

5i0

' 660

'

FIG. 17. A difference spectrum of the respiratory components of mitochondria isolated from baker's yeast cells, studied in collaboration with Dr. P. M. Nossal. The spectrum represents the difference between a succinate-reduced anaerobic sample and an aerobic substrate-free sample. The relative values of the cytochrome components observed are somewhat different from those of the intact cell. In the mitochondria, considerable cytochrome c has been removed, as is observed by a comparison of this figure with Fig. 16. The relative content of cytochromes b and a appears to be approximately the same as in the intact cell (experiment 595-C). cytochrome c and D P N H appears to have been considerably diminished by the method of preparation. This is indicated b y the appreciable band of cytochrome cl observed at 553 mtL and the lack of a cytochrome c band at 550 mt~. At wavelengths shorter than those shown, there is no absorption attributable to D P N H . Although it is to be expected t h a t the cytoplasmic D P N H would be lost, unpublished observations on the intact cell indicate t h a t as much as one-third of the total D P N H participates directly in the oxidation-phosphorylation system and therefore considerable ab55p. M. Nossal, B. Keech, and M. F. Utter, Federation Proc. 15, 321 (1956).

[19,]

299

TECHNIQUES FOR ASSAY OF RESPIRATORY ENZYMES

sorption would h a v e been expected in the region of 340 m~ if the mitochondria had been intact (eft Fig. 16). Excised Frog Muscle. T h e s p e c t r u m representing the difference between the oxidized and the reduced states of the respiratory p i g m e n t s of a 1-mm.-thick frog's sartorius muscle is illustrated in Fig. 18. I n this case, the absorption b a n d s of cytochromes a, b, and c are clearly shown in the visible region of the spectrum. T h e trough due to flavoprotcin and the Soret bands of cytochromes a3 and c are also clearly visible. Reduced pyridine nucleotide shows a relatively large peak slightly below 340 m#.5,22,56

"1" .02-

E -= +.01

~ "

Reducedpyridine

nucleolides

\Anaerobic

a~

'~teady-state

~t co¢

~, •~,

/

o

"'x.j"

- .ol

/ k/

A

Anaerobic

c -~ stendy- stole

flavoprotein

a

.~

o~I

A

\Aerobic

-.02

0 310

350

390

430

'

4"tO

5i0

550

5~)0

630

(m,u) Fro. 18. A spectrum representing the respiratory components of an excised frog sartorious muscle. The spectrum represents the differences between anaerobic and aerobic steady states of the muscle. The cytochrome, flavoprotein, and pyridine nucleotide components are indicated in the diagram. Note that all the optical density changes on this diagram are on the same scale, and hence the pyridine nucleotide content is relatively less than that recorded for the intact yeast cells. (Courtesy of Dr. A. M. Weber.) C y t o c h r o m e b is s o m e w h a t reduced in the aerobic steady state of the muscle and hence does not show a distinctive p e a k in the difference spectrum. P r e t r e a t m e n t of the muscle with Amytal, however, will cause oxidation of c y t o c h r o m e b, and this test shows clearly t h a t this component exists in the frog's sartorius muscle in the concentrations similar to t h a t of isolated sarcosomes. Isolated Muscle Sarcosomes. Although frog's sartorii h a v e not been available in sufficient q u a n t i t y to p e r m i t the p r e p a r a t i o n of sufficient sarcosomes for spectroscopic study, some d a t a on the sarcosomes from rat s6 C. M. Connelly and B. Chance, Federation Proc. 13, 29 (1954).

300

[12]

TECHNIQUES FOR METABOLIC STUDIES

heart are presented in Fig. 193s (see also ref. 34). Here are shown clearly the absorption bands of cytochromes a, b, c, and a3, together with appreciable absorption due to flavoprotein. T h e pyridine nucleotide content of the isolated sarcosomes is relatively low, and, in fact, Holton, in his recent paper, 84 was unable to p u t forward conclusive spectroscopic evidence for the existence of the pyridine nucleotide in the sarcosomes. We h a v e found,

• "t.06

÷.12Cyto(:hrome 0 3

7 E +.08

-1-.04~~

$1ote5

E÷.04 u

*.02

c

~

-o4

-o2

3'50

390

430

470

5i0

5~0

590

650

"),(my)

Fzo. 19. The respiratory components of isolated rat heart muscle sarcosomes. The spectrum represents the difference between anaerobic and aerobic sarcosomes, both samples being supplied with a-ketoglutarate as substrate, and with phosphate and phosphate acceptor. Note the scale change at 500 m~. In these particles, the characteristic absorption band of reduced pyridine nucleotide in the region of 340 mp is obscured by the large 320 m~ peak and is clearly shown in the state 3 to 4 transition which is included in the figure (labeled state 4). This graph is 3 times the scale of the other (experiment 661-B). however, t h a t reduction of the pyridine nucleotide with a specific D P N H linked s u b s t r a t e such as # - h y d r o x y b u t y r a t e gives appreciable change of absorption, with a m a x i m u m at 340 mp and t h a t addition of A D P causes this 340 mp absorption b a n d to diminish and then to intensify as the added A D P is exhausted (state 3 to 4 transition) as shown b y the dotted trace in Fig. 19. Thus, the b r o a d ultraviolet absorption b a n d in the reduced minus oxidized s p e c t r u m of Fig. 19 has a peak at a b o u t 320 mp which obscures the 340 mp b a n d of reduced pyridine nucleotide. 35 Isolated Rat Liver Mitochondria. A r a t h e r i m p o r t a n t material on which considerable spectroscopic d a t a are available is rat liver mitochondria, e.~7 These mitochondria are isolated according to the m e t h o d of L a r d y a n d ~7B. Chance and G. R. Williams, Nature 176, 250 (1955).

302

TECHNIQUES FOR METABOLIC STUDIES

[12]

Wellman. ~s The spectrum representing the differences of absorbancy between the oxidized and reduced forms of the respirator:/pigments is given in Fig. 9. In this case the difference spectrum of the cytochromes resembles that of the whole yeast cell, there being distinctive absorption bands of all the respiratory components, with an especially strong band due to reduced pyridine nucleotide.

f -F.02. E f~ c

+.ol!

~" 0

~ -.01

~(m,u) 460

sbo

660

6s'o

0

FIG. 22. The respiratory components involved in the photosynthetic purple bacteria, Rhodospirillum rubrum. This spectrum represents the difference between aerobic and anaerobic substrate-treated bacterial cells. Absorption bands due to cytoehromes of types b and c are seen in the visible region, and no bands attributable to cytochromes of type a are seen. These cells contain a pigment very similar to the "CO-binding pigment" of the cells of Figs. 20 and 21 (see also p. 276).

Bacterial Cells. As further examples, we present two spectra of whole bacterial cells. Figure 20 shows a pigment resembling, in some ways, cytochrome cl, with a sharp band at 554 m~. This type of spectrum is exhibited by Acetobacter suboxydans. The "CO-binding pigment" is the terminal oxidase 9~and its presence is indicated clearly by CO treatment2 A rather different type of difference spectrum is that of Aerobacter aerogenes, shown in the accompanying figure (Fig. 21). In this case, there is more than one terminal respiratory enzyme; these cells contain the "CO-binding pigment" with the absorption band noted on p. 277, cytochrome al absorbing a 590 m# and cytochrome a2 absorbing at about 630 m~. In addition, there are cytochromes of types related to b and c, absorbing in the region of 550 to 560 m#, together with the characteristic Soret bands of these pigments.%~s Photosynthetic Bacteria. As a last example, we present the cytochrome components of the photosynthetic purple bacteria, Rhodosprillum ~s H. Lardy and H. Wellman, J. Biol. Chem. 195, 215 (1952).

[19.]

TECHNIQUES FOR ASSAY OF RESPIRATORY ENZYMES

303

rubrum. 59 In this case, there is no distinctive band corresponding to cytochrome of type a. Two absorption bands are recorded in the region where peaks due to cytochromes of types b and c are expected. The region of the Sorer band is dominated by a peak at approximately 428 mp which is attributable to the "CO-binding pigment" (Fig. 22). This band usually masks the Sorer bands of cytochromes of types b and c contained by this microorganism, Rhodospirllum rubrum. There is very little absorption in the ultraviolet region that can be attributed to the reduced pyridine nucleotide.

Methods for Determining the Sequence of Reactions of Components of the Respiratory Chain Given a number of components which are spectroscopically identified by the methods described above, it is important to determine the order in which they react in the chain transferring electrons from the substrate to oxygen. The components fall into two general categories, external and internal enzymes. The external enzymes react with diffusible substrates, and the internal ones react with adjacent members of the respiratory chain. 6° External Enzymes: Substrate-Inhibitor Competition. For the enzymes that act at the end of the respiratory chain, the classic methods of competitive inhibition are applicable. For example, the competition of carbon monoxide with oxygen, combined with the fact that cytochrome a3 forms a spectroscopically distinct carbon monoxide compound, identifies it as the terminal respiratory enzyme. A similar test can be used for cytochrome al and for the "CO-binding pigment." Malonate is a competitive inhibitor of succinic dehydrogenase but does not form a spectroscopically distinct compound with the dehydrogenase. Addition of malonate to the succinic oxidase system does cause spectroscopic shifts in the cytochrome components; oxidation of the steady-state levels of all members of the chain will occur as evidence of the fact that the malonate is affectiag the lowest member. Isolation of Components of the Respiratory Chain. The DPN-linked dehydrogenases provide an example of enzymes that can be isolated, and whose activity can be demonstrated independently of the respiratory chain--for example, ~-hydroxybutyric~1 dehydrogenase. Isolation of DPN-linked cytochrome c reductases 42 without alteration of their specificity and the reaction mechanism has been more difficult, and it has not been adequately demonstrated that any DPN-linked cytochrome c ~9 B. Chance and L. Smith, Nature 175, 803 (1955). 60 B. Chance a n d G. R. Williams, Advances in Enzymol. 17, 65 (1956). 6~ A. L. Lehninger, J. Biol. Chem. 178, 625 (1949).

304

TECHNIQUES FOR METABOLIC STUDIES

[12]

reductase has been isolated without some change in the nature of the electron transport mechanism. The complex reaction mechanisms proposed for the flavoprotein enzymes 42 have not been shown to apply to the respiratory chain. Cytochrome c has, of course, been isolated, s2 and it appears to have a specificity similar to that which is contained in the intact system, s~ Enzymatic activity of isolated cytochromes a, cl, and b has not been demonstrated. Internal Enzymes: Substrate-Inhibitor Method. One way of determining the reaction sequence of internal enzymes is to add substrate which causes partial reduction of all the components, and to follow this by an inhibitor that breaks the electron transport system somewhere in the middle of the chain. Those components which lie on the substrate side of the inhibitor will be more reduced, and those components lying on the oxygen side of the point of inhibition will become more oxidized. Thus, the seven components of the respiratory chain can be divided into two groups. Ideally, there should be an inhibitor for each pair of respiratory enzymes because this would allow an exact determination of the sequence of the cytochromes. There are, unfortunately, only two classes of effective inhibitors for the internal enzymes. Amytal and antimycin A are examples of these two classes. Antimycin A divides the respiratory enzymes into the two groups: s° a~

b

a c

flavoprotein DPN

el

and, in intact mitochondria, Amytal gives the groups: 8~ a3 a c

DPN el

b flavoprotein A similar site for Amytal action is found in ascites tumor cells, although in nonphosphorylating particles it appears to act between cytochrome b and flavoprotein. 84 6~ D. Keilin, Proc. Roy. Soc. B106, 418 (1930). 8~L. Smith, J. Biol. Chem. 215, 833 (1955). 84 R. Estabrook, M. Rabinowitz, a n d B. deBernard, to be published.

[12]

TECHNIQUES FOR ASSAY OF RESPIRATORY ENZYMES

305

Such studies in themselves do not give complete information on the reaction sequence and need to be correlated with data obtained by the other methods described in this section in order to establish a reaction sequence. Kinetic Methods. On the addition of a small concentration of oxygen to a reduced respiratory chain, the sequence of oxidation and reduction reactions will be in the order in which enzymes act in the chain. Thus, the oxidase will be oxidized first, the second member in the chain second, the third member third, and so forth. After the steady state has been established, and the components begin to become reduced owing to lack of oxygen, the time sequence of the decrease of the steady-state levels of the components will again be in accordance with the sequence of their action in the respiratory chain, e° It is also possible to compare the steadystate concentrations of the intermediates at various times during their decay from the steady-state values, and in each case it will be found that the decay times will form a time sequence which is in accordance with their chemical sequence. Some methods for measuring these reaction sequences in suspensions of whole cells or in particulate preparations are described.

Rapid Reaction Methods Suitable for the Study of the Sequence of Action of the Components of the Respiratory Chain Mixing Methods. Mixing of the reactants with a stirring rod can be relatively effective for the study of the kinetics of a number of the reactions involving the respiratory components, provided that a sensitive spectrophotometric technique and low temperatures are employed. The minimal concentration which can be used effectively is of the order of lO-7 M for a 1-cm. path length, and with optical path lengths of greater than 1 cm. proportionally lower concentrations are usable, although the mixing time is longer. Operating temperatures of - 5 ° or - 1 0 ° are possible for enzyme systems that are resistant to reasonable concentrations of methanol, such as the cytochrome b~ system, and 0 ° is satisfactory for mitochondria and whole cells. Mixing times of a second or less can be obtained by adding substrate as a drop on the end of a stirring rod. Stirring is rapidly accomplished by twirling a glass rod between the finger tips. With differential spectrophotometry, such stirring does not seriously upset the spectrophotometric measurement. Thus, a second-order velocity constant of l0 s M. -1 see. -1 can be measured without great difficulty and over a reasonable range of concentration of the reactants. If a temperature coefficient of a factor of 10 is assumed between - 1 0 ° and room temperature, the method would be suitable for coping with a reaction whose room-temperature velocity constant was 10 ~ M. -~ see. -~.

[12]

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velocity constants of 800 sec. -1. This reaction requires an especially rapid flow apparatus, since the kinetics of the reaction must be evaluated over a wide range of substrate concentrations in order to determine what intermediate steps exist between the reaction of oxygen with the ferrous iron of the oxidase and the formation of the oxidized state. Details of Particular Flow Apparatuses. There has recently appeared a fairly complete description of the accelerated, stopped, and regenerative flow methods. 7° It is perhaps sufficient here to state that the methods have been used for study of the time sequence of reactions in the respiratory chain without significant modifications, 8~ except with respect to the optical system. The double-beam differential spectrophotometer which has been developed for studies of slower spectroscopic changes of the components of the respiratory system has now been adapted for use with the 1-mm.-capillary observation tube of the rapidflow apparatus. In this case, the image of the entrance slit is focused on the observation tube to make an illuminated area 3 ram. long and 1 mm. wide. The center of this illuminated portion is approximately 4 mm. downstream from the mixing chamber. If the particulate preparations are suspended in a high concentration of sucrose, the medium for studying the reactions in the flow apparatus should contain an equal concentration of sucrose in both syringes so that artifacts due to dilution of the sucrose do not arise.

Spectroscopic Methods Visual Spectroscopy. Hartree 49 has recently reviewed in detail the visual spectroscopic technique which has been used in Keilin's laboratory for a number of years. 71 As Hartree points out, the essentials of the method closely follow the early work of Sorby 7~ and of 1V[acMunn73 and differs only slightly from the final instrument of MaeMunn 1 (see Fig. 24). Nevertheless, considerable practice is required to obtain optimal results with this method, and the details given in Hartree's paper should be followed carefully. The important features of this method as applied to the study of respiratory enzymes of turbid materials are as follows: (1) An extremely intense light source should be used. Hartree recommends the Pointolite tungsten arc. We have had considerable success with the carbon arc and the open zirconium arc and strongly recommend light sources of comparable intensity for observation of absorption 7°B. Chance, in "Techniques in Organic Chemistry" (Weissburger, ed.), p. 690. Interscience Publishers, New York, 1953. 71 D. Keilin and Y. L. Wang, Biochem. J. 40~ 855 (1946). 7~H. C. Sorby, Proc. Roy. Soc. 15, 433 (1867). 73 C. A. MaeMunn, "Spectrum Analysis Applied to Biology and Medicine." Longmarts Green, London, 1914.

[12]

TECHNIQUES FOR ASSAY OF RESPIRATORY ENZYMES

/

309

•

FIG. 24. Mierospectroscopic apparatus as used by Keilin and Hartree. 7~ Part a represents the mierospectroscope ocular together with its illuminated scale. Part d is the prism for admitting the comparison spectrum obtained from a double-wedge trough, which is used to determine the concentration of the material under observation. This apparatus is especially useful in the rapid survey of the nature of the respiratory enzyme for many types of cell tissues. (Reproduced from Keilin and Wang, 71 permission of The Biochemical Journal.)

[12]

TECHNIQUES FOR ASSAY OF RESPIRATORY ENZYMES

311

steady-state oxidation-reduction levels of the cytochromes, coincident with the initiation of oxidative phosphorylation. 57 The visual method served to detect peroxidase complex I, but failed in the case of catalase complex I. Photoelectric Spectrophotometry. The development of a satisfactory technique for the spectroscopic examination of turbid biological materials has occurred slowly over a number of years. The first effective use of the photoelectric technique was probably that of Haas when he recorded the speed of reduction of cytochrome c on the addition of glucose to a suspension of cyanide-inhibited yeast cells. 76 Baumberger also recorded cytochrome c reduction in yeast.77 Millikan used a photoelectric technique for the recording of the disoxygenation of myoglobin in the intact cat's muscle, n Photography was used in recording the spectra of cytochromes at various times; for example, in Warburg's laboratory, photographs were taken of the cytochromes of the bee's muscle and it is believed that these data first clearly showed the Soret bands of the cytochromes. 79 Hill used this technique in 1936. 8° Later, Chaix and Fromageot obtained some clear recordings of the reduced cytochromes of bacteria. 81 Arvonitaki and Chalazonitis developed to a high degree the photographic recording of spectra in studies of pigments of muscles and nerves, especially those of invertebrates, s~ The development of photoelectric spectroscopy of turbid solutions in this laboratory came rather naturally as a consequence of the development of the sensitive spectrophotometric methods for the study of labile intermediates in the action of catalase and peroxidase, s3 These spectrophotometric methods were sensitive enough that the turbid suspensions of cytochrome preparations or of whole cells could be diluted sufficiently so that their opacity was not a serious factor. Thus, for examplel a Keilin and Hartree heart muscle preparation that absorbs 50% of the incident light will give an optical density change corresponding to the reduction of cytochrome a3 of approximately 0.07 cm. -~ but a yeast cell suspension of the same turbidity would give an absorbancy change of only about 0.002 cm. -~. The present spectrophotometric methods are sufficiently sensitive to record such small absorbancy changes and have been used to 76 E. Haas, Naturwissenschaften 22, 207 (1934). 77 j . p. Baumberger, Cold Spring Harbor Symposium Quant. Biol. 7, 208 (1938). 7s G. A. Millikan, Proc. Roy. Soc. B123, 218 (1937). 79 O. W a r b u r g a n d E. Negelein, Biochem. Z. 233, 486 (1931). 80 R. Hill, Proc. Roy. Soc. B120, 472 (1936). 81 p. Chaix and C. Fromageot, Bull. Soc. Chem. Biol. 24, 1259 (1942). 83 A. Arvonitaki and N. Chalazonitis, Arch. Internal Physiol. 54, 441 (1947). 83 B. Chance, Nature 161, 914 (1948).

312

TECHNIQUES FOR METABOLIC STUDIES

[12]

record difference spectra corresponding to the transition of the cytochrome system 19 from aerobiosis to anaerobiosis. As soon as the appropriate conditions for observing these spectroscopic changes were thoroughly understood, it became apparent that some commercially available spectrophotometers are satisfactory for recording the major absorption bands of the cytochrome system. 84 It is not often clearly understood that, in addition to high sensitivity of the spectrophotometer, some type of "bucking o u t " of the constant part of the transmitted light is needed. This " b u c k i n g o u t " may be done (a) electrically; (b) by a cuvette containing material of similar absorbancy; (c) by a second light beam of the same wavelength but which does not pass through the sample; or (d) by a second light beam of a different wavelength that does pass through the sample. In studies of mitochondrial suspensions and certain whole cells which exhibit changes of their light-scattering properties during the course of the experiment, or during the transition from aerobiosis to anaerobiosis, it has been desirable to develop techniques which are insensitive to nonspecific changes of light absorption and responsive to the appearance or disappearance of the specific absorption bands of the cytochromes. The use of an illuminometer sphere for the elimination of nonspecific lightscattering effects has been studied in this laboratory, and Bateman has published difference spectra of E. coli by this method. 8~ The sensitivity of the method is considerably reduced by the use of an illuminometer, and the double-beam method is preferred. In the latter method, the appearance of the absorption band of the cytochrome is recorded in terms of the difference of absorption at two closely spaced wavelengths28 These wavelengths are sufficiently close together that nonspecific changes, which vary only slowly with wavelength, give very little absorbancy change. This method has been highly successful for studies of mitochondria and of whole cells and, in addition, for studies of photosynthetic processes in various microorganisms (Fig. 25). It is often desirable to scan rapidly through the spectrum representing the difference between the aerobic and anaerobic states of an organism or between two otherwise chemically differentiated states of metabolism. In such a method, one uses two samples, each of which is characteristic of the two biochemical states. The spectrophotometer then plots the difference of absorption between the two samples. In our laboratory, Yang has developed a spectrophotometer which is highly efficient for this purpose 11'85 (Fig. 26). A more recent model of this is available which 84j. B. Bateman and G. W. Monk, Science 121, 441 (1955). 85C. C. Yang, Rev. Sci. Instr. 25, 807 (1954).

[12]

TECHNIQUES FOR ASSAY OF RESPIRATORY ENZYMES

313

is especially useful for measurements in extremely turbid material at very small spectral intervals. For some time, Keilin and Hartree have taken advantage of the lowtemperature sharpening and intensification of the absorption bands of

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cytochromes of types b and c. ~ Estabrook ~1 has had considerable success with the latter type of split-beam recording instrument in this study. The particular requirements are: (1) the use of very narrow spectral intervals because of the sharpness of the bands (about 0.5 m~); (2) the

314

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The Process and Instruments recorder automatically accomplishes functions similar to those of Lundeg•rdh's machine at the rate of 10 times per second and has been found suitable for recording the Sorer bands of cytochromes of intact yeast cells. The Aminco rapid scanning apparatus is unsuitable for turbid materials because the sample is placed at the entrance slit to the monochromator and the optical efficiency for such material is very low. Limitations to the Accuracy of Spectrophotometers. Commercially available spectrophotometers are usually not engineered for measurement of optical density changes of less than 0.02 with reasonable accuracy. The factors which prevent the obtaining of a higher degree of accuracy often represent limitations to more sensitive instruments, and are described here. The features which usually limit the performance of spectrophotometers for studies of this kind 88are (1) light source and stability, (2) phototube amplifier stability, (3) speed of indication and facilities for automatic recording, and (4) mechanical stability of optical and electronic components. Each one of these points will be commented on briefly. 1. Light source stability. With suitably maintained storage batteries, a very stable potential may be supplied to the lamp for brief periods. Considerable care and effort are required to ensure that the storage batteries are operating on the most efficient portion of their discharge curve, and often considerable intervals must be spent waiting until the storage batteries are either discharged to the optimal portion of their curve, or until they are recharged to this portion. There are two types of electronic circuits for stabilizing the potential applied to the lamp--those that monitor the light output from the lamp and regulate the potential applied to the lamp in response to changes of light output, and those that monitor the voltage or current of the lamp and maintain it constant. The former circuit is recommended as being a simple, straightforward control of the relevant variable. A number of simple circuits for accomplishing this have been published. 87,s8 On the other hand, there are commercially available a number of power supplies which give a reasonably constant output, but, in considering the use of these power supplies, it should be remembered that the light output of a tungsten lamp varies as the second, or higher, power of the applied potential; also, the degree of regulation of voltage to obtain the desired constancy of light output may be rather difficult to meet with commercially available stabilizers. It is often desirable to " f l o a t " a storage battery on a charging circuit so that it is maintained on the optimal portion of its discharge curve. If 87 B. Chance, Electronics 13~ 2 (1940). 88 B. Chance, Rev. Sci. Instr. 18, 601 (1947).

[19.]

TECHNIQUES

FOR ASSAY OF RESPIRATORY

ENZYMES

317

this is done, it is very important to ensure that connections are properly made to the battery so that the fluctuations of the charging circuit are not impressed directly on the lamp. It is desirable to solder the leads drawing current for the lamp directly to the battery terminals in order to minimize difficulties in this respect. Perhaps the least known and most objectionable source of fluctuation arises in tungsten lamps themselves, regardless of the constancy of the potential applied to them. ss A tungsten filament from which appreciable metal has been evaporated can be observed to have a number of cavities in it. This condition of the filament leads to generation of undesirable fluctuations, "noise," in the light. One explanation of the source of "noise" is that the temperature of the filament becomes nonuniform, and "craters" of material at considerably higher temperatures arise along the filament wire. The lifetime and intensity of such "craters" is variable, and hence fluctuations in the light intensity arise. Even the photoelectric control circuit has difficulty in coping with such fluctuations unless the light received by the photoelectric control circuit comes from an identical portion of the filament to that received by the measuring photocell. In practice, the easiest way to avoid this difficulty is to replace the lamp as soon as unusual fluctuations are observed. 2. Phototube and amplifier stability. The Cs-Sb-Ag surface has been of the greatest utility for spectrophotometry of the respiratory pigments, since it gives great sensitivity in the region of the Soret band where the emission from the tungsten lamp is decreasing. It may well be said that little of the work on the kinetics of the hemoproteins would have been possible without this surface. This surface is termed by most manufacturers S-4, but a number of variations of the surface have more or less the same properties with other " S " designations. The surface is used in most ultraviolet phototubes and has a performance exceeding that of the S-1 or Cs-O surface at wavelengths below roughly 620 m~. This S-4 surface is available in both ordinary phototubes, in photomultipliers, and iu so called "end-on" photomultipliers developed for use in scintillation counting. The choice of whether to use an ordinary phototube or an electron multiplier phototube is often a difficult one, and the factors involved are sometimes poorly understood. For measuring a small change in a large light intensity, corresponding to a primary photocurrent of between 0.1 and 1 ~a., there is no question but that the ordinary photosurface is required in order to give the most reliable results, because the electron multiplier systems show "fatigue" which is due to a decrease of the secondary emission ratio of the electron multiplier surfaces. This fatigue is very objectionable for most spectrophotometric measurements, since it causes a decrease of the output read-

[12]

TECHNIQUES FOR ASSAY OF R E S P I R A T O R Y ENZYMES

319

to rise from 10 to 90% of its final value and can be computed from the values of resistance and capacitance from the equation tr = 2.1RC. ~9 Correspondingly shorter times are needed for the flow apparatus, about 0.01 second being a reasonably lower limit. All these times are too short for manual registration, and some type of automatic recording is needed. For slower changes the EsterlineAngus meter is satisfactory (tr = 0.5 second), as is the slower Varian recorder, which records in rectangular coordinates. For more rapid recordings, faster instruments developed for electrocardiography or electroencephalography may be used (Grass, Brush, or Sanborn recorders), but we prefer the 12-cm. photographic oscillograph (General Electric, Hathaway, etc.) used simultaneously with a slower recorder. This is because we desire simultaneous recordings of slow and fast phases of enzyme reaction kinetics on appropriate time scales. 4. Mechanical stability of the optical and electronic components. Vibrations due to mixing of reagents or discharge of reactants with the flow apparatus must not affect the equipment. Some amplifier tubes, particularly the electrometer tube and phototube used in the past in the Beckman instrument, show large fluctuations in output in response to mechanical shock. Phototube Type 929 shows very little of this effect, as is the case with electron multiplier phototubes. Commercially Available Spectrophotometers. Holton 34 has recently published spectrophotometric observations on cytochrome components of heart muscle sarcosomes in which he has used the Hilger Uvispek. The error of his measurements is clearly indicated on each one of his graphs and corresponds to 0.01 cm. -l. Thus, this instrument is satisfactory for studies of the relatively clear suspension of heart sarcosomes. Since the Uvispek apparently contains a more stable electronic circuit than the Beckman spectrophotometer, its circuits need not be altered for such studies. Modifications of the Beckman Spectrophotometer. We 6s have published modifications that render the Beckman spectrophotometer suitable for the measurement of small changes in absorbancy. Holton's studies underline the need for such modifications; the standard instrument requires spectral intervals of about 10 m~ when used for studies of sarcosomes. ~4 These circuits s8 may be followed but have definite limitations. Two limitations of the basic electronic circuit of the Beckman spectrophotometer are microphonics and drift. Thus, vigorous mixing procedures are difficult to employ and the drift rate limits the sensitivity to values insufficient for some studies. 89 A m o r e d e t a i l e d d e r i v a t i o n i n d i c a t e s t h a t t h e f o r m J. M. Olson, D i s s e r t a t i o n , U n i v . of P e n n a . , 1957.

tr = 5 R C

is m o r e a c c u r a t e .

320

TECHNIQUES FOR METABOLIC STUDIES

[12]

Other Spectrophotometers. A spectrophotometer with excellent stability and sensitivity is the Eppendorf. Unfortunately, it operates only at wavelengths corresponding to the bright lines of the mercury arc. Other spectrophotometers observed have not had adequate sensitivity for studies of the changes that occur in correspondence to the oxidation and reduction of cytochromes. Double-Beam Spectrophotometer. For any detailed study of the nature of the respiratory chain, the double-beam spectrophotometer, developed especially for this purpose, is very nearly essential ~8 (Fig. 27). This spectrophotometer employs two light beams set at wavelengths appropriate to the peak and trough of the absorption band to be measured. For example, for the measurement of the reduction of cytochrome c, one wavelength is set at 550 m~ and the other at 540 m~. The instrument records the difference in optical density caused by the appearance of the absorption band of reduced cytochrome c because 550 m~ is near the peak in the difference spectrum and 540 m~ is near an isobestic point. There are a number of reasons for using the two beams. First, the light intensity fluctuations are minimized; two monochromators are illuminated from the same light source, and if the light increases for one light beam, it increases very nearly to the same extent on the other beam. If the wavelengths are the same, then of course the light intensity changes are equal, and there will be no change in the output of the photocell with light intensity fluctuation. If, however, the wavelengths differ, then the changes of photocell output will be described by the Wein law, which indicates that no great changes will be observed for wavelength differences of 5 or 10 m~ at 500 m~. Nevertheless, the spectrophotometer is provided with a photoelectric light-regulating circuit, since this is the simplest method of supplying regulated power to the light. The second reason for using a double-beam spectrophotometer is that it minimizes artifacts due to (1) stirring of the solution, (2) nonspecific changes in the solution by decreased light scattering as a function of time or on addition of various reagents, (3) oriented flow in the observation tube caused by rod shaped microorganisms, etc. Prism or grating monochromators have been used for this technique, and they both have certain advantages. The Beckman prism monochromator has low scattered light and operates with increasing efficiency in the ultraviolet region of the spectrum. The grating monochromator gives high resolution in the visible region of the spectrum, where it is needed in order to differentiate the a-bands of the cytochromes, particuIarly at low temperatures. Thus, we would consider the high-resolution grating spectrophotometer to be essential for low-temperature spectroscopy, whereas the prism monochromator has some advantages for studies of the ultraviolet region of the spectrum. The light intensity available

[12]

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from the grating spectrophotometer is considerably greater than that from the Beckman spectrophotometer. This is a factor oi considerable importance if rapid response for studying the reaction kinetics is needed. The light of the two different wavelengths is selected by a mirror mounted on top of a 60-cps. vibrating reed (Fig. 27). This method is superior to a rotating disk. The mirror may be interposed either between the light source and the entrance slit of the monochromator, or between the exit slit and the sample. Better illumination of the monochromators is obtained if the light chopper follows the exit slits (Fig. 25). It is, however, important to have the light sharply focused on the mirror in order that the chopping action be abrupt. The cuvette is illuminated, in the case of the grating monochromator, with an image of the grating, which can be adjusted to various sizes suitable for standard sizes of cuvettes. For studies of extremely small optical density changes in mitochondrial suspensions, a l ~ - c m . 2 light beam and cuvette have been used (Fig. 27). For studies of turbid suspensions it is extremely important to place the phototube as near the sample as possible, in order to gather as much of the light scattered by the particles as possible. Thus, spectrophotometers in which the sample is placed at the entrance slit will be quite unsatisfactory for this type of study. A theoretically perfect solution for gathering light scattered by the turbid sample is to place the sample in a reflecting sphere so that light scattered in any direction will finally reach the phototube. This technique has been tried in our laboratory and elsewhere, ~ but the losses in the multiple reflections from the sphere decrease the efficiency considerably, compared to that obtained with an "end-on" photomultiplier placed close to an ordinary rectangular cuvette. It has been found to be advantageous to use a cuvette area of at least 1 cm. ~, and preferably 2 or 3 cm. ~. When such a cuvette is placed less than ~ cm. away from the photo surface, the system operates very efficiently for measurement of small changes of absorbancy in a turbid suspension. A cuvette attachment providing a long optical path suitable for clear solutions of hemoproteins is shown in Fig. 28 and this extends the range of the apparatus towards solutions as dilute as 10-8 M. The attachment of a regenerative flow apparatus to the double-beam spectrophotometer is shown in Fig. 29. As mentioned above, the phototube may be supplied either by batteries or by a regulated power supply; the output is preferably amplified by means of a "chopper amplifier" and may be recorded in a variety of ways, depending on the "speed of response desired. We prefer to use a combination of slow and fast recorders in order to have the slow recorder

[12]

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FI(~. 29. Photograph of double-beam spectrophotometer with regenerative flow apparatus attached. The elements of this figure are clarified by reference to Fig. 23, except that in the actual apparatus two syringes are used for the enzyme solutions, but these are driven simultaneously by means of a mechanical connection. All the syringes are water-jacketed. The photocell has been removed in order that the observation chamber might be visible. as a c o n t i n u o u s m o n i t o r of t h e i n t e r v a l s a t w h i c h to use t h e r a p i d recorder.

Automatic Recording Spectrophotometers. As a n a l t e r n a t i v e to recording as a f u n c t i o n of t i m e t h e c h a n g e of a b s o r b a n c y i n a single s a m p l e a t a p a i r of fixed w a v e l e n g t h s , it is possible t o record, as a f u n c t i o n of w a v e l e n g t h , t h e difference of a b s o r b a n c y b e t w e e n t w o s a m p l e s t h a t are

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TECHNIQUES FOR METABOLIC STUDIES

[12]

identical in all respects except the state of oxidation of the respiratory pigments. We have developed a wavelength scanning spectrophotometer for this purpose and have used it for several years (Fig. 30). The electronic circuits are similar to those used in the earlier model of this instrument, 11 but the optical and mechanical parts differ considerably. This wavelength recording spectrophotometer has a number of essential features that distinguish it from those commercially available. First, a high sensitivity is available; a full-scale deflection of 10 inches corresponds to 0.02 in optical density. Second, the photocell is placed very near the sample for the reasons described above. Third, rapid scanning in wavelength is highly desirable in order to record transient changes such as those produced by the formation and disappearance o f labile intermediates. For example, a scanning rate of at least 10 m~/sec, is necessary to " c a t c h " some intermediates. Thus, the specifications of a recording spectrophotometer must fulfill the requirement of sufficient stability so that the error of optical density is about 10-4 . Further, it must record rapidly enough that the region of the Soret band (460 to 380 m~) is covered in about 10 seconds (although some error of recording will be acceptable under these conditions). The spectrophotometer should also give a sensitivity of 10-4 in optical density with a sample of turbiditysuch as that over 90% or more of the light is absorbed. The spectral interval for recording under these conditions should be about 2 m~ for room-temperature recording and as little as 0.5 m~ for low-temperature recording. The actual light chopping occurs when the monochromatic light beam sweeps by the partition between the two samples so that rectangular light pulses are obtained from the sinusoidal motion of the light beam. In this case, an " e n d - o n " photomultiplier is placed about ~ cm. from the cuvette holder, and an extremely efficient light gathering is thereby obtained. The electronic circuits follow closely those used for the earlier recorder. ~ This spectrophotometer is also useful for recording the spectra of the cytochromes at the temperature of liquid air. 49 In this case, the two cuvettes are made of Lucite and are placed a few inches above liquid air that is contained in an unsilvered Dewar flask about 3 inches in inside diameter (Figs. 31 and 32). This unfortunately increases the distance between the cuvette and the photocell so that less light is gathered. Nevertheless, the performance of the apparatus in recording such spectra is wholly satisfactory. A recording spectrophotometer employing a quartz prism was described by Yang.ll This spectrophotometer is advantageous for studies in the ultraviolet region of the spectrum by virtue of the low scattered light and increasing dispersion of the quartz monochomator. The light

[12]

327

TECHNIQUES FOR ASSAY OF RESPIRATORY ENZYMES

e'~e m

d

~

o

~ ~ ~ ~r

328

TECHNIQUES FOR METABOLIC STUDIES

[12]

i n t e n s i t y available is, however, low, and fairly wide slits are necessary for observations of highly turbid materials. This recorder does not e m p l o y an " e n d - o n " photomultiplier b u t r a t h e r the small cathode 1P28 t y p e of phototube. T h e d i s a d v a n t a g e of the small cathode is p a r t l y compensated b y the use of two mirrors which increase the light-gathering ability

adjus scr~

split light bearr from :50 c.p.s. oscillating mirr

ai

Dewar flask

FIG. 32. Drawing of the liquid air attachment for the split-beam recorder. The light path in this figure runs from left to right, rather than from right to left as in the previous figure. The level of the liquid air is no higher than the base of the cuvettes, although the cuvettes may be immersed in liquid air to provide for rapid freezing. The Dewar flask is unsilvered and approximately 3 inches in diameter. Flat surfaces are not provided on the side of the Dewar flask. An air blast prevents frosting of the outside surfaces. (Courtesy of The Journal of Biological Chemistry.) of the phototube. T h e scanning rate of the a p p a r a t u s is limited to some extent b y the speed of the s e r v o m e c h a n i s m t h a t linearizes the wavelength scale, but nevertheless the s y s t e m operates satisfactorily at 6 m~/sec. I n obtaining rapid recordings, it m u s t be emphasized t h a t the t i m e constant of the recorder m u s t be short enough to follow accurately the a b s o r b a n c y curve of the sample. T h u s for recording speeds of 10 m~/see.,

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ARTIFICIAL ELECTRONACCEPTORS

329

the time constant of 0.1 or 0.2 second is desirable, and even the speed of the Leeds and Northrup Speedomax is somewhat taxed. A fundamental limitation to the speed of recording with our instruments comes from the rate at which the light is flickered from sample to sample (usually 60 times per second). Thus, a wavelength change equal to the spectral interval should occur in a time interval of no less than one-sixtieth of a second (for a 1-mu spectral interval, less than 60 mu/sec.).

[13] Use of Artificial Electron Acceptors in the Study of

Dehydrogenases B y J. H. QUASTEL

A major advance in the study of cell metabolism took place with the introduction of artificial hydrogen acceptors for the study of biological oxidations. Studies with these substances made it possible to investigate enzymatic mechanisms concerned with the oxidative breakdown of nutrients and substrates of the cell, uncomplicated by considerations of oxygen access to the cell or oxygen activation in the cell. Since the introduction of the methylene blue technique by Thunberg I for animal dehydrogenases and by Quastel and Whetham 2 for bacterial dehydrogenases, a considerable variety of hydrogen acceptors has been used, some showing advantages over the others for the special purposes of the investigations. There has been an increasing tendency in recent years to study the reduction, by various substrates, of natural hydrogen acceptors of the cell such as diphosphopyridine dinucleotide (DPN) or triphosphopyridine dinucleotide (TPN), making use of the Beckmann absorption spectroscope, these substances often being added to enzyme systems or cell suspensions. The artificial hydrogen electron acceptors have, however, certain advantages, as they may be used for the study of the kinetics of hydrogen transport to various points along the normal respiratory chain of the cell; for example, ferricyanide may be used for the study of DPN-linked systems, methylene blue and other dyestuffs for the study of flavine-linked systems, and manganese dioxide for systems involving the reduction of ferricytochrome. A noteworthy feature of dehydrogenase systems is the fact that they will often accomplish the reduction of a variety of hydrogen acceptors. 1T. Thunberg, Skand. Arch. Physiol. 40, 1 (1920). 2j. H. Quastel and M. D. Whetham, Biochem. J. 18, 519 (1924).

[13]

ARTIFICIAL ELECTRONACCEPTORS

329

the time constant of 0.1 or 0.2 second is desirable, and even the speed of the Leeds and Northrup Speedomax is somewhat taxed. A fundamental limitation to the speed of recording with our instruments comes from the rate at which the light is flickered from sample to sample (usually 60 times per second). Thus, a wavelength change equal to the spectral interval should occur in a time interval of no less than one-sixtieth of a second (for a 1-mu spectral interval, less than 60 mu/sec.).

[13] Use of Artificial Electron Acceptors in the Study of

Dehydrogenases B y J. H. QUASTEL

A major advance in the study of cell metabolism took place with the introduction of artificial hydrogen acceptors for the study of biological oxidations. Studies with these substances made it possible to investigate enzymatic mechanisms concerned with the oxidative breakdown of nutrients and substrates of the cell, uncomplicated by considerations of oxygen access to the cell or oxygen activation in the cell. Since the introduction of the methylene blue technique by Thunberg I for animal dehydrogenases and by Quastel and Whetham 2 for bacterial dehydrogenases, a considerable variety of hydrogen acceptors has been used, some showing advantages over the others for the special purposes of the investigations. There has been an increasing tendency in recent years to study the reduction, by various substrates, of natural hydrogen acceptors of the cell such as diphosphopyridine dinucleotide (DPN) or triphosphopyridine dinucleotide (TPN), making use of the Beckmann absorption spectroscope, these substances often being added to enzyme systems or cell suspensions. The artificial hydrogen electron acceptors have, however, certain advantages, as they may be used for the study of the kinetics of hydrogen transport to various points along the normal respiratory chain of the cell; for example, ferricyanide may be used for the study of DPN-linked systems, methylene blue and other dyestuffs for the study of flavine-linked systems, and manganese dioxide for systems involving the reduction of ferricytochrome. A noteworthy feature of dehydrogenase systems is the fact that they will often accomplish the reduction of a variety of hydrogen acceptors. 1T. Thunberg, Skand. Arch. Physiol. 40, 1 (1920). 2j. H. Quastel and M. D. Whetham, Biochem. J. 18, 519 (1924).

330

TECHNIQUES FOR METABOLIC STUDIES

[13]

For example, xanthine oxidase ~ will bring about the reduction of hydrogen peroxide, nitrate, iodine, dinitrobenzene, picric acid, quinone, alloxan, guaiaeum blue, methylene blue, and a series of reduction potential indicators. Liver isocitric dehydrogenase 4 reduces methylene blue and dinitrobenzene but not nitrates. Potato aldehyde dehydrogenase ~reduces nitrate, dinitrobenzene, quinone, methylene blue, and many reduction potential indicators. The differences between the various dehydrogenase systems, so far as reduction of artificial electron acceptors is concerned, depend largely (1) on the chemical types of enzymatic and nonenzymatic components of the dehydrogenase systems, (2) on the magnitude of the redox potential of the reduction indicators used, and (3) on the accessibility of the hydrogen acceptor to the site of dehydrogenase activity.

Methylene Blue and Redox Indicators Methylene blue has been used extensively for the investigation of muscle dehydrogenases, 1 for the study of bacterial enzyme reactions, ~,e for the study of carrier-linked reactions, 7 and for a host of other reactions involving dehydrogenase activities. A convenient method of investigation of suspensions of resting bacterial cells has been to add to such a suspension of washed cells in 2 ml. of an appropriate sodium phosphate buffer (pH 7.4) solution in a Thunberg tube, 1 ml. of 1/5000 solution of methylene blue, a solution of the substrate under investigation, and a volume of boiled saline and distilled water to give a final volume of 7 ml. The tube is then exhausted at the pump, and the rate of reduction of the methylene blue i n v a c u o at 37 ° or 45 ° is estimated. It is a common practice to use a Thunberg tube with a hollow stopper into which the substrate is placed during the evacuation of the tube at the pump. When the air has been removed, the contents of the tubes are tipped into the suspension of cells, or the enzyme solution, containing the methylene blue and the rate of reduction of dye is measured. The practice is also varied by placing the dye in the hollow stopper and by tipping the dye solution i n v a c u o into the substrate-enzyme solution or suspension. The rate of reduction of dye is usually estimated visually by deterruination of the times taken to attain standard depths of color. Spectrophotometric methods s are also used, however--e.g., assay 9 of choline or s M. Dixon, Biochem. J. 20, 703 (1926) 4F. Bernheim, Biochem. J. 22, 1178 (1928). 5F. Bernheim, Biochem. J. 22, 344 (1928). s j. H. Quastel and M. D. Whetham, Biochem. J. 19, 520, 645 (1925). D. E. Green, L. H. Strickland, and H. L. A. Tarr, Biochem. J. 28~ 1812 (1934). 8j. S. Fruton and H. T. Clarke, J. Biol. Chem. 106, 667 (1934). 9J. N. Williams, J. Biol. Chem. 206, 191 (1954).

[13]

ARTIFICIAL ELECTRON ACCEPTORS

331

betaine aldehyde dehydrogenase by estimating rate of reduction of 2,6-dichlorophenolindophenol (2.3 mg. %). The reduction of the dye may be also estimated electrometrically by measurement of the potential at an electrode.l° It is important to note that the value of the equilibrium constant of the succinic-fumarie equilibrium, first established with resting bacteria,2 using methylene blue as indicator, is the same as that found with horse skeletal muscle and beef heart and diaphragm and equal to that expected on thermodynamic grounds.~X Such results indicate that the dye system acts as a measuring system which is thermodynamically reversible. Nevertheless , the utility of dyestuffs as hydrogen acceptors is marred by the fact that they may exert toxic effects on enzyme systems or that they may lack the ability to combine in an appropriate manner at the enzyme center so that reduction will occur. It is known ~2,~ that, whereas basic dyestuffs may be easily reduced by enzyme systems, acid dyestuffs of similar redox potential may only be reduced very slowly. This is due to the fact that the acid dyestuffs are not so accessible to, or so easily adsorbed by, the active centers of the enzymes involved as the basic dyes. The absorption of basic dyes may be so large, indeed, that irreversible inactivation of the enzymes may take place. ~4 This has been noted with hydrogen acceptors such as methylene blue and toluidine blue. The acid dyestuffs investigated have little or no inhibitory action on the dehydrogenases, a fact to be correlated with their low speeds of reduction.14

Ferricyanide The use of ferricyanide as an artificial electron aeceptor depends on the fact that when 1 mole of ferricyanide is reduced 1 mole of acid is formed, and in bicarbonate media this gives rise to 1 mole of CO2 which can be estimated. The relevant equations are: H -V Fe(CN)'8" --* H + + Fe(CN)~s''' H + + H C O ' 3 = H2CO~ = C O 2 + H 2 0 This method has been used for the manometric estimation of glutathione and D P N H , both of which are readily oxidized (nonenzymatically) by ferrieyanide. 15 10 j . Lehmann, Skand. Arch. Physiol. 58, 173 (1929). 11 H. Borsook, Ergeb. Enzymforsch. 4, 1 (1935). 12 j. H. Quastel and W. R. Wooldridge, Biochem. J. 2l, 166 (1927). ~ M. Dixon, Biochem. J. 20, 714 (1926). 14j. H. Quastel and A. H. M. Wheatley, Biochem. J. 25, 629 (1931). ~5E. Haas, Biochem. Z. 291, 79 (1937).

332

TECHNIQUES FOR METABOLIC STUDIES

[13]

Ferricyanide was introduced as a terminal hydrogen acceptor for the investigation of dehydrogenase systems by Quastel and Wheatley. I~ It is a substance with relatively little toxicity, and it has been shown T M that its presence at a concentration of 0.01 M has but little effect on anaerobic glycolysis and respiration of tumor tissue, though aerobic glycolysis is suppressed. Ferrocyanide is also relatively nontoxic at similar concentrations and has the advantage over most reduced dyestuffs of being nonautoxidizable. Experiments with rat liver slices have indicated that liver cells are more freely permeable to ferricyanide than to methylene blue or cytochrome c.19 The technique for the use of ferricyanide as artificial electron acceptor consists in placing within the main compartment of a Warburg manometric cup either tissue slices immersed in, or tissue extracts mixed with, a saline-NaHCOa (0.025 M) medium and placing in the side tube 0.2 ml. of a ferricyanide-NaHCO3 solution. The latter is made up by mixing 5 ml. of 10% Na3Fe(CN)6 with 1 ml. of 0.16 M NaHCO~. The vessel is then gassed with 95% N2 and 5% CO2, and after thermal equilibrium has been established the ferricyanide is tipped from the side tube into the main vessel and the velocity of gas output is followed. The manometric determination of the rate of reduction of ferricyanide may be roughly checked by estimating the resulting ferrocyanide colorimetrically, using the sensitive color reaction between ferrocyanide, acetic acid, and ammonium molybdate. ~ The fact that ferricyanide oxidizes D P N H nonenzymatically makes it possible to use ferricyanide for the study of DPN-linked dehydrogenases in systems which lack flavines and other carriers. With this method, the presence of lactic and malic dehydrogenases in red blood cells was detected, for example. 16 Ferricyanide has been introduced 2°,21 as an artificial electron acceptor in the study of the release of oxygen by illuminated isolated chloroplasts (Hill reaction). Hill 22 used potassium ferric oxalate as oxidant. Quinone ~° has also been used. Spikes 2~ used ferrieyanide, as it acts reversibly at the electrode and does not appreciably inhibit the Hill reaction. He estimated the reduction of ferricyanide potentiometrically. The rate of oxygen evolution was found to decrease as the initial ferrieyanide concentration 16 j. H. Quastel and A. H. M. Wheatley, Biochem. J. 32, 936 (1938). 17 B. Mendel and F. Strelitz, Nature 140, 771 (1937). is B. Mendel, Am. J. Cancer 80, 549 (1937). 19 R. M. ttoehster and J. H. Quastel, Arch. Biochem. and Biophys. 86, 132 (1952). ~0 D. I. Arnon and F. R. Whatley, Arch. Biochem. 25, 141.(1949). ~ J. D. Spikes, Arch. Biochem. and Biophys. 36, 101 (1952); see also Vol. IV [15]. 22 R. Hill, Proc. Roy. Soc. B127, 192 (1939).

[13]

A R T I F I C I A L ELECTRON ACCEPTORS

333

was increased. A concentration of 0.005 M ferricyanide was found to be most convenient for work in the Hill reaction. The over-all reaction was as follows: 4K3Fe(CN)6 + 4K + + 2H~O ~ 4KaFe(CN)~ + 4H + + O~

Manganese Dioxide Manganese dioxide has been introduced as an artificial electron acceptor, 19 as it possesses certain advantages over the well-known soluble electron acceptors. By means of the conventional Warburg manometric technique, experiments with MnO2 are carried out in an atmosphere of 93 % N2 + 7 % COs in 0.025 M N a H C Q solution. The following equations show the reactions involved, RH2 representing the substrate oxidized: RH2 + MnO~. = Mn(OH)., + R Mn(OH)2 + CO~ = MnC03 + H~O Thus, the principle of the method depends on the fact that the velocity of CO2 uptake is a measure of the rate of reduction of MnO2 by RH2. In practice, a suspension of MnO2 is added to the tissue or tissue extract in the main compartment of the Warburg vessel, and the solution of the substrate is tipped in the main compartment after thermal equilibrium. This procedure ensures oxidation of substances in the tissue, or tissue extracts, that may react with MnO2 nonenzymatically before the substrate is tipped in from the side tube. MnO2 appears to have no toxicity toward enzyme systems so far investigated. The MnO2 must be prepared from chemically pure materials. One liter of a solution of 0.1 M manganese sulfate is slowly poured into an equal volume of 0.1 M potassium permanganate solution which is stirred continuously. After the reaction is complete (about 45 minutes), the dark-brown precipitate is centrifuged and the supernatant discarded. The precipitate is washed as follows: four times with distilled water (to remove excess KMnO4), once with 150 ml. of 0.3 M sodium bicarbonate solution, and once with 150 ml. of 0.12 M sodium bicarbonate solution, the precipitate being centrifuged after each washing. It is then washed twice with distilled water and the second washing checked for pH which should not be less than 7.0. The precipitate is then given two thorough washings with acetone and filtered on the Biichner funnel where it is dried for 1 hour at room temperature. For experiment, 0.5 g. of MnO~

334

TECHNIQUES FOR METABOLIC STUDIES

[13]

powder is ground vigorously in a mortar with 2.5 ml. of distilled water, transferred to a test tube, and gassed with 93 % N2 -k 7 % CO2 just prior to use. The suspension is easily pipetted, 0.2 ml. being used per Warburg vessel. MnO2 may be used as an artificial electron acceptor to reduced cytochrome e, thus replacing cytochrome oxidase, z9 It is particularly useful in systems in which dehydrogenase activities have been supplemented by the addition of methylene blue or ferricyanide, only small quantities of these substances (e.g., 0.00008 M methylene blue or 0.001 M ferricyanide) being present as carriers. This is due to the fact that the reduced forms of these substances are quickly oxidized by MnO~ to the corresponding oxidized forms. The MnO2 technique allows the use of very small, nontoxic concentrations of artificial carriers in the respiratory systems. This technique has also been found useful in the study of dehydrogenase systems, in which the change in substrate concentration has to be followed spectrophotometrically. For example, in a study of anaerobic cholic acid oxidation, 23 terminal hydrogen acceptors other than MnO~ interfered with the spectrophotometric estimation of the product of cholic acid oxidation. In such experiments small quantities of a carrier (such as methylene blue) were added to the enzyme system, MnO2 being added as terminal electron acceptor. After centrifugation, a clear supernatant was obtained which was used for spectrophotometry. The relative insolubility of MnO2 makes it useful for the study of the permeability of the intact cell to hydrogen carriers. 19 MnO2 appears not to oxidize DPN-linked or flavine-linked dehydrogenase systems in the absence of carriers. Tetrazolium Tetrazolium salts represent a class of compounds which are being used in increasing amount owing to their high aqueous solubility and to the fact that the normally colorless or pale-colored salts have the property of being easily changed into highly colored, water-insoluble formazans on reduction. They have a low redox potential in the neighborhood of - 0 . 0 8 volt, ~4 making them useful as reduction indicators in biological systems. They seem to be of particular value in histochemical studies, as the formazan is deposited in close proximity to the site of its formation. 25 ~ A. H. Halperin, J. H. ,Quastel, and P. G. Scholefield,Arch. Biochem. and Biophys. 52, 5 (1954). 24D. Jerchel and W. Mohle, Ber. 77B, 591 (1944). 2~F. E. Smith, Science 113, 751 (1951).

[13]

ARTIFICIAL ELECTRON ACCEPTORS

335

Observations 26-29 may, therefore, be made both of the location of enzymatic reduction as well as its intensity. In a typical experiment, 29 the basic incubation medium for tissue slices consists of 3 ml. of 1% aqueous solution of 2,3,5-triphenyltetrazolium chloride plus 1 ml. of 0.9% NaC1 buffered to p H 7.2. This medium is used for the study of endogenous dehydrogenous activities, the saline being replaced b y substrate, etc. Histochemical and quantitative comparisons are made between the endogenous activity and t h a t observed in the presence of substrates. In other studies, 3° slices 1 mm. thick are placed in ice-cold tetrazolium-saline solution and incubated anaerobically or aerobically for 1 to 2 hours at 37 °. Tissues are fixed in 10% neutral formalin, sectioned at 15 to 20 ~ with a freezing microtome, mounted in glycerin, and examined. I t was found, for example, t h a t neotetrazolium penetrated adrenal slices poorly, whereas triphenyltetrazolium penet r a t e d well and showed uniform reduction t h r o u g h o u t the cortex. The formazan was deposited intracellularly, particularly in the lipid droplets. I t seems t h a t the tetrazolium salts are useful indicators for the study of changes in tissue metabolism during changes in functional state. '~° Tetrazolium salts are not without toxicity, neotetrazolium having lethal effects on influenza and other viruses. 3~ According to Antopol e! al., 32 fibroblast cultures reduce neotetrazolium in the presence of mammse, glucose, a-glycerophosphate ( + D P N ) , succinate, and lactate ( ~ - D P N ) . Brodie and Gots 33found in an enzymatic reducing system consisting of triosephosphate dehydrogenase, D P N , and a flavoprotein t h a t the flavine is the immediate electron donor to 2,3,5t r i p h e n y l t e t r a z o l i u m chloride. According to Rosenfeld 34 there is close parallelism between the intensity of formazan deposition in tissues under anaerobic conditions and their oxygen consumption. The formazan may be quantitatively extracted from weighed dried tissue by solvents such as ethyl acetate and accurately assayed in the spectrophotometer. Studies of perfused adrenal glands by solutions of 2,3,5-triphenyltetrazolium chloride containing various substrates showed that glucose, 2~ M. M. Black and I. S. Kleiaer, Science 110, 660 (1949). 27 M. M. Black, S. R. Opler, and F. D. Speer, Am. J. Pathol. 26, 1097 (1950). 28 M. M. Black, B. W. Zweifach, and F. D. Speer, Am. J. Clin. Pathol. 23, 332 (1953). 23 M. M. Black, Trans. N. Y. Acad. Sci, [21 17, 398 (1955). 30E. Kivy-Rosenberg, J. Casetrano, and B. W. Zweifach, Trans. N. Y. Acad. Sci. [21 17, 402 (1955). 31H. Kodza and W. Antopol, Trans. N. Y. Acad. Sci. [2] 17, 389 (1955). 3~W. Antopol, G. H. Fried, and H. Kodza, Trans. N. Y. Acad. Sci. [2] 17, 385 (1955). 3~A. F. Brodie and G. S. Gots, Science 114, 40 (1951). 34G. Rosenfeld, Arch. Biochen. and Biophys. 62, 125 (1956).

336

TECHNIQUES FOR METABOLIC STUDIES

[14]

hexosephosphates, and members of the citric acid cycle bring about enhanced rates of formation of formazan in the tissue, whereas the amino acids and fatty acids studies had but little effect.

[14] S t u d y of Soil M e t a b o l i s m w i t h the Perfusion T e c h n i q u e

By

J. I-~. QUASTEL and P. G. SCHOLEFIELD

It has been found essential, for an accurate study of the metabolic events taking place in soil, to have an apparatus that will secure standardization of soil conditions and hence reproducibility of results. Many observations have shown how difficult it is to secure such reproducibility even with the most careful control of soil conditions. There are difficulties due to the heterogeneity of the soil, to variations of the water content within the soil, to variations of rates of oxygen penetration to various parts of the soil, even to alteration of conditions in the soil due to its handling for analytical purposes. A fresh method of approach was made by Lees and Quastel, i using an apparatus in which a column of soil, in the form of sieved air-dried crumbs, is perfused with oxygenated, or aerated, fluid by a circulatory technique. This enables the same soil solution or perfusate to percolate through the soil for an indefinite period. The fluid contains the substance whose transformation in soil is being studied. The underlying principle is the treatment of soil as a biological whole and the perfusion of fluid through it as in the preparations of intact isolated organs familiar to the physiologist. The soil perfusate is adequately aerated, and the perfusion is intermittent so that waterlogging of the soil does not occur. The apparatus used at present is a modification of that used by Lees and Quastel, as the latter suffers from the defect that it is somewhat complicated and involves the use of a continuous stream of water to produce the intermittent perfusion. The modification, devised by Audus, 2 is shown in Fig. 1. The soil, 30 g. of air-dried sieved crumbs (2 to 4 mm. in diameter), is contained in a glass tube between pads of glass wool. The perfusing solution is in the separatory funnel, F. A constant small suction is applied at A. This suction is transmitted back through the lengths of thermometer tubing, R1 and R2 (or other suitable resistances), and the soil column in P 1H. Lees and J. H. Quastel, Chemistry & Industry 26, 238 (1944); Biochem. J. 40, 803, 815, 824 (1946). L. J. Audus, Nature 158, 419 (1946).

336

TECHNIQUES FOR METABOLIC STUDIES

[14]

hexosephosphates, and members of the citric acid cycle bring about enhanced rates of formation of formazan in the tissue, whereas the amino acids and fatty acids studies had but little effect.

[14] S t u d y of Soil M e t a b o l i s m w i t h the Perfusion T e c h n i q u e

By

J. I-~. QUASTEL and P. G. SCHOLEFIELD

It has been found essential, for an accurate study of the metabolic events taking place in soil, to have an apparatus that will secure standardization of soil conditions and hence reproducibility of results. Many observations have shown how difficult it is to secure such reproducibility even with the most careful control of soil conditions. There are difficulties due to the heterogeneity of the soil, to variations of the water content within the soil, to variations of rates of oxygen penetration to various parts of the soil, even to alteration of conditions in the soil due to its handling for analytical purposes. A fresh method of approach was made by Lees and Quastel, i using an apparatus in which a column of soil, in the form of sieved air-dried crumbs, is perfused with oxygenated, or aerated, fluid by a circulatory technique. This enables the same soil solution or perfusate to percolate through the soil for an indefinite period. The fluid contains the substance whose transformation in soil is being studied. The underlying principle is the treatment of soil as a biological whole and the perfusion of fluid through it as in the preparations of intact isolated organs familiar to the physiologist. The soil perfusate is adequately aerated, and the perfusion is intermittent so that waterlogging of the soil does not occur. The apparatus used at present is a modification of that used by Lees and Quastel, as the latter suffers from the defect that it is somewhat complicated and involves the use of a continuous stream of water to produce the intermittent perfusion. The modification, devised by Audus, 2 is shown in Fig. 1. The soil, 30 g. of air-dried sieved crumbs (2 to 4 mm. in diameter), is contained in a glass tube between pads of glass wool. The perfusing solution is in the separatory funnel, F. A constant small suction is applied at A. This suction is transmitted back through the lengths of thermometer tubing, R1 and R2 (or other suitable resistances), and the soil column in P 1H. Lees and J. H. Quastel, Chemistry & Industry 26, 238 (1944); Biochem. J. 40, 803, 815, 824 (1946). L. J. Audus, Nature 158, 419 (1946).

[14]

STUDY OF SOIL METABOLISM WITH PERFUSION TECHNIQUE

337

to the perfusing solution in tube T. This causes air to be drawn in at the bottom of the side tube, S, thereby detaching a column of solution which is drawn up tube T and falls on the top of the soil column. This discharge releases the partial vacuum, and the solution again rises above the base of S until it reaches the level of the solution in F. More air is then drawn

A'-

FIG. 1. Soil perfusion apparatus (Audus modification).

in, through S, and the whole process is repeated. The liquid discharged from tube T perfuses through the column of soil, which thus becomes saturated with fluid and is intermittently aerated. The apparatus may be insulated against aerial organisms by a cotton plug, but this has not been found to be necessary. It may be linked with any source of gas (oxygen, nitrogen, helium, carbon dioxide, or mixtures of these gases) by connecting with an appropriate gas cylinder. For analysis the suction is

338

TECHNIQUES FOR METABOLIC STUDIES

[14]

stopped, and fluid is allowed to rise in S, whence aliquots are removed. After such removal the suction is restored and perfusion recommences. This goes on indefinitely. The soil is not handled except at the end of the experiment, so as not to interfere with the equilibria established in the soil. The soil may of course be removed at any time for analysis of adsorbed constituents. Experiments are run in a thermostatically controlled room (21 °) and in the dark. As the apparatus is inexpensive and easy to handle, experiments may be run in duplicate or triplicate. The authors have had a batch of 100 units in constant use. A substance whose metabolism in the soil is being investigated is dissolved in the perfusion fluid or mixed with the column of soil. The volume of perfusate is kept constant by additions of water to replace the amount lost by evaporation. This technique for investigating soil metabolism has many advantages including the following: 1. The water content of the soil is kept constant and it is homogeneously distributed in the soil throughout the experiment. 2. Maximal aeration of the soil is effected. 3. Gases entering the apparatus can be controlled, and metabolic events in any atmosphere may be studied. 4. Substances such as biological poisons or inhibitors may be added to the soil solution (or perfusate) during the course of the experiment, at any period corresponding to a known metabolic activity of the soil. 5. The soil solution can be replaced at any time by a solution of any metabolite whose transformations by a soil with known metabolic activity are the subject of study. 6. Ionic equilibria between soil and solution are quickly secured and are not further affected except in so far as the equilibria are disturbed by the products of metabolism in soil. 7. The soil is not handled in any way during the experiment, analysis being confined to the constituents of the perfusate. The soil may be examined after any arbitrary time for analysis of ions and other substances adsorbed on to the soil. 8. Gases leaving the apparatus may be analyzed for COs, etc. 9. The apparatus minimizes biological variation between one sample of soil and another and lends itself to quantitative kinetic studies which may be reproduced with considerable accuracy. The Lees and Quastel perfusion unit has been modified by Lees, 3 by Temple 4 and by Collins and Sims. 4a 8 H. Lees, J. Agr. Sci. 37, 27 (1947); Plant and Soil 1, 221 (1949). 4 K. L. Temple, Soil Sci. 71, 209 (1951). 4~ F. M. Collins and C. M. Sims, Nature 178, 1073 (1956).

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The Soil Experience has shown that the ideal soil for the perfusion apparatus is one that has been air-dried for a week at room temperature and consists of particles 2 to 4 mm. in diameter. A garden soil or agricultural soil of good structure is to be preferred. If the soil is frozen when obtained and forms a mud on thawing, then the crumbs obtained on drying usually disintegrate in the soil perfusion apparatus. The crumbs, however, may be stabilized with an inert soil conditioner, such as Krilium. If the soil is caked and dry when obtained, it is apt to form many small particles ( < 2 mm. diameter) on crumbing and sieving, which, if included, will block perfusion in the apparatus. The soil dust or particles should not be placed in the apparatus. A heavy soil of poor structure may be treated with sand or with a suitable inert soil conditioner. Many soils, slowly dried and of the right crumb size, have been used successfully in the soil perfusion apparatus for six months before sufficient deterioration occurs to disturb the perfusion process. The Perfusate In all experiments reported to date in which the soil perfusion technique has been utilized, no nutrients other than the substrate under consideration have been added. Soils do not seem to require the addition of minerals, vitamins, essential amino acids, or any accessory growth factors for metabolism to take place. For example, to obtain good proliferation of the nitrifying organisms in soil it is necessary only to perfuse ammonium chloride solution. However, sufficient experiments have not yet been carried out to establish optimal nutrient conditions in soil for the metabolism of any given substrate. The concentration of a substrate may be varied within wide limits, while ensuring good rates of metabolism. High concentrations produce only an increase in the lag period preceding proliferation, and low concentrations suffer only from the drawback that most of the substrate is oxidized or metabolized before the proliferating stage is reached. In general, a concentration of 10-~ M substrate has been found convenient. Enriched Soils When a metabolizable substrate, e.g., ammonium ions, nitrite, o: thiosulfate ions, is perfused through soil, there occurs an initial lag perio~ before metabolis~a commences, whose length varies with the type of soft concentration of substrate, etc., but which diminishes with subsequen perfusions. Eventually, after repeated perfusions, each of which is carrie

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[14]

out until the metabolite perfused has been broken down completely, a constant rate of breakdown of substrate or other metabolite occurs. With each perfusion an increased enrichment of the soil with those microorganisms that attack the substrate takes place, and finally a state of saturation of the soil occurs after which no further increase in the velocity of breakdown of the metabolite takes place. Such a soil is termed an enriched or bacterially saturated soil. With many forms of metabolism in soil, and especially those involving the autotrophs, the entire metabolism seems to occur at the surfaces of the soil particles. When the soil is removed, no further changes take place in the soil perfusate, which is usually water-clear and shows remarkably few organisms under the microscope. The organisms are adsorbed by, and probably proliferate at, the soil crumb surfaces. An enriched or saturated soil may be used experimentally for a variety of purposes. It may be used for the metabolic events under study, or it may be used directly for kinetic and stoichiometric studies. Experiments show that such an enriched soil acts in every way as though it were a preparation of resting cells of the responsible organisms. Samples of such an enriched soil may be placed in a conventional Warburg manometric apparatus, and the oxygen uptake of the soil in the presence or absence of the substrates or inhibitors may be estimated. 5,6 In this way, for example, it is easily shown that for every molecule of thiosulfate added to a suitably enriched soil four atoms of oxygen are taken up, corresponding to complete oxidation of thiosulfate to sulfate. The fact that the soil organisms are not proliferating under these conditions is shown by the absence of any inhibitory effect of a growth inhibitor such as sulfanilamide. Many other properties of the resting organisms in soil may be studied in a similar manner. Enriched soil crumbs may be used to inoculate fresh samples of soil and so diminish, or avoid, lag periods. It is known now that a soil may become saturated at the same time with at least two different sets of organisms, 7 it being apparent that specific organisms adhere to, or proliferate at, specific sites on the soil crumb surfaces. When wet "enriched" soils (e.g., those capable of oxidizing thiosulfate) are quickly dried in a current of cold air, they may retain their high oxidizing activities for several months if they are stored between 0 ° and 4 ° . However, if the soils are kept at room temperature, this rapid oxidizing power may be lost in a few days. The retention of oxidizing 5 j. H. Quastel and P. G. Scholefield, Bacteriol. Revs. 15, 1 (1951). e H. Gleen and J. H. Quastel, Appl. Microbiol. 1, 70 (1953). 7 j. H. Quastel and P. G. Scholefield, Soil Sci. 75~ 279 (1953).

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ability of stored "enriched" soils depends on the efficiency, and temperature, of drying and on the nature of the organisms involved.

Studies of Soil Metabolism by the Perfusion Technique The perfusion technique was primarily designed for the study of metabolic patterns in soils. It has the advantage over techniques involving the use of pure cultures of organisms in that it enables studies to be made of soil metabolic processes, and the factors influencing proliferation of responsible organisms, under conditions approximating as closely as possible those in the field. Investigations are confined to studies of organisms proliferating in contact with the many species of microorganisms normally present in soil. Thus, it may be shown that a compound normally without effect on an organism grown in a pure culture is highly inhibitory to the development of the organism in soil. This may be due to the compound's giving rise in soil to other substances highly inhibitory to the organism under investigation. It also happens that a substance highly inhibitory to an organism grown in pure culture has but little effect on the organisms developing in soil. This may be due to rapid decomposition of the substance in soil, or to the development of resistant or adapted strains of the organisms under study. Problems of adaptation or mutation may be conveniently investigated with the soil perfusion technique. Metabolic Studies in Soil

The following are the main metabolic studies in soil that have been carried out so far with the perfusion technique: 1. Nitrification in soil and nitrate formation from organic nitrogen compounds. 1.s.s-10 2. Effects of chlorates on soil nitrification. H 3. Effects of alkylthio compounds on soil nitrification.~2 4. Effects of urethans on soil nitrification and metabolism of urethans. ~ 5. Conversion of a-keto acid oximes to nitrites. 14 8[j. H. Quastel and P. G. Scholefield, Nature 164, 1068 (1949). 9j. H. Quastel and P. G. Scholefield, Nature 166, 239 (1950). ~0 F. E. Chase, Sci. Agr. 28, 315 (1948); F. E. Chase and G. Barker, Can. J. ¥ierobiol. 1, 45 (1954); D. J. Greenwood and It. Lees, Plant and Soil 7, 253 (1956). 11 H. Lees and J. H. Quastel, Nature 16§, 276 (1945). 12 W. T. Brown, J. H. Quastel, and P. G. Scholefield, Appl. Mierobiol. 2, 235 (1954). 13j. tI. Quastel and P. G. Seholefield, Appl. Microbiol. 1, 282 (1953). ~4j. H. Quastel, P. G. Scholefield, and J. W. Stevenson, Nature 166, 940 (1950); Biochem. J. 51, 278 (1952).

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6. Manganese metabolism.Z6 7. Thiosulfate and tetrathionate metabolism.6 8. Arsenite conversion to arsenate. 7 9. Iron metabolism. ~ 10. Breakdown of indoleacetic acid, coumarin, and herbicides such as 2,4-dichlorophenoxyacetic acid. 17.~8 11. Metabolism of bile acids in soil. 19 12. T h i o c y a n a t e oxidation in soil. 2° 16p. j. G. Mann and J. H. Quastel, Nature 1581 154 (1946). ~eH. Gleenl Nature 166, 871 (1950). 17L. J. Audus and J. H. Quastel, Nature 169, 320 (1947). 18L. J. Audus, Plant and Soil 2, 31 (1949). ~9A. H. Halperin, J. H. Quastell and P. G. Scholefield, Arch. Biochem. and Biophys. 52, 5 (1954). ~0H. Gleen, Nature 168, 117 (1951).

[15] Methods for Study of the Hill Reaction

By WOLF VISHNIAC For the purposes of this chapter the Hill reaction is defined as a lightdependent oxidation-reduction reaction, catalyzed b y chloroplasts, chloroplast fragments, or, in some instances, intact plant cells, in which (disregarding intermediate mechanisms) water is the hydrogen donor, oxygen is evolved, and some substance other than carbon dioxide (Hill reagent, oxidant) is the hydrogen acceptor. The reaction bears the name of R o b e r t Hill, who first observed the reduction of ferric oxalate b y illuminated chloroplasts. ~ The Hill reaction has been studied by following the evolution of oxygen, the change in hydrogen ion concentration, the change in oxidation-reduction potential, and the reduction of an oxidant. Application. Hill reactions are generally carried out with whole or fragmented chloroplasts. 2 However, with p-quinone a Hill reaction can be demonstrated in intact Chlorella or Scenedesmus. Assay of Chlorophyll. ~ T h e chlorophyll concentration in a suspension of chloroplasts, fragments, or intact algae can be determined b y measuring the optical density of a methanol extract. Centrifuge 1.0 ml. of a dark1R. Hill, Nature 139, 881 (1937). For the preparation of chloroplasts and chloroplast fragments, see Vol. I [5].

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6. Manganese metabolism.Z6 7. Thiosulfate and tetrathionate metabolism.6 8. Arsenite conversion to arsenate. 7 9. Iron metabolism. ~ 10. Breakdown of indoleacetic acid, coumarin, and herbicides such as 2,4-dichlorophenoxyacetic acid. 17.~8 11. Metabolism of bile acids in soil. 19 12. T h i o c y a n a t e oxidation in soil. 2° 16p. j. G. Mann and J. H. Quastel, Nature 1581 154 (1946). ~eH. Gleenl Nature 166, 871 (1950). 17L. J. Audus and J. H. Quastel, Nature 169, 320 (1947). 18L. J. Audus, Plant and Soil 2, 31 (1949). ~9A. H. Halperin, J. H. Quastell and P. G. Scholefield, Arch. Biochem. and Biophys. 52, 5 (1954). ~0H. Gleen, Nature 168, 117 (1951).

[15] Methods for Study of the Hill Reaction

By WOLF VISHNIAC For the purposes of this chapter the Hill reaction is defined as a lightdependent oxidation-reduction reaction, catalyzed b y chloroplasts, chloroplast fragments, or, in some instances, intact plant cells, in which (disregarding intermediate mechanisms) water is the hydrogen donor, oxygen is evolved, and some substance other than carbon dioxide (Hill reagent, oxidant) is the hydrogen acceptor. The reaction bears the name of R o b e r t Hill, who first observed the reduction of ferric oxalate b y illuminated chloroplasts. ~ The Hill reaction has been studied by following the evolution of oxygen, the change in hydrogen ion concentration, the change in oxidation-reduction potential, and the reduction of an oxidant. Application. Hill reactions are generally carried out with whole or fragmented chloroplasts. 2 However, with p-quinone a Hill reaction can be demonstrated in intact Chlorella or Scenedesmus. Assay of Chlorophyll. ~ T h e chlorophyll concentration in a suspension of chloroplasts, fragments, or intact algae can be determined b y measuring the optical density of a methanol extract. Centrifuge 1.0 ml. of a dark1R. Hill, Nature 139, 881 (1937). For the preparation of chloroplasts and chloroplast fragments, see Vol. I [5].

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green suspension for 20 minutes at 20,000 X g, and discard the supernat a n t fluid. Suspend the sediment in 5.0 ml. of absolute m e t h a n o l at room t e m p e r a t u r e , centrifuge, and decant the s u p e r n a t a n t m e t h a n o l extract. E x t r a c t the sediment twice more with 5.0 ml. of absolute methanol, combine the extracts, and dilute to a final volume of 20 ml. with methanol. Method of Warburg. 3 D e t e r m i n e in a 1.0-cm. cell the optical density at 578 m~ of the m e t h a n o l extract. Calculation: Optical density at 578 m~ = mg. chlorophyll per ml. 7.4 Method of Mackinney. 4 D e t e r m i n e in a 1.0-cm. cell the optical density of the m e t h a n o l extract at 665 mu (OD 665) and at 650 m~ (OD65°), the absorption m a x i m a for chlorophyll a and chlorophyll b. Calculation: 0.0338 X OD 85° - 0.0125 × OD 665 = mg. chlorophyll b per ml. 0.0165 X O D 665 - 0.0083 X OD 65° = mg. chlorophyll a per ml. 0.0255 X ODeS° -b 0.0040 X OD 66~ = mg. total chlorophyll per ml. 1.0 mg. chlorophyll per ml. = 1.11 × 10 -3 M

Manometric Techniques Evolution of 02, if of sufficient magnitude, is most easily measured in a W a r b u r g respirometer which has been fitted with a source of illumination. Figure 1 shows a few useful designs for a rectangular W a r b u r g bath. A circular model in which the reaction vessels are illuminated is commercially available (Bronwill W a r b u r g Apparatus, Model UVL). T h e intensity of illumination is usually between 5000 and 50,000 lux at the level of the vessels, b u t the particular experimental a r r a n g e m e n t will depend on individual requirements. W h e n Hill solution is used as an oxidant the light is filtered through a Corning No. 349 filter (removes wavelengths below 535 mu) to p r e v e n t photodecomposibion of the oxidant.5 T h e o p t i m u m t e m p e r a t u r e for experiments on the Hill reaction is generally 10 ° and 15 °. 30. Warburg and W. Liittgens, Biokhimiya 11, 303 (1946). 4 G. Mackinney, J. Biol. Chem. 140, 315 (1941). 5 K. A. Clendenning and P. R. Gorham, Can. J. Research C28, 78 (1950).

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a

b

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c

Fro. 1. Several designs used to illuminate Warburg vessels. (a) Cross section through a bath in which light enters the glass bottoms of the "bMeonies." Vessels are shaken directly above the glass plates. (b) Cross section, above, and longitudinal section, below, showing the position of "lumiline" bulbs sealed water tight in glass tubes. Vessels are shaken 1/~ to 1/~ inch above the lights. (c) Cross section through a Warburg bath showing a window through which light enters on the left, and a mirror inclined at 45° on the right. Vessels are shaken directly above the middle of the mirror.

Hill Solution The original oxidant used b y Hill was a mixture of ferric oxalate and ferricyanide. Ferric oxalate is the hydrogen acceptor in the reaction light H~O + 2Fe(C~O4)3 ~- ch'\roplasTs ) t o t 2Fe(e~O4)a4- -b 2H + + 1~O,

(1)

The ferrous oxalate is then reoxidized by ferricyanide: 2Fe(C204)s 4- Jr 2Fe(CN)6~---~ 2Fe(C204)33- -~ 2Fe(CN)84-

(2)

The net reaction is therefore light H20 + 2Fe(CN)83- chloroplast: 2Fe(CN)64- -}- 2tt+ + 1/~O2

(3)

Reagents 6

Oxidant. K3Fe(CN)6, 0.02 M; FeNH4(SO4)~, 0.01 M; K~C20,, 0.5 M ; K2HPO4-KH2P04 buffer, 0.02 M ; KC1, 0.0067 M ; sucrose, 0.2 M. Dissolve in 60 ml. of H~O:K3Fe(CN)6, 0.66 g,; FeNH4(SO4)2.12H20, 0.48 g.; K2C204.H~O, 9.20 g.; KH2P04, 0.27 g.; KC1, 0.05 g.; and sucrose, 6.84 g. Adjust to p H 6.5 with 2.0 N KOH, and dilute with H20 to 100 ml.

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Chloroplast preparation. Suspend chloroplasts or fragments in 0.02 M phosphate buffer, pH 6.5, containing 0.05% KC1, to make a concentration of 3.0 mg. of chlorophyll per milliliter. Procedure. Place 0.20 ml. of green suspension in the side arm of a Warburg vessel. Place 2.0 ml. of oxidant in the main compartment. Flush the vessel and manometer with N2. Equilibrate in dim light, and then start the reaction by tipping chloroplast preparation into the oxidant and turning on the light. Controls should include a vessel wrapped in tin foil (dark control), a vessel with boiled green suspension, and a vessel without oxidant but with phosphate buffer of the same pH and volume. Stoichion~etry. According to equation 3, 1 mole of 0~ should be evolved per 4 g. atoms of Fe 3+ reduced. Using limiting amounts of Fe(CN)63-, Holt and French 6 found 92 to 95% of the calculated amount of 0~ evolved. Anaerobic conditions favor quantitative evolution of O~ because some photoSxidation occurs in air.

p-Quinone Warburg and Liittgens 3 found p-quinone to serve as an oxidant in the Hill reaction. light H20 + p-quinone chloroplast->shydroquinone -b ~ 0 2

(4)

Reagents Oxidant. p-Quinone deteriorates rapidly, forming products which inhibit the Hill reaction. A quinone solution must therefore be prepared immediately before use. Commercial p-quinone is steam-distilled to remove major impurities and kept cool and dry, but not in a vacuum. Immediately before use 20 to 40 mg. of steam-distilled p-quinone is sublimed in a microsublimation apparatus (Scientific Glass Apparatus Co., No. JM-3525, or similar equipment). The sublimed crystals are dissolved in0.01 M H:SO4 to make a 1% (0.0926 M) solution, which is protected from bright light until used. Plant cells or derivatives. Chloroplasts, fragments or algal cells are suspended in 0.02 M phosphate buffer, pH 6.5, containing 0.05% KC1, to make a concentration of 0.90 mg. of chlorophyll per milliliter. A. S. Holt and C. S. French, Arch. Biochem. 9, 25 (1946).

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Procedure. Place 2.0 ml. of green suspension in the main compartment of a Warburg vessel. If algal cells are used, place 0.2 ml. of 5 N KOH in the center well to absorb C02. Place 0.10 ml. of the oxidant in the side arm. Flush the vessel and manometer with N2. Equilibrate in dim light, and then start the reaction by turning on the light and tipping the oxidant in the main compartment. Stoichiometry. According to equation 4, I mole of 02 should be evolved per 2 moles of p-quinone reduced. The author has observed 98 % of the calculated amount of 02 evolved. Quantitative evolution of 02 is favored by anaerobic conditions and by short reaction times, not exceeding 20 minutes. For this reason it is advisable to use no more than 10 micromoles of p-quinone and to employ the optimum concentration of grana. At 10,000 lux and 15° the rate of 02 evolution is directly proportional to the density of particles up to 0.90 mg. of chlorophyll per milliliter. Beyond this concentration the O3 yield declines.

Spectrophotometric Techniques The Hill reaction can be followed photometrically if it is coupled to a substance which changes its absorption spectrum in the course of the reaction. The conversion of hemoglobin into oxyhemoglobin has been used as an indicator of 03 evolution. Alternatively, the use of a colored oxidant which is reduced to a colorless compound (e.g., 2,6-dichlorophenolindophenol) affords a direct measure of the progress of the reaction.

Hemoglobin Hill 7 introduced hemoglobin as a reagent for 02 in his studies on the light-dependent evolution of 02 from cell-free leaf preparations. Originally ox muscle hemoglobin was used; later Davenport s employed hemoglobin from the perienteric fluid of Ascaris which has a higher affinity for oxygen. Although Ascaris hemoglobin is a more sensitive reagent for 02, its use is complicated by the difficulty of deoxygenating it. Application of reduced pressure alone is not adequate, and special treatment is required. Apparatus. The reaction is carried out in a Thunberg tube in which the reagents can be deoxygenated before the experiment. A single light beam (18,000 lux at the tube) serves as the energy source for the photochemical reaction and as light source for the spectroscopic measurements. Hill 9 developed an apparatus, built of a Zeiss microspectroscope and a 7 R. Hill, Proc. Roy. Soc. B127, 192 (1939); R. Hill and R. Scarisbrick, ibid. B129, 238 (1940). a H. E. Davenport, Proc. Roy. Soc. B156, 281 (1949). 9 R. Hill, Proc. Roy. Soc. B120, 472 (1936).

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Dubosq colorimeter, in which a collimated light beam passes through the Thunberg tube, which is immersed in a transparent constant-temperature bath (20°), into the aperture of the microspectroscope. The comparison prism is used to compare light transmitted through the T h u n b e r g tube to light transmitted through standard hemoglobin-oxyhemoglobin mixtures. These standard mixtures are prepared optically b y passing light through layers of hemoglobin and oxyhemoglobin solutions of varying depth. An alternative procedure is to measure the transmitted light, after selecting the proper wavelength, by the use of a p h o t o t u b e and a galvanometer or automatic recorder.

Reagents Muscle hemoglobin. Four pounds of shin beef are freed from fat, cut, rolled in 100 g. of CaCO3 (precipitated chalk), and minced. Then 700 ml. of H20 is added. The suspension is stirred interm i t t e n t l y for 10 minutes and is then squeezed rapidly in portions through a cloth bag. The strained fluid is immediately mixed with 30 g. of diatomaceous earth and filtered rapidly through a layer of diatomaceous earth on a large B~tchner funnel. The filtrate (700 ml.) is at once treated with 92 ml. of basic lead acetate, i° filtered, adjusted to pH 7.5 to 8.0 with solid Na2HPO4, and centrifuged. The supernatant fluid is then dialyzed against distilled H20 for 24 to 36 hours. At 0 ° the preparation will keep free of methemoglobin for several days. If the preparation is carried out too slowly, or if insufficient lead acetate is added, methemoglobin forms. This preparation usually contains 1.8 X 10-~ g. atom of Fe per liter. An optical density of 1.0 at 581 ms measured through a 1.0-cm. cell corresponds to 6.25 × 10-~ g. atom of Fe per liter. The solution is adjusted to 1.5 × 10-4 g. atom of Fe per liter. Ascaris hemoglobin. Ascaris lumbricoides is collected from a slaughterhouse as fresh as possible. The worms should be kept near 38 ° in a Dewar flask containing a balanced saline medium 11 until ready for use. In prolonged storage the medium should be renewed every 24 hours. The worms are slit longittidinally and the perienteric fluid drained. The fluid is dialyzed against dis~0Dissolve, with boiling, 250 g. of (CH3COO)~Pb.3H20 in 750 ml. of H20, add 175 g. of PbO, and continue to boil with occasional stirring for 30 minutes. Cool, store for 48 hours, filter, and dilute the filtrate to 1000 ml. ~i Dissolve in 1000 ml. of 0.0005 M NaH2PO4-Na2HPO~ buffer, pH 6.4: NaC1, 8.0 g.; KCI, 0.2 g.; CaCl~, 0.2 g.; MgSO4-7H20, 0.1 g. [E. Baldwin, Parasitology 35, 89 (i9 t3)].

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tilled H~O for 24 hours and the precipitate centrifuged off. By dialysis the supernatant fluid is brought to 0.53 saturation with neutral (NH4)~S04, the precipitate discarded, and the bright red supernatant fluid dialyzed against 0.73 saturated neutral (NH4)2S04. The precipitated hemoglobin is centrifuged off, dissolved in a minimum of 0.05 M phosphate buffer, pH 7.0, and dialyzed against distilled H~O. Ascaris hemoglobin can be estimated as the cyanmethemoglobin, an optical density of 1.0 at 544 m~ measured through a 1.0-cm. cell corresponds to 8.63 X 10-5 g. atom of Fe per liter. The solution is adjusted to 1.2 X 10-4 g. atom of Fe per liter. 'Leaf extract. Two grams of leaf acetone powder 1~ is stirred into 20 ml. of H20, squeezed through cloth, centrifuged, and the sediment discarded. Chloroplast preparation. Chloroplasts or fragments are suspended in a 0.02 M phosphate buffer, pH 7.4 or 7.9, as required by the procedure, to make a chlorophyll concentration of 3.0 Y 10-4 M. The suspension is freed from 02 by being subjected to reduced pressure in a Thunberg tube. A vacuum is applied and the tube shaken until vigorous frothing ceases.

Procedure with Muscle Hemoglobin. Place in a Thunberg tube (15 mm. in diameter) 3.0 ml. of oxyhemoglobin stock solution, 0.45 ml. of 0.2 M phosphate buffer, pH 7.9, containing 0.5% KC1, 0.5 ml. of green suspension, and dilute with H.20 to 4.95 ml. Place into the bulb of the Thunberg tube 0.05 ml. of Hill solution or an equivalent amount of another oxidant. Evacuate the tube with gentle shaking until less than 5 % oxyhemoglobin remains, as determined by observation at 581 m~. Mix the oxidant in the bulb with the contents of the tube, and observe the formation of oxyhemoglobin. A muscle hemoglobin solution of 9.0 × 10-~ g. atoms of Fe per liter represents 2.0 ~1. of O~ per milliliter when fully saturated at 20 ° and pH 7.9. Half-saturation corresponds to 1.2 ~1. of 02 per milliliter. If the Thunberg tube is not disturbed during the experiment there is almost no loss of 02 to the vapor phase; virtually all 02 is captured as oxyhemoglobin. Vigorous shaking will equilibrate the mixture in about 3 minutes. Procedure with Ascaris Hemoglobin. Place in a Thunberg tube (15 ram. in diameter) 3.0 ml. of oxyhemoglobin stock solution, 0.45 ml. of 0.2 M phosphate buffer, pH 7.4, containing 0.5% KC1, and 1.5 ml. of leaf extract. Evacuate with gentle shaking until vigorous frothing ceases, 15 In the preparation of acetone powder Hill used leaves of Lamium album, the European dead-nettle. Presumably leaves from other plants will also be efficaceous.

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and incubate at room temperature for 18 hours or until 80 to 90% of the hemoglobin is deoxygenated. Add 0.5 ml. of chloroplast or grana suspension by filling the side arm of the Thunberg tube with the suspension and attaching a 1.0-ml. pipet filled with the suspension. Carefully manipulate the stopcock so that the desired amount of green suspension is sucked into the Thunberg tube without admitting any air. Mix the components of the reaction mixture, illuminate, and observe the formation of oxyhemoglobin at 578 mu. The affinity of Ascaris hemoglobin for 02 is such that no measurable dissociation of the oxyhemoglobin occurs during the experiment, hence oxyhemoglobin is formed in stoichiometric amounts until all hemoglobin is saturated. No oxidant need be added to this reaction mixture, since the leaf extract required for the deoxygenation of the hemoglobin also provides the oxidant for the Hill reaction. Therefore Ascaris hemoglobin, although a desirable indicator because of its extreme sensitivity, is limited in use unless other means of deoxygenation can be found.

2,6-Dichlorophenolindophenol Holt and French 13 observed that 2,6-dichlorophenolindophenol can serve as an oxidant and developed a method TM for a rapid spectrophotometric measurement of the Hill reaction. Since O2 evolution is not measured in this procedure, it is important to rule out reactions which may reduce the dye independent of photochemical 02 evolution. Extensive washing or dialyzing of the chloroplast preparations is required to remove natural reducing agents such as ascorbic acid. No significant dye reduction must occur in the dark, and none in the light with boiled chloroplasts. Apparatus. The slow, spontaneous oxidation of 2,6-dichlorophenolindophenol permits the use of an open cuvette (1.0-cm. light path, 3.0to 5.0-ml. capacity) as a reaction vessel. A beam from a 1000-watt bull) (25,000 to 40,000 lux at the cuvette) is focused on the cuvette which is immersed in a transparent constant-temperature water bath (15°). The light passes through the reaction mixture, then through a neutral filter (Corning No. 2424), and enters a phototube connected to a Brown automatic recorder.

Reagents 2,6-Dichlorophenolindophenol. The Na salt of the dye is dissolved to make a 1.1 X 10-4 M solution in 0.04 M phosphate buffer, pH 6.5, containing 0.01% KC1. L3 A. S. Holt and C. S. French, Arch. Biochem. 19, 368 (1948). 14 A. S. Holt, R. F. Smith, a n d C. S. French, Plant Physiol. 26~ 164 (1951).

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[15]

Chloroplast preparation. Chloroplasts or fragments are suspended to a density of 1.0 mg. of chlorophyll per milliliter in 0.02 M phosphate buffer, pH 6.5, containing 0.05 % KC1.

Procedure. Mix 5.0 m]. of dye solution with 0.1 ml. of green suspension, and dilute with H~O to 10 ml. Fill two cuvettes, and to one of them add enough solid Na~S204 or ascorbic acid to reduce the dye. This reduced solution serves to obtain a zero reading on the instrument. For the experiment place the reaction vessel in the constant-temperature bath and allow 5 minutes in the dark for establishment of thermal equilibrium. To start the reaction turn on the light and observe the deflection on the recorder. The difference between the initial reading and the zero reading is proportional to the change in optical density when all the dye is reduced. Since the amount of dye is known, the recorder can be calibrated in terms of micromoles of dye reduced. Acidometric Techniques It is evident from reaction 3 that the Hill reaction can be followed by measuring the change in hydrogen ion concentration when Hill solution is used as oxidant. Other oxidants which liberate hydrogen ions when reduced can be used in similar studies.

Volumetric Titration Holt and French 6 measured the time required for known amounts of KOH to be neutralized during the Hill reaction. No significant production of acid must occur in the dark.~5 Apparatus. The reaction takes place in a 50-ml. beaker cooled to 10° by cold water circulating around it in a glass-bottomed container. The beaker contains the electrodes of a pH meter, a thermometer, a stirrer, and the tip of a 5-ml. buret graduated to 0.01 ml. The buret is jacketed and cooled to 10° by the same circulating water which cools the beaker. The beaker is illuminated from below by a 1000-watt bulb, the light from which is filtered through a Corning No. 349 orange filter 5 (to avoid photodecomposition of the Hill solution) and through 12 cm. of H~O. The light intensity at the beaker bottom is 40,000 lux.

Reagents Oxidant. The Hill solution is made up as described above, except that no KH~PO4 is added. Chloroplast preparation. Chloroplasts or fragments are suspended in 0.05 % KC1 to make a concentration of 0.5 mg. of chlorophyll per milliliter. 16 K, A, Clenderming and P. R. Gorham, Can. J. Research C28, 102 (1950).

[15]

METHODS FOR STUDY OF THE HILL REACTION

351

Procedure. In dim light mix 10 ml. of Hill solution with 1.0 ml. of green suspension, start the stirrer, wait for thermal equilibrium to become established, and rapidly adjust the mixture to pH 7.0 with a few drops of 0.1 N KOH or 0.1 N HC1. To start the reaction turn on the light, start a stopwatch, and add 0.1 ml. of 0.015 N KOH from the buret. Note the time at which the pH returns to 7.0, add another 0.1 ml. of 0.015 N KOH, and continue to take readings in this fashion. Each mole of KOH consumed corresponds to a gram atom of Fe 3+ reduced and 0.25 mole of 02 evolved.

Direct Acidity Measurement Instead of determining the acidity produced in the course of the Hill reaction by volumetric titration, the change in pH over a small range may be recorded. In a procedure similar to the one above, the rate of change of pH can be observed directly with the pH meter. Alternatively, an automatic recorder may be substituted for the galvanometer.

Potentiometric Technique The reduction of an oxidant in the Hill reaction leads to a change in the redox potential of the reaction system. This change can be measured by determining the output of a platinum and calomel electrode pair immersed in the reaction mixture. ~6 This technique is sensitive, rapid, precise, and lends itself easily to the automatic recording of results. It has been described in detail by Spikes et al. 17 Apparatus. The reaction can be carried out in a specially constructed reaction cell which embodies a pair of electrodes and a stirrer, 16,17 or in a modified Warburg vessel which has been fitted with electrodes. 17 The output of the electrodes can be fed directly into suitable pH meter (Beckman Model G)16 or through an impedance-matching circuit into a Brown automatic recorder.17 Procedure with Special Reaction Cell. The composition of the reaction mixture used in this technique is similar to that of other techniques, but the total volume is only 0.5 ml. It contains chloroplast fragments equivalent to 0.1 mg. of chlorophyll in a medium of the following composition: K~Fe(CN)6, 0.001 M; KC1, 0.01 M; sucrose, 0.17 M; and K~HPOdKH2PO4 buffer of pH 6.4, 0.05 M. Other oxidants can be used instead of K3Fe(CN)6. The illumination is red light, filtered to eliminate wavelengths below 580 m~, of 8000 lux. The reaction is started by turning on the light, and the change in potential per unit time is recorded. 16 j. D. Spikes, R. Lumry, H. Eyring, and R. E. Wayrynen, Arch. Biochem. 28, 48 (1950). 17 j. D. Spikes, R. Lumry, J. S. Rieske, and R. J. Marcus, Plant Physiol. 29, 161 (1954).

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TECHNIQUES FOR METABOLIC STUDIES

[15]

Procedure with Modified Warburg Vessel. The use of a modified Warburg vessel fitted with electrodes has obvious advantages: better mixing, better equilibration with the gas phase, and greater versatility. Thus it permits the use of different gases and makes it possible, by using a glass electrode in addition to the platinum electrode, to determine in one experiment gas changes, pH changes, and potential changes. The reaction mixture is essentially the same as in any other manometric determination of the Hill reaction, except that no buffer should be used if pH changes are to be recorded. Interpretation of Results. Potentiometric measurements of the Hill reaction are given as change in potential (in volt) per unit time. These data can be converted to amount of oxidant reduced per unit time by applying the Nernst equation. With ferricyanide as an example: E

=

Eo °

R T , [ferricyanide] -- m F [ferrocyanide]

(5)

where E is the observed absolute potential, Ec° the standard half-cell potential for the ferricyanide-ferrocyanide couple uncorrected for the calomel reference potential, F a faraday, R the gas constant, and T the absolute temperature. The fraction of oxidant reduced at any potential can be expressed as [ferrocyanide]

[ferrocyanide] + [ferricyanide]

=

1 e-Iv(E-Ec°)/RT ~- l

(6)

For a discussion on the use of this expression as applied to ferricyanide and the Hill reaction, see Spikes et al. is

Enzymatic Techniques PYRIDINE NUCLEOTIDES AS OXIDANTS Pyridine nucleotides can be reduced in the Hill reaction, but until recently this reduction had not been observed directly.18 San Pietro and Lang 18" found that chloroplast fragments (0.434 rag. chlorophyll per milliliter) incubated with DPN in substrate amounts (50 micromoles per milliliter) will in the light reduce up to 85% of the DPN. Prior to this observation the reduction of DPN and TPN was judged by following a sensitive indicator reaction the occurrence of which depended on reduced pyridine nueleotides. 19 Hendley and Conn ~° made use of the GSSGis A. H. Mehler, Arch. Biochem. and Biophys. 33~ 65 (1951). 18~ A. San Pietro and H. M. Lang, Science 124, 118 (1956). 19 W. Vishniac and S. Ochoa, J. Biol. Chem. 195, 75 (1952). ~0 D. D. Hendley and E. E. Conn, Arch. Biochem. and Biophys. 46, 454 (1953).

[15]

METHODS FOR STUDY OF THE HILL REACTION

353

reductase reaction. The partial reactions are H20 + T P N +

light

--* T P N H + H + + 1/60,,

chloroplasts

T P N H + H + + GSSG--* T P N + + 2GSH

(7) (8)

The observed reaction is H20 + GSSG--* 2GSH + ~ 0 2

(9)

The experiment can be carried out in an illuminated Warburg bath at 15 °. Reagents

Chloroplast preparation. Chloroplasts or fragments are suspended in 0.05 M sorbitol-borate buffer, pH 7.0, to a density of 0.5 nag. of chlorophyll per milliliter. GSSG reductase. 21 GSSG. 22 0.1 M solution. TPN. 0.001 M solution. Metaphosphoric acid. 30% solution. Procedure. Mix in the main compartment of the Warburg vessel 2.0 ml. of chloroplast preparation, 0.1 ml. of GSSG, and an excess of GSSG reductase. In one side arm place 0.1 ml. of TPN, and in the other 0.2 ml. of metaphosphoric acid. Flush the vessel and manometer with purified N2 or H2. Start the reaction by turning on the light and tipping T P N from the first side arm. Follow 02 evolution manometrically. Stop the reaction by tipping metaphosphoric acid from the second side arm. The formation of GSH can be determined by titration with KIO3, 23 by the nitroprusside test, 24 or by Dische's sugar test. 25 Stoichiometry. According to equation 9 the formation of I mole of GSH corresponds to the evolution of 0.25 mole of 02. Hendley and Conn 2° found 50 to 80% of the calculated amount of 02, depending on a number of factors which included the purity of the GSSG reductase and the extent to which 02 could be excluded from the atmosphere. Related Procedures. Vishniac and Ochoa 19followed a variety of pyridine nucleotide-linked reactions, none of which was as effective as GSSG reduction: reduetive carboxylation of pyruvate, reductive carboxylation of a-ketoglutarate, reduction of pyruvate, reduction of oxalacetate, 21 For the preparation of GSSG reductase, see Vol. I I [126, 127].

52GSSG can now be obtained commercially. 2aA. Fujita and I. Numata, Biochem. Z. 299, 249 (1938). 24R. R. Grunert and P. H. Phillips, Arch. Biochem. $0, 217 (1951). ~5Z. Dische and H. Zil, Proc. Assoc. Research Ophthalmol. 19, 104 (1950).

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TECHNIQUES FOR METABOLIC STUDIES

[15]

reduction of fumarate, reduction of 1,3-di-PGA, and reductive amination of a-ketoglutarate.

Oxygen as Oxidant Mehler is observed that in the absence of other oxidants 02 itself can be reduced by illuminated grana. The reaction is presumably light * H~O2 q- 1/~O2 H20 -~- O~ chloroplasts

(10)

This reaction can be followed by measuring the amount of acetaldehyde formed -08 in the presence of ethanol and catalase, since catalase will peroxidize ethanol: catalase --* CHA?HO q- 2H20

H202 -[- CHsCH~OH-

(11)

The observed reaction is then CH3CH20H + 1~O2

light chloroplasts

CH3CHO + H20

(12)

catal~se

Cytochrome C as Oxidant Mehler Is observed the reduction of cytochrome c by illuminated grana when the reoxidation of the reduced cytochrome was inhibited by KCN. The reaction was carried out in a Beckman cell (3.0 ml.) containing a dilute suspension of grana (0.01 rag. of chlorphyll per milliter) in 0.001 M of KCN, and 0.3 micromole of cytochrome c. The cell was illuminated for a selected period of time, rapidly transferred to the Beckman spectrophotometer, and the change in optical density determined at 550 m~. Tolmach (unpublished experiments) observed 02 evolution from illuminated grana with cytochrome c as oxidant when NaN~ was used instead of KCN.

Additional Techniques Study of the Hill reaction and related phenomena frequently requires the determination of small amounts of 03. The following methods have been employed: polarography, 27 fluorescence quenching, 2s and absorption of 03 by CrCI2 followed by back titration.19 It is also possible to adapt Strehler's technique 29 to the microdetermination of 02, by using an ~8For the determination of acetaldehyde, see Vol. III [54]. ~7F. S. Brackett, R. A. Olson, and R. G. Crickard, J. Gen. Physiol. 36, 529 (1953). ~8j. Franck and P. Pringsheim, J. Chem. Phys. 11, 21 (1943). 89B. L. Strehler, Arch. Biochem. and Biophys. 43, 67 (1953).

[16]

METHODS FOR MEASUREMENT OF NITROGEN FIXATION

355

O~-dependent luminescent system. The paramagnetic susceptibility of O2 makes possible the continuous determination of the partial pressure of 02. 30 30 L. Pauling, R. E. Wood, and J. H. Sturdivant, Science 108, 338 (1946).

[16] Methods for M e a s u r e m e n t of N i t r o g e n F i x a t i o n

By R. H. BURRIS and P. W. WILSON The traditional method for detection of biological nitrogen fixation had been the ocular assay based on observation of growth of the agent on what is alleged to be a N-free medium. Though not a quantitative procedure, this method deserves some consideration because of its usefulness in screening agents and because historically it has led to the investigation of many agents known or alleged to fix nitrogen. In addition to the obvious limitation that the experiments must be made in the absence of any source of combined nitrogen, not always a desirable limitation, others may be cited. First, it is extremely difficult to free a medium completely from traces of combined nitrogen, and many organisms (filamentous fungi, capsulated or gum-producing bacteria) will make what appears to be noteworthy growth on mere traces of such compounds. Again, in the long time trials frequently employed, such agents may absorb sufficient combined nitrogen from the air, particularly ammonia, not only to grow but also to allow the much more impressive demonstration of continuous transfer in the "N-free" medium. Control of these sources of error, however, can be readily achieved by comparison of the growth of the agent in two controlled atmospheres, both free of combined nitrogen compounds, and one free of N2, e.g., for aerobic agents growth in a gas mixture of N2-O2 versus that in one of H2-O~. If ocular assay suggests that a particular organism can fix N2, N~fixing ability can be established by showing that its final content of nitrogen is greater than that supplied from the medium and surrounding air as fixed nitrogen during the period of testing. Although this is simple in principle, errors inherent in methods, such as the Kjeldahl method, for the determination of total nitrogen and difficulties in obtaining uniform samples make the technique applicable only to agents which fix substantial quantities of N~. The use of N15-enriched N2 (designated hereafter as N~s) provides a much more sensitive and reliable test for N2 fixation.

[16]

METHODS FOR MEASUREMENT OF NITROGEN FIXATION

355

O~-dependent luminescent system. The paramagnetic susceptibility of O2 makes possible the continuous determination of the partial pressure of 02. 30 30 L. Pauling, R. E. Wood, and J. H. Sturdivant, Science 108, 338 (1946).

[16] Methods for M e a s u r e m e n t of N i t r o g e n F i x a t i o n

By R. H. BURRIS and P. W. WILSON The traditional method for detection of biological nitrogen fixation had been the ocular assay based on observation of growth of the agent on what is alleged to be a N-free medium. Though not a quantitative procedure, this method deserves some consideration because of its usefulness in screening agents and because historically it has led to the investigation of many agents known or alleged to fix nitrogen. In addition to the obvious limitation that the experiments must be made in the absence of any source of combined nitrogen, not always a desirable limitation, others may be cited. First, it is extremely difficult to free a medium completely from traces of combined nitrogen, and many organisms (filamentous fungi, capsulated or gum-producing bacteria) will make what appears to be noteworthy growth on mere traces of such compounds. Again, in the long time trials frequently employed, such agents may absorb sufficient combined nitrogen from the air, particularly ammonia, not only to grow but also to allow the much more impressive demonstration of continuous transfer in the "N-free" medium. Control of these sources of error, however, can be readily achieved by comparison of the growth of the agent in two controlled atmospheres, both free of combined nitrogen compounds, and one free of N2, e.g., for aerobic agents growth in a gas mixture of N2-O2 versus that in one of H2-O~. If ocular assay suggests that a particular organism can fix N2, N~fixing ability can be established by showing that its final content of nitrogen is greater than that supplied from the medium and surrounding air as fixed nitrogen during the period of testing. Although this is simple in principle, errors inherent in methods, such as the Kjeldahl method, for the determination of total nitrogen and difficulties in obtaining uniform samples make the technique applicable only to agents which fix substantial quantities of N~. The use of N15-enriched N2 (designated hereafter as N~s) provides a much more sensitive and reliable test for N2 fixation.

356

TECHNIQUES FOR METABOLIC STUDIES

[16]

Methods for Growing the Organisms As biological nitrogen-fixing agents include higher plants and their associated microorganisms, blue-green algae, and a variety of aerobic and anaerobic bacteria, it is not feasible to discuss here optimum methods for cultivating them. This information must be sought in original literature describing cultural conditions. It should be pointed out, however, that it is not obligatory that the organisms under test be grown on a nitrogen-free medium. Although a nitrogen-free medium is desirable with some agents, others will not initiate growth unless a limited supply of combined nitrogen is furnished. As a vigorously growing culture exhausts its supply of combined nitrogen, it may start to fix N2.

Determination of N2 Fixation by an Increase in Total N This method is adequate for active N~ fixers, but it will not detect increases of less than about 1% in the total nitrogen even when uniform samples can be taken. The organism should be grown under the most favorable conditions except that its supply of combined nitrogen should be limiting. Ammonia and nitrogen oxides should be excluded from the culture room as completely as possible. Several vessels of the culture medium minus and plus the agent under test should be analyzed for total N at the start and the end of the experiment. A statistical treatment of the results is usually obligatory to indicate what confidence can be placed on limited fixation. Wilson 1,~ discusses how an inherent error in the Kjeldahl method led to the assertion that aseptic germinating peas fixed N2, and how adequately controlled and statistically analyzed experiments revealed the occurrence and source of the error. Hiller et al. 8 have studied carefully the requirements for rapid and accurate Kjeldahl analysis; the method described here departs but little from their recommendations and utilizes samples which are conveniently handled with semimicro equipment. Kjeldahl Method (see also Vol. I I I [145]): Place a sample containing 0.6 to 6.0 rag. of N in a 100-ml. Kjeldahl flask, and then add 1.5 g. (1/~ level teaspoon) of K2S04 through a long-stemmed powder funnel. Add 1.5 ml. of mercuric sulfate solution (dilute 12 ml. of concentrated H2SO4 to 100 ml., and dissolve 10 g. of red mercuric oxide), 3 ml. of concentrated H2SO4, and 2 glass beads or Alundum chips. Heat gently 1p. W. Wilson, "The Biochemistryof Symbiotic Nitrogen Fixation," pp. 100-107. University of Wisconsin Press, Madison, 1940. 2p. W. Wilson,in "Bacterial Physiology" (Werkmanand Wilson,eds.), pp. 471-473. Academic Press, New York, 1951. 3A. Hiller, J. Plazin, and D. D. Van Slyke, J. Biol. Chem. 176, 1401 (1948).

[16]

METHODSFOR MEASUREMENT OF NITROGEN FIXATION

357

on a digestion rack 4 until the water is removed and charring is complete, and then increase the heat so the solution boils constantly with slight motion. After complete clearing, continue to boil gently for 30 minutes. Cool for 5 to 10 minutes, and then add 25 ml. of water to the flask. Cool

~L~.(::~ jAi~ ~. ---j

Fro. 1. Apparatus for distillation from 100-ml. Kjeldahl flasks. Standard Pyrex flasks are attached to the apparatus with rubber stoppers. The glass tubing from the Iowa State-type spray heads which passes through the rubber stopper should be 8 ram. o.d. ; smaller tubing gives excessive holdup in the spray head. Air is passed from needle valves on the manifold to the bottom of the individual flasks. This aeration combined with heating of the flasks by a strip heater or bare nichrome coils speeds the distillation and prevents sucking back of the distillate. The distillate is condensed in stainless steel tubing passing through a 4-inch-deep stainless steel box through which cooling water is circulated.

under the water tap, and add 0.6 g. of ammonia-free Zn dust with a glass or plastic spoon, followed by 12 ml. of 13 N NaOH flowed down the wall of the inclined K]eldahl flask. Attach the flask to the still (Fig. 1), and aerate the solution at a rate of 3 to 5 bubbles per second. Apply heat, and distill into 10 ml. of 2% boric acid until the flask starts to bump. 4 p. W. Wilson and S. G. Knight, "Experiments in Bacterial Physiology," pp. 58--59. Burgess Publishing Co., Minneapolis, 1952. A suitable digestion rack also is made by the American Instrument Co., Silver Spring, Md.

358

TECHNIQUES FOR METABOLIC STUDIES

[16]

During the last 30 seconds of distillation, tilt the receiving flask so the delivery tube is not immersed; rinse the end of the delivery tube. Titrate the ammonia with standard H~SO~ (about 0.02 N). Use 2 drops of Tashiro's indicator (0.25 g. of methylene blue, 0.375 g. of methyl red, 300 ml. of 95% ethanol); this turns from green through gray to purple, and the gray serves as a satisfactory end point.

Determination of N2 Fixation by Gasometric Analysis Because of the limitation of the Kjeldahl technique, particularly in the presence of heterogeneous material high in organic nitrogen (nodules, seeds), several workers have developed direct methods for demonstration of fixation by gasometric analyses (Allison et al., 5 Virtanen6). Usually these methods depend on gas analysis at the beginning and end of the experiments; these procedures are not only tedious and exacting in execution, but they are not very precise. The gas mixture changes in composition during the experiment, and the quantity of N~ that disappears is extremely small in comparison with changes in O2; usually the N~ is determined by difference after analysis for the other gases. To overcome some of these limitations, Hurwitz and Wilson 7 developed a manometric method in which both 02 consumed and N2 assimilated are replaced with 03. Fixation is accordingly measured by increase in O~ in the gas mixture. This method was originally designed for macrotrials (500-ml. Erlenmeyer flasks, 100 ml. of medium, Novy-Soule gas apparatus); we have modified it for semimicrotrials by using special 125-ml. flasks with the Warburg respirometer. Even more improvement in precision could be introduced through further refinements, e.g., absorption of 02 directly in the experimental vessel by use of a double side arm of the Siamese type; however, except for special instances neither the precision nor accuracy would compare with the available isotopic methods.

Determination of N2 Fixation with N~5 By extensive replication and statistical analysis an increase in nitrogen of 1.0% may be established with the Kjeldahl method under favorable conditions. With a mass spectrometer 0.003 atom % of N 15 excess can be detected and 0.015 atom % of N 15 excess will serve as a conservative level for establishing fixation of N~6. If an agent be supplied N2 with 60 atom % of excess N 15, the accumulation of 0.015 atom % excess would require an increase of only about 0.025% in the total nitrogen of the agent. The isotope method thus is at least forty times, and in actual 5 F. E. Allison, S. R. Hoover, and F. W. Minor, Botan. Gaz. 104, 63 (1942). s A. I. Virtanen, Trans. 3rd Comm. Intern. Soc. Soil Sci. A, 4 (1939). C. Hurwitz and P. W. Wilson, Ind. Eng. Chem., Anal. Ed. 12, 31 (1940).

[16]

METHODS FOR MEASUREMENT OF NITROGEN FIXATION

359

practice probably closer to one hundred times, as sensitive as the Kjeldahl method for establishing N2 fixation. Moreover, the isotope method is direct and is not subject to the sampling problems of the Kjeldahl method. Whereas the sampling of a heterogeneous material, such as leguminous root nodules, presents formidable difficulties in the analysis of total nitrogen, the mere increase in N 15 concentration above the normal abundance in agents supplied N~~ constitutes positive evidence of biological nitrogen fixation. Preparation of N~s. Distillation Products Industries, Rochester 3, New York, supplies Nl~-enriched NH4NOa (enriched in ammonium ion only), HNO3, KNO3, and potassium phthalimide. The N~ gas can be prepared conveniently by passing NH3 over hot CuO in the apparatus sketched in Fig. 2. Place 2.25 g. of N15H4NO3 in tube A, which is fastened to the apparatus with a well-fitted rubber stopper, B. Fill column C with NaOH pellets, retained by a perforated porcelain disk, D, such as forms the bottom of a Gooch or Caldwell crucible. Lubricate 10/30 joint E, and attach unit F to the Toepler pump. Lubricate and attach inner 14/35 joint G; hold it in place with springs or rubber bands. Place sealing wax (Consolidated Engineering Co., 300 N. Sierra Madre Villa, Pasadena 8, California, furnishes a highly satisfactory wax under catalog No. A-18792) on inner joint H; warm this joint and outer joint H, and join them. While the wax is warm, line up outer joints I and J by placing a 1-1. gas bulb, K, in position on the apparatus. Now place sealing wax on inner joints I and J, and seat them in their warmed outer joints; support the bulb, K, from a ring stand or support rod. Attach the manometer with wax at ball joint L, and the NaOH pellet column with wax and a ball joint clamp at joint M. A suitable oxidizing tube, N, can be made as follows: To approximately 20-mm.-diameter Vycor tubing about 200 ram. long, seal the two outer 10/30 Vycor joints; only these two outer ioints need be Vycor. Cover the Vycor tube with wet, 1/~6-inch asbestos sheet, and then wind 12 or 13 feet of No. 22 nichrome wire on it; cover the wire with a ~ - to 1/~-inch layer of wet asbestos sheet and mold to form. Attach a Variac or similar variable-voltage source to the nichrome leads from tube N, and at increasing voltages measure the temperature inside the tube with a thermocouple. Select a voltage setting for subsequent use which will give a temperature of about 650 °. Pack the tube with wire form CuO, which is furnished in lengths of about ~ inch; confine the packing to position with Pyrex glass wool. Evacuate the system through stopcock 0, and immediately apply a counter vacuum with a water pump attached to mercury reservoir P; check the system for leaks with a Tesla coil type tester. Outgas the sys-

360

TECHNIQUES FOR METABOLIC STUDIES

[16]

tern by evacuating it for about an hour while the oxidizing tube N is being heated. Shut off the vacuum line with stopcock 0 (mark the level of the mercury in the manometer), and see if the mercury in the manometer maintains its level for 10 minutes. If this check on the vacuum is satisfactory, close stopcocks Q and R'. Allow about 5 ml. of 13 N NaOH

Qq

FIG. 2. Apparatus for converting NHa to N~ by passing it over hot CuO. See text for explanation. solution to flow from reservoir S into chamber A ; leave some solution in S so that no air is introduced into A. Warm the mixture in A, and pump the NH3 generated into K by closing stopcock T, opening stopcock U to position 2, and closing stopcock V immediately after mercury has passed into it; now return stopcock U to position 1, and when mercury has been pulled back into chamber W, reopen stopcock T. Repeat the pumping operation, but subsequently when the mercury rises do not open V until the mercury has just risen into chamber X. To force the

[16]

M E T H O D SFOR MEASUREMENT OF NITROGEN FIXATION

361

gas past V, it may be necessary to apply air pressure in P by pumping with an atomizer bulb attached to the lower outlet of stopcock U; U should be in position 2. As the pumping progresses, heat chamber A more vigorously. Usually before the transfer of NH3 to chamber K is complete the column of NaOH pellets in C largely dissolves; do not allow the column of pellets to cake by interrupting the heating of A. Discontinue the pumping before the column's drying capacity is destroyed by its complete solution. Close stopcock Y, and immediately remove column C at the ball joint; dissolve the residual, and warm NaOH in the column before it cakes. Open stopcocks R' and Q, and immediately mark the level of mercury in the manometer before any appreciable NH~ can be oxidized (all measurements on the manometer should be made with the mercury pulled down into P or with it up against stopcock V). Circulate the NH3 over the hot CuO with the Toepler pump as described, being careful not to pull any out through it. The gas volume will decrease to approximately half its original volume as the NH3 is converted to N2; heating of the gas in the furnace and the presence of water vapor may alter the pressure from the theoretical 50% value. Pump the gas until the pressure registered by the manometer is constant, and then continue the pumping for an additional 30 minutes to be sure that conversion is complete. Close stopcock R', and pump the gas from the left side of the apparatus into K with the Toepler pump. Finally allow the mercury to rise through V, attach an atomizer bulb to the bottom lead on stopcock U (stopcock in position 2), and pump the mercury up almost to the side arm on unit F. Close V and then R. Remove reservoir K after warming joints J and I. If you wish to have dry N2, before removing K place a Dewar flask of liquid air around tube Z and circulate the gas with the Toepler pump; this also should freeze out oxides of nitrogen if any are present (introduction of 02 will convert NO, melting point - 161 °, to NO2, melting point -9.3°). If 20% Na2S04 in 5 vol. % of H2SO4 is used to displace the N2 from flask K when the gas is used subsequently, the acid solution will capture any unreacted NH3 that remains in the gas bulb. Exposure of the Biological Agent to N~ 5. Aerobic and anaerobic gas mixtures can be prepared from commercial cylinder gases and the N~5 prepared as described. The mixtures can be made by displacing measured volumes of water or other fluid from a vessel of known volume or by evacuating the vessel and adding measured pressures of the individual gases. The techniques have been described in more detail by Umbreit et al. 8 It is seldom necessary to use a N2 atmosphere in the mixtures comparable to that in air, for known nitrogen-fixing agents fix nearly as 8 W. W Umbreit, 1%. H. Burris, and J. F. Stauffer, "Manometric Techniques and Tissue Metabolism," pp. 45-46. Burgess Publishing Co., Minneapolis, 1949.

362

TECHNIQUES FOR METABOLIC STUDIES

[16]

rapidly at a pN2 of 0.1 as 0.8. Usually 0.1 atm. of N2 and 0.9 atm. of He is suitable for anaerobes, and for aerobes 0.1 atm. of N2, 0.2 atm. of 02, and 0.7 atm. He is suitable. The biological agents can be exposed to N~5 conveniently under aseptic conditions in the apparatus described by Burris et a l 2 The culture under test is placed in a cotton-plugged flask equipped with a ground joint and is furnished with 02 from a reservoir through a capillary tube. Enhanced diffusion of gas is achieved by placing vessels of this type on a shaking apparatus. If the material under test has a sufficiently high initial content of N it can be added directly to conventional Warburg flasks shaken in a respiromcter bath. The flasks are readily modified for aseptic trials (Umbreit et al.Da). Ordinary respirometer vessels will serve for short runs which will be little affected by contaminants. For example, the fixation of N~6 by excised nodules from leguminous plants has been tested by placing a few nodules or nodule slices in 8-ml. Warburg flasks, gassing them, and shaking them in the respirometer bath for an hour. As the rate of fixation decreases with time, ~° it is highly unlikely that it arises from soil contaminants. Contamination should always be considered, and in critical experiments plate counts of viable organisms should be taken; however, it requires a readily detectable number of contaminating organisms to fix an appreciable amount of N~ 5. For example, assume that organisms of 3 t~3 volume per cell, 75% water content, and 2.5% N on a wet basis are being tested for their ability to fix N~5. As the analysis requires about 1 mg. of N, 40 mg. of wet cells would be needed, or about 13.3 X 109 cells. If the agent under test did not fix N2 supplied at 30 atom % of N ~5 excess but a million cells of an N2-fixing organism of similar size and composition developed in the culture during the test period, these cells would enhance the N 15 concentration of the complete culture by only about 0.002 atom %. As such a culture might occupy a volume of 10 ml., a population density of 100,000 contaminants per milliliter should be detectable by microbiological techniques if several selective media were employed in the search for contaminants. When relatively large quantities of a nitrogen-fixing organism must be grown, it may be necessary to circulate the gas through the culture to achieve adequate growth. A system such as that described by Burris ~1 9 R. H. Burris, F. J. Eppling, H. B. Wahlin, and P. W. Wilson, J. Biol. Chem. 148, 349 (1943). ,a W. Umbreit, R. H. Burris, and J. F. Stauffer, "Manometric Techniques and Tissue Metabolism," pp. 62-63. Burgess Publishing Co., Minneapolis, 1949. lo M. H. Aprison and R. H. Burris, Science 115, 264 (1952). 11R. H. Burris, J. Biol. Chem. 143, 509 (1942).

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is applicable, but it should be modified by making the culture vessel deeper and narrower and by substituting a pump of greater capacity. For low aeration rates a small Sigmamotor pump or for greater rates a large-capacity Sigmamotor pump will serve (Sigmamotor pumps are distributed by Schaar and Co., Chicago). A highly satisfactory pump also can be made from an automobile fuel pump driven by a cam placed on a shaft driven at about 500 r.p.m, with a belt or gear drive from a X-horsepower electric motor. To prevent leakage, the housi~lg of the pump diaphragm should be sealed at the edges and over the bolts with a heated mixture of equal parts beeswax and rosin. Such a pump will circulate about 15 1. of gas a minute. Recovery of Gas. If an experiment demands that the gas be recovered very quickly, most of it is captured merely by allowing it to expand into an evacuated bulb with a volume greater than that of the exposure chamber. The gas also can be recovered by pumping it into an evacuated bulb with a Sigmamotor pump or with a Toepler pump. By keeping the volume of the exposure chambers small and the pN~ low it may be possible to avoid the nuisance of recovering gas. For example, in a 10-ml. vessel with gas at a pN2 of 0.1 atm., the value of the N~5 with 30 atom % of N ~5 excess is about 8 cents exclusive of the cost of generating it from the ammonium salt. Digestion of Samples. Samples may be digested in the same manner as described for analysis of total nitrogen. It is desirable, however, to continue the digestion for 2 hours after clearing to be sure that no methylamine remains. Methylamine survives Kjeldahl digestion with copper and selenium catalysts, but we have never experienced difficulty with it in samples digested with a mercury catalyst. ]~.iethylamine distills with the ammonia from basic solution and passes into the sample bulbs during conversion of NH3 to N~ with alkaline hypobromite. In the electron beam of the mass spectrometer the methylamine decomposes to yield a product of mass 29 which "sticks" in the spectrometer and gives a steadily increasing 29/28 mass ratio. The instrument must be "baked o u t " after contamination with methylamine. Distillation of Samples and Colorimetric Analysis for Total N. Transfer the digested sample to a Kjeldahl flask with an outer 19/38 standard taper joint as pictured in Fig. 3. Cool the flask, add powdered Zn, add NaOH, and rinse the ground joint so that it will not stick. Attach the flask to the distillation apparatus, and distill with steam for about 5 minutes (ca. 30 ml. of total distillate) into 10 ml. of 0.1 N H2SO4 (caution: do not use more acid than this). Note that a Teflon plug is used to avoid the necessity for greasing stopcock A. As Teflon has a greater coefficient of expansion than glass, it is necessary to loosen the plug

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when the s t e a m generator is started a n d to allow s t e a m to escape around the plug until it is heated; after heating, the plug m a y be seated for the remainder of the operations. Transfer the distillate f r o m the 125-ml. E r l e n m e y e r flask to a 50-ml. volumetric flask, and m a k e to volume. W i t h d r a w duplicate 0.5- or 1.0-ml. samples, place t h e m in tubes standardized for colorimetry, and add w a t e r to 3.0-ml. volume. R e t u r n the residual distillate to its E r l e n m e y e r Steam-

B

)

.°

i

Fro. 3. Glass apparatus for steam distillation of NHs. Teflon stopcock plug A is turned with a ~ 0 taper and drilled in the T form to fit a standard taper Pyrex stopcock shell. The apparatus is connected at B with 12/~ ball and socket joints and joint clamps. The steam line and the area from the flasks to the condensers is covered with asbestos molded into place while wet. flask. Place the colorimeter t u b e in a 20 ° w a t e r bath, agitate, and add slowly, in order, 2 ml. of Nessler's reagent and 2 ml. of 2 N N a O H . (Nessler's reagent according to Johnson: 1~ dissolve 4 g. of K I and 4 g. of HgI2 in 25 ml. of water; reflux 1.75 g. of light-colored g u m ghatti in 750 mh of w a t e r until " d i s s o l v e d " ; add the K I - H g I ~ solution to the g u m ghatti solution and m a k e to 1 1. ; allow to settle and then decant.) Allow the t u b e to stand at a b o u t 20 ° for 20 minutes, and read in a colorimeter with a 490-m~ filter; compare with a reagent blank and a standard coni~ M. J. Johnson, J. Biol. Chem. 157, 575 (1941).

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365

taining 25 ~/of ammonia N. The useful range of the method is 10 to 40 of N. Calculate the total N of the digested sample from these analytical data. Check the distillate in the Erlenmeyer flask with blue litmus paper to be sure that it is acid. Add 2 glass beads and boil the sample down over a gas burner to a volume of about 3 ml., exercising care to avoid boiling the flask dry. Conversion of NH3 to N2. Rittenberg et al. 13,~4have described a satisfactory apparatus for conversion of NH3 samples to N2. (The Toepler pump section of this apparatus can also be used for the oxidation of NHa to N2 over CuO as shown in Fig. 2.) The sample boiled to a small volume is transferred to the apparatus, evacuated, and treated with alkaline hypohromite (to 8 ml. of Br2 and 70 ml. of H:O add 40 ml. of 13 N NaOH while shaking the flask in a cooling bath) to oxidize the ammonia to N2. The N2 is transferred with the Toepler pump to a sample bulb provided with a stopcock and a standard taper 10/30 joint that will fit the manifold of the mass spectrometer. The method yields clean samples, but for generating large quantities of N2 from ammonia we have found it less satisfactory than the oxidation over CuO with the apparatus shown in Fig. 2. Mass Analysis. The samples from the converter can be analyzed with any suitable isotope ratio type of mass spectrometer; the ConsolidatedNier instrument is very satisfactory. It is beyond the scope of this article to outline the operation of the spectrometer, but it should be noted that it is desirable to immerse the sample bulb in a liquid air or dry ice freezing bath before and during the introduction of the gas sample into the mass spectrometer. Water vapor gives spurious ratios in the analysis of N2• especially on small samples which must he analyzed at low pressure. For a detailed description of the analysis, see Vol. IV [21].

Determination of N2 Fixation by Isotope Dilution Although the isotopic method based on assimilation of N~5 remains the method of choice because of its greater sensitivity, a variation based on isotopic dilution developed by Newton 1~ has specific advantages that recommend its use for special problems. In this procedure the agent to be tested is labeled by growing it in a medium to which a compound has 13 D. Rittenberg, in "Preparation and Measurement of Isotopic Tracers" (Wilson, Nier, and Reimann, eds.), pp. 31-42. Edwards Bros., Ann Arbor, 1946. 14 D. Rittenberg, A. S. Keston, F. Rosebury, and R. Schoenheimer, J. Biol. Chem. 127, 291 (1939). ~6j. W. Newton, Isotopic Studies on Biological Nitrogen Fixation, M. S. Thesis, University of Wisconsin, 1952.

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been added containing the tracer. Usually a small amount of N15H+ is added, but any other suitable (it need not be assimilable by the agent) source of the label can be used. An aliquot of the total culture is taken for analysis in the mass spectrometer by the methods already described; then the agent is exposed to normal N~4 under any conditions the experimenter wishes. After exposure another aliquot is analyzed for N 15, and from the decrease in atom % of N 15excess, fixation of the N~4 is estimated. In a detailed examination of the most suitable experimental conditions Newton 1~ concluded that neither accuracy nor precision was materially affected by the level of the label used; 1 to 5 atom % of N 15 excess appeared to be most convenient. The sensitivity of the method was only about one-tenth that of a suitably designed experiment using N~5, but certain advantages may be cited. The chief one is that the test can be made under conditions best suited to the physiology of the agent and the convenience of the experimenter rather than those dictated by the closed system required for trials made with N~~. This advantage is noteworthy for experiments made with pathogenic agents with which special safety precautions must be employed. Loss of expensive N 15, particularly in long time experiments, is avoided as well as the necessity of recovering and storing the gas mixtures used. Finally, the method should be of particular value for cooperative experiments. Since the apparatus and skill required for actually running the experiment are those to be found in any laboratory, the isotopic method need not be restricted to individuals possessing both expensive physical equipment and the experience in its operation, All that the inexperienced worker need do is to arrange for a cooperating laboratory possessing a mass spectrometer to analyze the sample. This courtesy is much more readily obtained than that involving making the entire experiment with some agent of interest to only one party. An illustration of the use of the dilution method is given by Newton et al. le 16j. W. Newton, P. W. Wilson, and R. H. Burris, J. Biol. Chem. 204, 445 (1953).

[17] M i c r o m e t h o d s f o r

the Assay of E n z y m e s

B y OLIVER H. LOWRY

In this section will be described techniques for measuring enzyme activities on a smaller scale than may be common at present (50 to 200 ~, of tissue). Several major advantages can be had from small-scale methods. There is conservation of material, either of the enzyme itself or an

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been added containing the tracer. Usually a small amount of N15H+ is added, but any other suitable (it need not be assimilable by the agent) source of the label can be used. An aliquot of the total culture is taken for analysis in the mass spectrometer by the methods already described; then the agent is exposed to normal N~4 under any conditions the experimenter wishes. After exposure another aliquot is analyzed for N 15, and from the decrease in atom % of N 15excess, fixation of the N~4 is estimated. In a detailed examination of the most suitable experimental conditions Newton 1~ concluded that neither accuracy nor precision was materially affected by the level of the label used; 1 to 5 atom % of N 15 excess appeared to be most convenient. The sensitivity of the method was only about one-tenth that of a suitably designed experiment using N~5, but certain advantages may be cited. The chief one is that the test can be made under conditions best suited to the physiology of the agent and the convenience of the experimenter rather than those dictated by the closed system required for trials made with N~~. This advantage is noteworthy for experiments made with pathogenic agents with which special safety precautions must be employed. Loss of expensive N 15, particularly in long time experiments, is avoided as well as the necessity of recovering and storing the gas mixtures used. Finally, the method should be of particular value for cooperative experiments. Since the apparatus and skill required for actually running the experiment are those to be found in any laboratory, the isotopic method need not be restricted to individuals possessing both expensive physical equipment and the experience in its operation, All that the inexperienced worker need do is to arrange for a cooperating laboratory possessing a mass spectrometer to analyze the sample. This courtesy is much more readily obtained than that involving making the entire experiment with some agent of interest to only one party. An illustration of the use of the dilution method is given by Newton et al. le 16j. W. Newton, P. W. Wilson, and R. H. Burris, J. Biol. Chem. 204, 445 (1953).

[17] M i c r o m e t h o d s f o r

the Assay of E n z y m e s

B y OLIVER H. LOWRY

In this section will be described techniques for measuring enzyme activities on a smaller scale than may be common at present (50 to 200 ~, of tissue). Several major advantages can be had from small-scale methods. There is conservation of material, either of the enzyme itself or an

[17]

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expensive substrate or coenzyme. There is usually substantial saving of time, since it is faster to manipulate small volumes. Perhaps more useful than either of these is the consequence that sensitive methods permit high tissue dilution during enzyme action. Such dilution greatly reduces the danger of disturbances from inhibiting substances, from side reactions, or from removal of some of the product by other enzymes present. This often permits the measurement of an enzyme in a crude tissue homogenate when this might otherwise not be possible. All the methods given here are applicable directly to the whole tissue indicated, as well as to purified preparations. Because of the high dilutions employed, with highly purified enzyme it may in some instances be necessary to add other protein such as serum albumin to protect the activity. As a further means to save time as well as decrease chances for errors the number and complexity of analytical operations have been reduced wherever possible. An optimal scale of operations has been given more consideration than highest sensitivity. In fact, most of the procedures are macro-adaptations of methods designed for histochemical purposes. Even so, the methods are fifty to one thousand times as sensitive as the Warburg apparatus, for example. Unfortunately, measuring an enzyme is not like measuring chloride or ATP in that a given reaction may be catalyzed by different enzymes in different tissues or species. These enzymes may conceivably differ in regard to pH optimum, substrate optimum, temperature coefficient, stability under incubation conditions, cofactor requirements, or possible presence of destroying or inhibiting factors. The methods given are therefore guaranteed only for the tissue indicated. No attempt has been made to adapt as many enzyme methods as possible to a microscale. Rather, a cross section is given to illustrate a sufficient variety of analytical possibilities with the thought that when necessary the reader can easily adapt other methods in a similar manner.

I. General Techniques and Tools Colorimetry Most of the colorimetric measurements are made with the Beckman spectrophotometer fitted with quartz cells of 10-mm. light path but only 3.5-mm. inner width. These cells have a thick bottom and thick walls so that the outer dimensions are those of the standard Beckman cuvette. They are used with a 2-mm. pinhole in the path of the incident light beam. Both pinhole attachment and cells are obtainable from the Pyrocell Mfg. Co. (207 E. 84th Street, New York). The pinhole is adjusted to the center of the cells, and the positioning of the cells is checked with

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0.3 ml. of water in each cell. If the pencil of light from the pinhole strikes the meniscus, the cells are raised with low blocks. By an extension of the same principle the Beckman may be adapted to still smaller volumes. 1 After use, the cells are emptied into a trap with suction through a tube tipped with a short length of polyethylene tubing which prevents scratching of the cell walls. If care is taken to remove liquid from the corners, rinsing is usually unnecessary between samples that are not too dissimilar.

Fluorimetry Measurements are made in a Farrand fluorimeter (Farrand Optical Co., New York) ~ with at least 1 ml. in Pyrex test tubes, 10 X 75 mm. (3 ml.). These tubes are selected on the basis of freedom from stria and uniformity of diameter. Tubes are chosen which slip easily into the instrument with a minimum of play. For very precise measurements tubes are further selected on the basis of uniformity of reading with an appropriate quinine solution in 0.1 N H2SO~. Fluorimetry has not been used and exploited as much as it may deserve, particularly for microchemical applications. It is inherently much more sensitive than colorimetry, since the emitted fluorescence is directly related to the substance measured and the illumination. Therefore, sensitivity can be increased almost without limit by increasing the amplification, the phototube sensitivity (photomultiplier), or the intensity of the light source. Fluorimetry also may be used over a much wider concentration range than colorimetry. Thus colorimetric readings are ordinarily restricted to the range of 5 to 90% transmission, a 25-fold concentration range, but fluorimetric measurements may be satisfactory over a 1000- to 10,000-fold range. There are certain peculiarities to fluorimetry which are not always appreciated and which do not seem to have been systematically treated in the literature. Therefore, a brief statement on some analytical considerations may be desirable. Proportionality. Since fluorescence is directly proportional to the exciting light intensity, proportionality between emitted light and the substance measured is achieved only if the absorption of the exciting wavelengths is negligible. Therefore fluorimetry begins at dilutions too great for accurate colorimetry. Quenching and Related Phenomena. Fluorescence is affected by many factors. (1) The exciting light may be partially absorbed by substances present in the sample. As indicated, this may be the substance itself. 10. H. Lowry and O. A. Bessey, J. Biol. Chem. 168, 633 (1946). O. H. Lowry, J. Biol. Chem. 178, 677 (1948).

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Since ultraviolet wavelengths are frequently used for excitation, absorption may be unsuspected. (2) The emitted light may h~ partially absorbed by colored substances present. (3) Fluorescence has a large negative temperature coefficient. A rise in temperature of 1 degree will usually decrease fluorescence by about 1%. (4) Depending on the particular substance, small changes in the solution may have large effects on the fluorescence; e.g., traces of chloride ion will decrease quinine fluorescence, and alcohol enhances thiochrome fluorescence. Standardization. Uncertainties due to quenching, etc., may be overcome by dilution and internal standards. The inherent sensitivity usually permits dilution to a point where there is no essential difference between sample and standard. As an added check, standard can be added directly to part of the sample, and the increment in galvanometer deflection will provide a reliable basis for calculation. Setting Standards. All readings have to be made by comparison with some reference solution, since there is no initial light beam with which to adjust the instrumental sensitivity. Readings might be made against one of the standards, but many fluorescent substances are unstable to the intense amount of light usually employed, and the standard would change with repeated readings. Therefore a stable working standard is ordinarily utilized, and all readings including the true standards are read against it. For reasons given, the working standard should not substitute for an actual standard, and care should be taken that the temperature of the working standard does not change during a series of readings. To exploit fluorimetry to the fullest, working standards over a 100- to 1000-fold concentration range may be desirable. Specificity. The specificity of fluorimetry can often be improved through suitable choice of filters. Both the primary filters (exciting light) and secondary filters can be varied with advantage. The exciting wavelengths have usually been limited to major mercury lines, but this is no longer necessary with more sensitive equipment. Constriction P i p e t s

All volumes less than 1 ml. are measured with Lang-Levy pipets 3 which are simple to construct 4 or which may be purchased from Microchemical Specialties Co. (Berkeley, California), Carlsberg Laboratories (Copenhagen), or Arthur H. Thomas Co. (Philadelphia). Some commercial pipets, particularly in the smaller sizes, have been unsatisfactory owing to either (1). the constriction's being too wide, which makes it 3 M. Levy, Compt. rend. tray. Lab. Carlsberg, S~rie chim. 21, 101 (1936). 40. A. Bessey, O. H. Lowry, and M. J. Brock, J. Biol. Chem. 164, 321 (1946).

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difficult to fill the pipet accurately, or (2) the tip's being too blunt, which results in too much of the sample clinging to the tip, or (3) the delivery's being too fast, which results in inaccurate delivery and leaves too much of the sample on the pipet walls. A 10-~l. pipet, for example, ought to deliver in not less than 3 seconds. Delivery time is easily lengthened by cautiously fire-polishing the tip. Calibration. Larger pipets are calibrated by delivery of water into a narrow-mouthed vial which is not more than twenty times as large as the pipet. To correct for possible error due to evaporation, a sham delivery is performed and any weight loss is noted and used in calculating the pipet volume. Smaller pipets are calibrated with 0.6% p-nitrophenol. 5 This is pipetted into an exactly measured volume of 0.01 N NaOH of about 1000 times the volume of the pipet. The reading at ~,400m, is compared to the readings of two standards prepared the same day by adding, respectively, 1.000 and 1.100 ml. of the 0.6 % p-nitrophenol to 1-1. volumes of 0.01 N NaOH. The volume of the pipet is calculated by interpolation if the optical densities are not linear. Calibration to within 0.3% is easily attainable. It will be noted that in most of the methods given the pipet volumes do not enter into the calculation of the analytical results, because standards are measured with the same pipets. Test Tubes

The only special tubes required are 7 X 70-mm. 1-ml. tubes Kimble No. 45060-5181, obtainable from A. S. Aloe Co. (St. Louis) or in lots of 15 gross from Kimble Glass Co. (Vineland, New Jersey). For centrifuging in these tubes eight-place aluminum centrifuge carriers No. 381 of the International Equipment Co. (Boston) are convenient. Mixers

When tapping with the finger is inadequate for mixing, some variety of "buzzer ''5'e is required. For 1-ml. tubes a flattened nail is mounted in the chuck of a commercial high-speed hand drill (5000 to 10,000 r.p.m.). When the tube, not more than half full, is held against the rotating nail, a violent whirling motion is imparted to the liquid contents without spilling. A commercial massage vibrator is a fairly good substitute. For 3-ml. tubes or larger, a bigger rod turning at 2000 to 5000 r.p.m, in a heavier motor is used. 50. H. Lowry, N. R. Roberts, K. Y. Leiner, M.-L. Wu, and A. L. Farr, J. Biol. Chem. 207, 1 (1954). 60. A. Bessey, O. H. Lowry, M. J. Brock, and J. A. Lopez, J. Biol. Chem. 166, 177 (1946).

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Homogenizers Thorough homogenization is necessary for reproducibility with tissue samples. Small glass homogenizers, v available from Macalaster Bicknell Co. (Cambridge, Massachusetts), are suitable for preparing 0.05 to 0.2 ml. of homogenate with a few milligrams of tissue. Somewhat larger homogenizers are supplied by Microchemical Specialties Co. (Berkeley, California). After freezing and thawing, rehomogenization is frequently required in order to obtain fair sampling. Tube Racks It is convenient for incubation to have 4 × 8-inch metal racks to hold fifty tubes each. They are made from one piece of heavy stainless steel screen (for the bottom) and two pieces of stainless steel or Monel sheet metal drilled with five rows of ten 7/(6-inch holes (2/~ inch between centers). The three decks are held 3~ inch apart by brass bolts and nuts at the corners. II. Specific Procedures Alkaline Phosphatase 8 Principle. The substrate, p-nitrophenyl phosphate, is split to free nitrophenol which in alkaline solution has strong absorption at a wavelength (410 m~) at which the substrate has little or no absorption. Reagents

16 mM disodium p-nitrophenyl phosphate. This is a 0.42 % solution of the 70% product of Sigma Chemical Co. (St. Louis). 1 M buffer, pH 10.0. This is prepared from 8.9 g. of 2-amino-2methyl-l-propanol (Distillation Products Industries), 45 ml. of 1 N HC1, and water to 100 ml. The buffer base (liquid) has a pK of 9.9. Complete buffer-substrate. This consists of equal volumes of the first two reagents plus 0.2% of the total volume of 1 M MgCl~. The complete reagent may be stored frozen for some time. Procedure. The sample, 5 to 25 ~l. (equivalent in activity to 50 to 200 ~ of brain or 5 to 20 ~ of kidney), is placed in a 7 X 70-mm. tube in a metal rack in an ice bath. Ice-cold reagent, 100 ~l., is added rapidly to all samples (mixing by tapping with the finger). The entire rack is transferred to a 38 ° water bath at zero time. After 30 minutes the rack is

7 H. H. Hess and A. Pope, J. Biol. Chem. 204, 295 (1953). 80. H. Lowry, N. R. Roberts, M.-L. Wu, W. S. Hixon, and E. J. Crawford, J. Biol. Chem. 207, 19 (1954).

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[17]

returned to the ice bath, and 300 ~l. of 0.25 N NaOH is added rapidly to each tube with mixing by buzzing. Readings are made at 410 m~ at any time within 6 hours. Standards and blanks, consisting of 10 ~1. of 1 mM p-nitrophenol or water, are carried through the entire procedure with the samples. After correcting all readings for the blank, if the same volume of sample and standard have been used, the activity of the sample in millimoles per liter per hour is 2 X sample reading/standard reading. Since protein is not removed, in exceptional circumstances the turbidity of the sample may contribute significantly to the readings and a separate blank with sample but no reagent may be needed. With a 30-minute incubation, the total time in ice water after substrate addition and before alkali addition is kept under 10 minutes to avoid significant positive error. At pH 10, alkaline phosphatases tested are slowly destroyed and therefore incubations longer than 60 minutes are undesirable. Comment on the Method. The buffer is used in high concentration to avoid pH change from CO2 in the air. This is a special danger with small volumes. The buffer usea, unlike glycine, does not inhibit the enzyme even in 1 M concentration. The enzyme activity with this buffer is about double that found in 0.1 M glycine. Phosphatase Activity at Other pH's. By changing the buffer, phosphatase activity may be measured with the same substrate at other pH's. Succinate (0.1 M final strength) is a suitable buffer in the range pH 5 to 6; acetate (0.1 M final strength) is satisfactory at more acid pH's. The substrate becomes more unstable in acid solutions, and therefore substrate and buffer are combined just before use. With these weaker buffers the final alkali strength may be reduced to 0.1 N (see also Vol. I [16]). Adenosine Triphosphatase s

Principle. Inorganic phosphate liberated is measured with molybdate at pH 4 with ascorbic acid as reducing agent. Reagents ATP, 5 mM. Buffer, pH 8.4 ± 1. The composition is Tris, 0.03 M; 2-amino-2methyl-l,3-propandiol, 0.03 M; HC1, 0.03 M; and MgC12, 2 mM. Buffer-substrate. Equal parts of buffer and substrate are mixed and may be stored frozen until the blank is too large. Molybdate reagent. 9 Two milliliters of 2.5 % ammonium molybdate, 90. H, Lowry and J. A. Lopez, J. Biol. Chem. 162, 421 (1946); see also Vol. I I I [114].

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46 ml. of acetate buffer (0.1 M acetic acid, 0.065 M NaAc), and (just before use) 2 ml. of 1% ascorbic acid, fresh or frozen.

Procedure. The incubation conditions are as given for alkaline phosphatase except for the change in buffer-substrate. An amount of enzyme equivalent in activity to 100 to 200 ~, of brain is appropriate. After the rack of incubated samples is returned to the ice bath, 20 ~1. of 30% TCA is added to each tube, which is then centrifuged. Within an hour, the liberated inorganic P is measured by adding a 100-~l. aliquot of supernatant fluid to 1 ml. of molybdate-ascorbic acid reagent in a 3-ml. tube with very prompt and thorough mixing. The color is read at 870 m~ after 15 to 45 minutes. Since ATP is slowly split by molybdate, all readings are made after the same time interval ( _ 10 minutes). Standards and blanks consisting of 10 ~1. of 15 mM KH2PO4 or water are carried through from the beginning. After correcting for the blanks, if an equal volume of enzyme solution and standard have been used, the activity of the sample is 30 mM per liter per hour X sample reading/ standard reading. Proportionality with time or amount of enzyme may not be perfect, since the adenosine diphosphate formed is inhibitory.

Inorganic Pyrophosphatase Principle. The enzyme is allowed to act on magnesium pyrophosphate in the presence of BAL. The liberated phosphate is measured under conditions which yield maximum color (~ about 25,000) with minimum hydrolysis of pyrophosphate. Reagents BAL, 65 mM. One milliliter of 10% BAL in peanut oil is extracted with 10 ml. of water (85% extraction) and stored frozen. Buffer-substrate. To 10 ml. of 2 mM sodium pyrophosphate in 0.02 M Veronal buffer at pH 7.5 is added 0.25 ml. of 65 mM BAL on the day to be used. This is chilled in ice water, and within 15 minutes of use 0.4 ml. of 0.1 M MgC12 is added. Molybdate reagent. A stock solution of 0.25% ammonium molybdate in 1.0 N H2SO4. Just before use, to 7 ml. of this is added 3 ml. of fresh or frozen 10% ascorbic acid. The final concentration after addition of sample is designed to be 0.15 % ammonium molybdate in 0.7 N H2SO4 with nearly 3% ascorbic acid.

Procedure. The incubation conditions are the same as those for alkaline phosphatase except for the change in buffer-substrate. Activity equivalent

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TECHNIQUES FOR METABOLIC STUDIES

[17]

to that of 50 to 200 ~, of brain is appropriate. After the incubation is arrested in ice water, 20 ~l. of 30% TCA is added per tube. After centrifuging, and within 2 hours, 20 #1. of supernatant fluid is added to 1 ml. of molybdate reagent in a 3-ml. tube. The mixture is heated 20 minutes at 38 °, cooled to room temperature, and read within the next half hour at 820 m~. In hot weather after the samples are heated they are kept at 25 ° or below until read. Comment on Pyrophosphatase Measurement. The properties of pyrophosphatase are rather specialized, and without further studies the procedure given can be guaranteed only for brain. Pyrophosphatase in brain homogenates is not stable in water, but in 0.02 M MgC12:0.01 M BAL the activity may be preserved for some time. The concentration of pyrophosphate cannot exceed 1 mole per 2 moles of Mg without inhibition, although excess Mg is not inhibitory. Magnesium pyrophosphate is very insoluble, and the reagent must be used before precipitation proceeds too far. An increase in the concentration of substrate accelerates precipitation, and lower results are obtained after the first few minutes. Since the concentration of substrate is ]ow, the permissible degree of splitting is limited to 1 mM. The presence of BAL interferes with the measurement of inorganic phosphate at pH 4 or pH 2, although with addition of slightly more than 1 mole of CuS04 per mole of BAL this inhibition may be overcome. The phosphate method given circumvents this difficulty by permitting greater final dilution. Other Phosphatases. Any phosphatase which acts on a substrate more stable than pyrophosphate may be measured in a similar manner with appropriate changes in the buffer-substrate. When stability of the substrate is not of first consideration, a somewhat more stable phosphate reagent may be substituted. This is 1% ascorbic acid and 0.25% ammonium molybdate in 1 N sulfuric acid. 5 With this reagent 2 hours at 38 ° is required for full color development. The molar extinction coefficient is about 26,000. Fumarase 8

Principle. Malate formed is measured by the fluorescent product formed with ~-naphthol in 65% sulfuric acid. The procedure is based on an unpublished method of the late Dr. John F. Speck. Reagents Buffer-substrate, pH 6.8 _+ 0.1. This is 0.02 M fumaric acid in 0.04 M NasHPO4. It is stored frozen to discourage bacterial growth. Fluorescence reagent. This is a fresh mixture of 1 vol. of 56 mg. %

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375

/3-naphthol in 0.004 N NaOH with 25 vol. of 7:1 sulfuric acid (875 ml. of concentrated H2S04 and 125 ml. of water). The stock #-naphthol may be preserved frozen for a long time and is discarded when it turns quite yellow. The viscous final reagent is conveniently handled with a syringe pipet. Procedure. Other than the change in buffer-substrate, the incubation conditions are identical to those given for alkaline phosphatase. Enzyme activity equivalent to that of 50 to 250 ~ brain is appropriate. After enzyme action is arrested by placing the rack of samples in an ice bath, a 10-~l. aliquot of each sample is added to 1 ml. of fluorescence reagent in a 3-ml. fluorimeter tube. Vigorous mixing is necessary because of the viscosity. For this, a large buzzer is very convenient. The tubes are then stoppered with corks covered with aluminum foil and heated for 30 minutes in a shallow boiling water bath. A delay up to an hour is permissible before heating. The tubes are cooled just to room temperature and wiped with a clean towel dampened with distilled water. They are mixed again and read in the fluorimeter with a primary filter which isolates the Hg line at X~65m, (Corning glass No. 5860) and a secondary filter with transmission maximum at 465 m~ (Corning glass No. 5543 plus No. 3387). Readings are made against working standards of quinine in 0.1 N H2SO4. Comment on the Fumarase Method. The fluorescence does not change for 3 hours or longer after heating. The time of heating is not critical, since doubling the prescribed period does not affect the readings. The sulfuric acid concentration is critical. The substitution of 6:1 sulfuric acid for 7:1 lowers the fluorescence by 14%, and concentrated sulfuric acid gives even lower results. The concentration of f~-naphthol is less critical for optimal readings but must be kept within 20% of the concentration recommended. Most of the blank fluorescence is contributed by the f~-naphthol. A l d o l a s e S , 10

Principle. Triose phosphates, formed from FDP, are trapped with hydrazine and then converted with alkali to methylglyoxal, or something very similar. This is treated with dinitrophenylhydrazine in HC1 and finally diluted with alkaline Methyl Cellosolve to yield a violet solution which is read at 570 mu (see also Vol. III [32]). Reagents Substrate reagent. This is a fresh mixture of equal parts of 0.02 M magnesium F D P and 0.12 M hydrazine of pH 8.2 ___ 0.2. Each 10 j . A. Sibley and A. L. Lehninger, J. Biol. Chem. 177, 859_'(1949).

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TECHNIQUES FOR METABOLIC STUDIES

[17]

component may be preserved for long periods if frozen. The hydrazine is prepared either from hydrazine sulfate with 1.5 moles of NaOH per mole of hydrazine or from hydrazine hydrate with 0.5 mole of HC1 per mole of hydrazine. Reagents prepared from 100% hydrazine hydrate (Fairmount Chemical Company, Inc., 136 Liberty Street, New York) have given lower blank values. TCA, 30% solution. NaOH, 0.6 N. Dinitrophenylhydrazine, a 0.075% solution in 1.5 N HC1. This is stable for long periods at 4 ° but is centrifuged if not crystal-clear. Alkaline ~Iethyl Cellosolve. A fresh mixture of 1 vol. of 1 h r NaOH and 2 vol. of ethylene glycol monomethyl ether. Procedure. The incubation conditions are as described for alkaline phosphatase except for the change in substrate reagent. However, the sample volume is kept within the limits of 10 to 20 ~l. in order to keep the conditions more nearly constant in the subsequent steps. Enzyme equivalent to 50 to 200 ~/of brain is appropriate for a 30-minute incubation. After the incubation is arrested by placing the rack of tubes in an ice bath, 20 ~l. of 30% TCA is added to each sample. However, it is unnecessary to remove precipitated protein unless more than 500 ~, of tissue has been used. 11 To each sample is added 100 ~l. of 0.6 N NaOH with prompt mixing. This gives a total volume of 230 to 240 ~I. A 50-~1. aliquot is transferred to the bottom of a 3-ml. test tube (10 X 75 mm.). After the sample has stood at room temperature for 30 _ 5 minutes from the time of alkali addition, 50 ~l. of dinitrophenylhydrazine solution is added to each sample with careful mixing. After 30 to 60 minutes at room temperature, 1 ml. of alkaline Methyl Cellosolve is added. Readings are made after 30 to 45 minutes at 570 m~. There is no convenient standard at present. Occasional standardization may be made, if necessary, by means of alkali-labile P, or with conversion of the triose phosphates formed to methylglyoxal. 8 By such means a molar extinction coefficient of 58,000 for the splitting of F D P has been obtained, and for most purposes it may be satisfactory to use this value. (For example, if the volume of sample used was 10 t~l. and a final optical density of 0.580 was obtained with a 30-minute incubation, the activity of the sample would be (0.580/58,000) X (60/30) X (1100/50) X (230/10) --- 10.1 X 10-3mole/1. of original sample per hour.) The nonphosphorylated trioses do not give as much color as the triose phosphates, nor do they give color proportionality. H If too much protein is present, the sample is centrifuged and I00 ~1. of the supernatant fluid is transferred to another tube before proceeding.

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Comment on Aldolase Method. Hydrazine acts both as a buffer and triose trap. Its concentration is not critical during incubation but it is critical later. Acidification (with TCA) for some unknown reason is required for full color development. If necessary, the analysis may be interrupted at this point overnight at 4 ° . The most critical step is the first alkali treatment during which the triose phosphates are probably converted to methylglyoxal. With too little hydrazine or too much alkali the reaction goes too far and lactate is formed. No chromogen at all is formed in the absence of hydrazine. The reaction with dinitrophenylhydrazine presents no problem. In the final alkaline step the Methyl Cellosolve is necessary to stabilize the color. The final net alkali concentration must be at least 0.15 N to prevent fading and a high blank. With high levels of triose phosphate, precipitation occurs during the reaction of dinitrophenylhydrazine and final mixing must be adequate to dissolve any precipitate. Lactic D e h y d r o g e n a s e 12

Principle. Lactate is oxidized to pyruvate by DPN at pH 9.9. The pyruvate is measured by forming the 3-quinolylhydrazone, which has an intense absorption at 305 mu. Although the reaction is measured in the less favorable direction as far as equilibrium is concerned, the high substrate level and high pH result in an initial velocity of the same order of magnitude as in the reverse direction. Reagents

Buffer-substrate. The buffer is 0.1 M 2-amino-2-methyl-l-propanol in 0.05 M HC1 (see Alkaline Phosphatase section). To this is added 2.5 ml. of 4.5 M sodium lactate (50%) per 100 ml., and the mixture is preserved in the frozen state. Just before use DPN equivalent to 0.8 mg. of pure DPN is added as a dry powder to each milliliter of buffer-lactate, and this is kept in ice water. (DPN concentration is about 1 raM.) 3-Quinolylhydrazine. The stock solution is a 25 mM (0.4%) solution in 0.1 N HC1. This is stable for some months at 4 °. For use this is diluted to 1.5 mM with 0.45 N HC1. The dilute solution is discarded after 1 week at 4 °. HC1, 0.01 N. Procedure. Except for the change in substrate the incubation steps are those given for alkaline phosphatase. For a 15-minute incubation 1~E. Robins, N. R. Roberts, K. M. Eydt, O. H. Lowry, and D. E. Smith, J. Biol. Chem. 218, 897 (1956).

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lactic dehydrogenase activity equivalent to that of 15 to 75 ~ of brain is satisfactory. The sample volume may be 5 to 20 ~l. After the incubation is arrested in an ice bath, 20 ~l. of 1.5 M quinolylhydrazine in HC1 is added to each sample, a n d the rack of tubes is allowed to sit for 60 minutes at room temperature. Each sample is then diluted with 0.5 ml. of 0.01 N HC1 and mixed by inversion against Parafilm. Readings are made at 305 m~ within 10 to 30 minutes (see below). Standards of 10 ~1. of 0.75 and 1.5 mM pyruvate and blanks of 10 ~l. of water are carried through all steps. The molar extinction coefficient is about 22,000. However, to this must be added a contribution from D P N H of about 6000. (Although the 340-m~ band of D P N H disappears at once on acidification, a new band appears at shorter wavelengths. This falls with time and has the indicated value under the prescribed conditions.) For most purposes it is sufficient to use the standard pyruvate readings but to multiply the result by 22,000/28,000. Thus if the 0.75 mM standard was used at a volume equal to that of the sample, and if the incubation time was 15 minutes, the activity of the sample would be (60/15) X (22/28) X 0.75 X reading of sample/reading of standard. C o m m e n t s on Method. Pyruvate formation, as measured, may not be strictly linear with time or amount of enzyme if the net optical density is more than about 0.3 (about 0.06 millimole oxidized per liter of incubation mixture). The falloff is largely due to accumulation of DPNH and pyruvate. Standard readings are essentially linear with pyruvate concentration. Glycine buffer may be substituted for the buffer recommended, but in this case the blank value increases greatly on storage. The D P N H contribution decreases from about 6000 (molar extinction coefficient) 10 minutes after dilution to about 4000 two hours later. Therefore, if there is a long delay in reading, suitable correction is necessary. Malic and Glutamic D e h y d r o g e n a s e s ~

These enzymes may be measured in the same manner as lactic dehydrogenase, since the oxalacetate or a-ketoglutarate produced react with quinolylhydrazine to give products of very similar absorption in the ultraviolet. For malic dehydrogenase the substrate reagent is 50 mM malate and 2 mM DPN in 0.1 M 2-amino-2-methyl-l-propanol buffer at pH 10.5 to 10.6. For glutamic dehydrogenase the substrate reagent is 90 mM glutamate and 5 mM DPN in 0.05 M Veronal buffer at pH 8.0 to 8.1. The activity of this enzyme, at least in brain, falls off rapidly at 38 °. Therefore it is measured at 30° .

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379

Malic D e h y d r o g e n a s e 1~

Principle. Malate is oxidized with DPN, and the D P N H produced is measured at 340 m~. The reaction is carried out in selected 3-ml. tubes held in a special adapter for the Beckman spectrophotometer. 8 Reagents Buffer-substrate. The buffer is 0.1 M 2-amino-2-methyl-l-propanol in 0.015 M HC1 (see Alkaline Phosphatase section). To this is added 5 ml. of 1 M potassium malate made from malic acid which has been recrystallized (from warm ethyl acetate with the addition of petroleum ether). The mixture is stored frozen. Within an hour or two of use dry D P N equivalent to 1.6 mg. of pure D P N is added per milliliter of buffer-malate (to give about 2 mM DPN). The complete reagent is kept in ice water until the beginning of the incubation.

Procedure. One milliliter of complete buffer substrate is placed in a 3-ml. tube in a 30 ° water bath. Enzyme equivalent in activity to 5 to 50 "~ of brain is added ill a volume of 10 to 50 ~1. After mixing a zero time reading is made. Subsequent readings are made at appropriate intervals with the tube returned to the bath between readings. With a light path of 0.8 cm. an optical density change of 0.3 in 20 minutes would correspond to (0.3 × 10-~ X 60/20)/(6270 X 0.8) = 0.179 micromole of D P N reduced per hour (~ = 6270). The rate is not linear with optical density change of more than 0.4 (0.075 m M D P N H ) . Phosphogluconic Dehydrogenase (method of Dr. R. W. Albers, to be published),

Principle. T P N is reduced by 6-phosphoglueonate, and the T P N H is measured at 340 m# after the reaction is stopped with Methyl Cellosolve. Reagents Buffer-substrate. The final composition is 10 mM MgCl~, 1 mM disodium ethylenediaminetetraacetic acid (Versene), 22 mM potassium-6-phosphogluconate, and 1 mM T P N in 0.08 M 2-ami~m-2-methyl-l,3-propandiol (HC1) buffer at pH 9.0. This may be stored frozen without the T1)N which is added as a powder just before use (about 1 mg./ml.). Alternatively the T P N may be prepared as a 10 mM solution in 0.01 M succinate buffer, pH 6, and stored frozen. 13 j . Strominger and O. H. Lowry, J. Biol. Chem. 213, 635 (1955).

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TECHNIQUES FOR METABOLIC STUDIES

[17]

Diluent. A mixture of 1 vol. of 1 M KC1 with 2 vol. of ethylene glycol monomethyl ether (Methyl Cellosolve). The organic solvent must not contain free acid, which would destroy D P N H .

Procedure. The sample, equivalent in activity to 25 ~/of adrenal gland or 100 ~, of brain, in a volume of not more than 10 ~l. is placed in a 7 X 70-mm. tube in a metal rack in an ice bath. To each sample is added 50 ~l. of ice-cold buffer-substrate. The rack of tubes is transferred to a 38 ° bath for 60 minutes. After enzyme action is arrested by placing the rack of tubes in ice water, 300 ~l. of Methyl Cellosolve diluent is added. The protein precipitated by the salt and organic solvent is removed by centrifuging, and the optical density of the supernatant fluid is read at 340 m~. If 50 -y of tissue in 10 ~l. (final volume 360 ~l.) was incubated for 30 minutes and a net increase of 0.25 in optical density occurred, the activity of the original tissue would be (360,000/50) X (60/30) X (0.25/6270) = 0.575 moles per kilogram and hour (6270 is taken to be the molar extinction coefficient of TPNH). X a n t h i n e O x i d a s e ~4,~5

Principle. The enzyme is allowed to pteridine le in the presence of phosphate fluorescence of the substrate. The product measured by its fluorescence, which is little

oxidize 2-amino-4-hydroxybuffer which quenches the formed, isoxanthopterin, is affected by phosphate.

Reagents Buffer-substrate. A 5 X 10-4 M (8.15 rag. %) stock solution of 2-amino-4-hydroxypteridine is prepared in water and frozen to preserve. On the day of use this is diluted 100-fold with 0.2 M phosphate buffer, pH 7.2 to 7.3.

Procedure. One milliliter of buffer-substrate in a fluorimeter tube is brought to 30 ° in a water bath, and 10 to 100 ~l. of enzyme sample is added with activity sufficient to oxidize half the substrate in 30 to 90 minutes (e.g., 1 mg. of rat liver, 2 mg. of rat kidney). An initial reading is made as soon as possible at an instrumental setting with quinine sulfate in 0.1 N H2SO~ which will permit approximately full-scale reading with 100% oxidation of substrate. (The fluorimeter filters are Corning No. 5860 14 O. II. Lowry, O. A. Bessey, and E. J. Crawford, J. Biol. Chem. 180, 399 (1949). 15 H. B. Burch, O. H. Lowry, A. M. Padflla, and A. Marshall, in preparation for early publication. le This m a y be obtained from Lederle Laboratories, Pearl River, New York.

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HmTOCHEM~CAL METHODS FOR ENZYMES

381

as primary, and Nos. 5113 plus 3389 as secondary.) The tube is returned to the water bath at 30 °, and two or three subsequent readings are made in the interval between 0 and 50% oxidation. The sample is mixed before each reading. A final reading is taken after oxidation is complete, i.e., after an interval three or four times as long as that required for 50% oxidation. If an active xanthine oxidase preparation is available, it may be used to complete the reaction in a few minutes. Care is taken that the working standard (see Introduction) does not change temperature over the experimental period. If the fluorescence change is not linear with time, the initial velocity may be calculated graphically. One milliliter of substrate contains 5 X 10-6 millimole. Therefore, if ] rag. of tissue caused a fluorescence change of 1 galvanometer division per minute and the total change for 100% oxidation was 80 divisions, the tissue enzyme activity would be (l/80) × 5 X 10-6 X 60 × 106 = 3.75 millimoles per kilogram and hour. Comment on Xanthine Oxidase. The Michaelis constant for xanthine oxidase with aminohydroxypteridine is quite small so that the enzyme is saturated throughout most of the course of the reaction. The rate may fall off, however, because of marked product inhibition. The velocity expressed as per cent of total substrate oxidized will of course vary with the substrate concentration. The level chosen is low enough to permit a good rate without requiring an amount of tissue which would produce too much turbidity and consequent quenching. It is also low enough to prevent self-quenching. On the other hand, the substrate level is high enough to provide strong fluorescence which avoids troublesome optical blanks. The procedure used and method of calculation, whereby each sample is its own standard, automatically corrects for quenching or fluorescence contribution from the enzyme sample.

[18] H i s t o c h e m i c a l M e t h o d s for E n z y m e s B y GEORGE GOMORI

I. Oxidative Enzymes Succinic Dehydrogenase Principle. In the presence of substrate, succinic dehydrogenase reduces colorless tetrazolium salts to highly colored, insoluble formazans 1,~ which precipitate at the sites of enzymatic activity. Since oxygen (probA. M. Rutenburg, R. Gofstein, and A. M. Seligman, Cancer Research 10, 113 (1950). 2 A. M. Seligman and A. M. Rutenburg, ,.~cience 115, 317 (1951).

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H I S T O C H E M I C AMETHODS L FOR ENZYMES

381

as primary, and Nos. 5113 plus 3389 as secondary.) The tube is returned to the water bath at 30 ° , and two or three subsequent readings are made in the interval between 0 and 50% oxidation. The sample is mixed before each reading. A final reading is taken after oxidation is complete, i.e., after an interval three or four times as long as that required for 50% oxidation. If an active xanthine oxidase preparation is available, it may be used to complete the reaction in a few minutes. Care is taken that the working standard (see Introduction) does not change temperature over the experimental period. If the fluorescence change is not linear with time, the initial velocity may be calculated graphically. One milliliter of substrate contains 5 × 10-6 millimole. Therefore, if 1 mg. of tissue caused a fluorescence change of 1 galvanometer division per minute and the total change for 100% oxidation was 80 divisions, the tissue enzyme activity would be (1/80) × 5 X 10-6 × 60 × 10 e = 3.75 millimoles per kilogram and hour. Comment on Xanthine Oxidase. The Michaelis constant for xanthine oxidase with aminohydroxypteridine is quite small so that the enzyme is saturated throughout most of the course of the reaction. The rate may fall off, however, because of marked product inhibition. The velocity expressed as per cent of total substrate oxidized will of course vary with the substrate concentration. The level chosen is low enough to permit a good rate without requiring an amount of tissue which would produce too much turbidity and consequent quenching. It is also low enough to prevent self-quenching. On the other hand, the substrate level is high enough to provide strong fluorescence which avoids troublesome optical blanks. The procedure used and method of calculation, whereby each sample is its own standard, automatically corrects for quenching or fluorescence contribution from the enzyme sample.

[18] Histochemical Methods for Enzymes By GEORGE GOMORI

I. Oxidative Enzymes Succinic Dehydrogenase Principle. In the presence of substrate, succinic dehydrogenase reduces colorless tetrazolium salts to highly colored, insoluble formazans 1,~ which precipitate at the sites of enzymatic activity. Since oxygen (probA. M. Rutenburg, R. Gofstein, and A. M. Seligman, Cancer Research 10, 113 (1950). 2 A. M. Seligman and A. M. Rutenburg, Science 115~ 317 (1951).

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TECHNIQUES FOR METABOLIC STUDIES

[18]

ably through the cytochrome system) may compete with tetrazolium as a hydrogen acceptor, the experiment should be run under anaerobic conditions, 3,4 or, simpler, the cytochrome system should be poisoned with cyanide, s

Reagents 0.1% solution of neotetrazolium chloride. 0.1 M phosphate buffer, pH 7.0 to 7.2. 0.25 M Na succinate. 0.1 M NaCN.

Procedure. Use razor blade slices (1/~ to 1 ram. thick) of fresh tissues, or teased preparations. Fresh frozen sections (30 to 40 ~ thick) are also suitable. Prepare the substrate solution by mixing 3 ml. each of neotetrazolium, buffer, and succinate; add 1 ml. of cyanide. Incubate the tissue slices at 37 ° for 30 to 60 minutes. Fix in 10 to 20% formalin for 24 hours. Cut on the freezing microtome, and mount the sections in glycerin jelly. The slides should be inspected promptly because the dye has a tendency to migrate into fat droplets. Results. The sites of activity are in purple. Specificity. The pattern of localization obtained is a composite of the activities of succinic dehydrogenase and of the so-called endogenous dehydrogenases which require no added substrate. The difference between the pictures obtained by the regular technique and in the absence of added succinate shows the true localization of succinic dehydrogenase. In frozen sections, all activity is due to succinic dehydrogenase because endogenous dehydrogenases are destroyed. 5~ Other Dehydrogenases Dehydrogenases requiring the presence of coenzyme I (alcohol, lactic, malic, glutamic dehydrogenases, etc.) can be demonstrated by substituting the corresponding substrate for succinate and adding about 2 to 3 mg. of D P N to the incubating mixture. Cytochrome Oxidase Principle. Cytochrome oxidizes dimethyl-p-phenylenediamine to a quinone; the latter couples with ~-naphthol to form an insoluble blue 8 E. Kun, Proc. Soc. Exptl. Biol. Med. 78, 195 (1951). 4 H. H. H i a t t a n d E. Shorr, Proc. Soc. Exptl. Biol. Med. 82, 543 (1953). 5 C. G. Rosa a n d J. T. Velardo, J. Histochem. Cylochem. 2, 110 (1954). 5~ E. Shelton a n d W. C. Schneider, Anat. Record 112, 61 (1952).

[18]

HISTOCHEMICAL METHODS FOR ENZYMES

383

dye. Reduced cytochrome, in turn, is reoxidized by cytochrome oxidase in the presence of oxygen. 6 Reagents

1% solution of a-naphthol in 40% ethanol. 1% aqueous solution of dimethyl-p-phenylenediamine-HC1. 0.1 M phosphate buffer, pH 7.4. Procedure (So-Called G-Nadi ReaclionT). Use small fragments of fresh tissue or razor blade slides. Frozen sections of unfixed tissue may also be used, but they are distinctly less active. Prepare the substrate solution just before use by adding 1 ml. each of the naphthol and of the dimethyl-p-phenylenediamine solution to 25 ml. of the buffer. Incubate the tissue at room temperature for 5 to 10 minutes. Counterstaining with alum carmine or lithium carmine is permissible. Mount in glycerin jelly. Results. The sites of activity are in blue. The dye has a tendency to migrate into fat droplets which will be stained purplish. After a while, the preparation will fade. Specificity. At the pH given and with an incubation time not exceeding 10 minutes, the method is reasonably specific. Note. The enzymatic nature of the reaction obtained at much higher pH levels (M-nadi reaction 7) is open to doubt.

Dopa Oxidase Principle. 1-Dioxyphenylalanine is oxidized by dopa oxidase to insoluble brown-black melanin, s Reagents

Dopa (dioxyphenylalanine; the commercial dl grade is satisfactory). 0.1 M phosphate buffer, pH 7.3 to 7.4. Procedure2 Use frozen sections fixed briefly (for about 4 to 6 hours) in 5 % formalin. Rinse in distilled water. Prepare the substrate solution by dissolving 0.1% dopa in the buffer. Incubate the sections in an open dish at 20 to 37 ° for 4 to 5 hours. If the solution darkens, transfer the sections to a fresh solution. Rinse, counterstain lightly with hematoxylin, dehydrate, and mount.

e D. Keilin, Ergebn. Enzymforsch. 2, 239 (1933). 7 E. V. Gierke, Miinch. Med. Wochschr. 58, 2315 (1911). B. Bloch and P. t~yhiner, Z. ges. exptl. Med. 5, 179 (1916-17). 9G. F. Laidlaw and S. N. Blackberg, Am. J. Pathol. 8, 491 (1932).

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TECHNIQUES FOR METABOLIC STUDIES

[18]

Results. Melanin, both performed and produced by enzymatic activity, stains brown to black. Specificity. For all practical purposes, the method is absolutely specific. Peroxidase

The only peroxidase identifiable histochemically is hemoglobin. The enzymatic nature of the peroxidase-like activity of myeloid granules is open to doubt. Principle. In the presence of H202 and peroxidase, some leuco dyes are reoxidized to their colored form, and benzidine into a brown or blue quinoid dye. The Z i n c - L e u c o - D y e Method ~°

Reagents Patent blue (C.I. No. 712). Acetic acid. Zinc dust. H202 (commercial 3%).

Procedure. Fix the tissue in neutralized formalin. Frozen sections are preferable, but the method will work reasonably well in paraffin sections also. Smears should be fixed in formalin-alcohol (1 : 10). Dissolve 1 g. of Patent blue in 100 ml. of a 2% solution of acetic acid; add about 5 to 10 g. of zinc dust, and boil the mixture until decolorized to an amber shade. Add 2 ml. more of acetic acid. Keep the solution in the icebox. In case of recolorization, heat for a few minutes until the color is gone. For use, filter about 10 ml., add 1 ml. of H202, and place the tissue in the mixture (or pour the mixture over the slide). Hemoglobin will stain intense green-blue in a matter of 10 to 20 seconds. Counterstain as desired, dehydrate, and mount. The Benzidine Method H,12

Reagents Benzidine (reagent grade). 95 % ethanol. Na nitroprusside. H202 (commercial 3%).

Procedure. Preparation of the tissue is the same as for the zinc-leucodye method. ~0L. Lison, Compt. rend. soc. biol. 106, 1266 (1931). 1~A. H. Washburn, J. Lab. Clin. Med. 14~ 246 (1928). ~ E. E. Osgood, "Atlas of Hematology." Stacey, San Francisco, 1937.

[18]

HISTOCHEMICAL METHODS FOR ENZYMES

385

Dissolve about 200 rag. of benzidine in 100 ml. of ethanol. Add about 500 rag. of Na nitroprusside dissolved in a few milliliters of water. Keep the reagent in the icebox. For use, mix equal volumes of the mixture and of a 1 : 5 dilution of H~O~. Place the tissue in the mixed reagent for about 5 minutes. Wash the tissue, counterstain with alum carmine, dehydrate, and mount. Hemoglobin stains in shades ranging from dark greenish brown to greenish blue. Specificity. The zinc-leuco-dye method stains only native or slightly altered hemoglobin; the benzidine method stains also considerably degraded hemoglobin and may actually depend on nonenzymatic heine catalysis.

II. Hydrolytic Enzymes Alkaline Phosphatase The Calcium-Cobalt Method. 13,1~ Principle. Phosphatase hydrolyses glycerophosphate around pH 9; the phosphate ions liberated are precipitated in the form of calcium phosphate. The latter is transformed first into cobalt phosphate and, in a second step, into black cobalt sulfide.

Reagents 0.1 M solution of Na glycerophosphate (any commercial brand). 0.2 M solution of calcium chloride. 0.05 M solution of magnesium chloride. Na barbital. 2 to 3 % solution of cobaltous acetate. Light yellow ammonium sulfide.

Procedure. Use paraffin sections of tissue fixed in 80 to 100% ethanol or acetone. Mix 20 ml. each of the glycerophosphate and CaCl~ solutions, and add 10 ml. of the MgC12 solution. Stir in about 0.5 g. of Na barbital. If the solution is turbid owing to the presence of inorganic phosphate, it should be filtered. Incubate the slides for 30 to 90 minutes at 37 ° . Rinse in distilled water, and place in the cobalt solution for 5 minutes. Wash under the tap for 1 minute. Transfer to a dilute solution of ammonium sulfide (2 to 3 drops to a Coplin jarful of distilled water) for 2 minutes. Wash under the tap, counterstain as desired, dehydrate, and mount. Results. The sites of activity are in black or shades of gray. Specificity. The reaction itself is, for all practical purposes, absolutely specific; however, preformed calcifications may be mistaken for sites of 13 H. Takamatsu, Trans. Soc. Pathol. Japon. 29~ 492 (1939). 14 G. Gomori, Proc. Soc. Exptl. Biol. Med. 42~ 23 (1939).

386

TECHNIQUES FOR METABOLIC STUDIES

[18]

activity. Such calcifications can be removed before incubation with a citrate buffer, pH 4.5, in about 10 minutes. T h e Azo-Dye Method. 15,16 Principle. Phosphatase hydrolyzes ~naphthyl phosphate; the naphthol liberated is coupled with a diazonium salt to form an intensely colored azo dye.

Reagents Na a-naphthyl phosphate.17 0.05 M solution of magnesium chloride. Na barbital or borax. Diazo Red T R Salt.ls

Procedure. Preparation of the tissue is the same as for the calciumcobalt method. Dissolve about 10 rag. of naphthyl phosphate in 40 ml. of distilled water. Add 10 ml. of MgC12 solution. Add about 0.5 g. of Na barbital or borax and 50 mg. of diazonium salt. Stir until dissolved. Filter into a Coplin jar. Incubate the sections at room temperature until, under the microscope, the sites of activity show up in a rich red-brown shade (usually 10 to 30 minutes). Counterstain with hematoxylin, and mount in glycerin jelly. Specificity. The method is absolutely specific. 5 - N u c l e o t i d a s e 19

The procedure is identical with the calcium-cobalt method for alkaline phosphatase, except that glycerophosphate is replaced by about 20 mg. of 5-adenylic acid in 20 ml. of water. Incubation may have to be extended to about 3 hours. Acid P h o s p h a t a s e ~°

Principle. Acid phosphatase hydrolyzes glycerophosphate around pH 5; the phosphate ions liberated are precipitated in the form of lead phosphate; the latter is transformed into brown-black lead sulfide. Reagents 0.1 M solution of Na glycerophosphate (any commercial brand). 1~ G. Gomori, J. Lab. Clin. Med. 37, 526 (1951). 1~ E. Grogg and A. G. Everson Pearse, Nature 170, 578 (1952). 17 Obtainable from Dajac Laboratories, 511 Lancaster St., Leominster, Massachusetts. is Obtainable from General Dyestuff Corp. 19 G. Gomori, Proc. Soc. Exptl. Biol. Med. 72, 449 (1949). ~0 G. Gomori, Stain Technol. 25, 81 (1950).

[18]

HISTOCHEMICAL METHODS FOR ENZYMES

~87

0.05 M acetate buffer, pH 5. Lead nitrate. Light yellow ammonium sulfide.

Procedure. Use frozen sections of tissues fixed in ice-cold neutralized formalin for not longer than 24 hours. Paraffin-embedded material after fixation in cold absolute acetone may give good results, but partial and total failures are common. After fixation in alcohol or in formalin, paraffin sections are practically inactive. Dissolve 0.2 g. of lead nitrate in 200 ml. of acetate buffer. Gradually stir in 20 ml. of glycerophosphate solution. The mixture, which is somewhat turbid, can be kept in the icebox for about 2 months. For use, filter 40 ml. into a Coplin jar; add 10 ml. of distilled water. Incubate the sections at 37 ° for 30 minutes to 16 hours, depending on their activity. Human prostate may require only 10 to 15 minutes; other tissues, several hours. Rinse the sections for 1 minute in 2 % acetic acid, followed by distilled water. Immerse in a dilute solution of ammonium sulfide (see under Alkaline Phosphatase, the calcium-cobalt method). Counterstain as desired. Dehydrate in alcohols; clear in gasoline or tetrachloroethylene, and mount in a synthetic resin dissolved in one of the solvents mentioned. Results. The sites of activity are in brown-black. Specificity. Preformed calcifications and nerve tissue (axons) may be impregnated by lead and give a spurious reaction. A section run in the presence of 0.005 M fluoride may serve as a control; any coloration obtained in the presence of this inhibitor is due to nonenzymatic impregnation. Partial and complete failures (negative reactions in spite of the known presence of enzyme) are not rare with this technique. Phosphamidase 2~

Principle. Phosphamidase hydrolyzes phosphamides around pH 5.5; the phosphate ions liberated are precipitated in the form of lead phosphate; the latter is transformed into brown-black lead sulfide. Reagents 0.05 M maleate buffer, pH 5.5 to 5.6. 0.1 M lead nitrate. 0.1 M chloranilidophosphonate. 2-° 31 G. Gomori, Proc. Soc. Exptl. Biol. Med. 69, 407 (I948). ~2 A soluble salt (exact formula not declared), very suitable as a substrate, is obtainable under the n a m e " M y 2 1 " from the firm Bayer in Leverkusen, G e r m a n y (0.22-g. ampoules, to be dissolved in 10 ml. of distilled water).

388

TECHNIQUES FOR METABOLIC STUDIES

[18]

0.1 M solution of manganous chloride. 0.1 M citrate buffer, pH 4.5.

Procedure. Use paraffin sections of acetone or alcohol-fixed material. Blow 1.5 ml. of the lead nitrate solution into 50 ml. of buffer, and heat slightly until the initial precipitate disappears; add 1 to 2 ml. of the phosphonate solution. Pour the solution into a Coplin jar which should be supported at an angle so that the tissue sections face downward. Incubate at 37 ° for 8 to 16 hours. Wipe the precipitate from the back of the slides and around the tissue; rinse the slides in distilled water. Differentiate carefully in citrate buffer until the slide around the tissue appears completely clear. Immerse in ammonium sulfide, etc., as in the method for acid phosphatase. Results. The sites of activity are in brown-black. Specificity. Specificity and reliability of the reaction are essentially the same as those of the method for acid phosphatase. Note. The differentiation in citrate buffer is a delicate step on which success or failure may depend. Insufficient differentiation will leave a coarse black precipitate all over the slide; overdifferentiation may remove the phosphate deposited by enzymatic action. It is advisable to incubate several slides of the same material and to determine the optimal length of differentiation by trial and error. Phosphorylase 2'~

Principle. Phosphorylase produces glycogen from glucose-l-phosphate. The reaction is accelerated in the presence of glycogen as a primer, and of adenylic acid and insulin as activators. Freshly formed glycogen stains with iodine purple-blue, very much like starch, in contrast to preformed glycogen, which stains brown. Reagents Glucose-l-phosphate. Glycogen. Muscle adenylic acid (adenosine-5-phosphate). Regular insulin for injections (40 to 80 units/ml.). 0.2 M acetate buffer, pH 5.7 to 6 (1 part of 0.2 M acetic acid and 20 parts of 0.2 M Na acetate). Gram-Lugol's iodine solution (1 g. of Is and 2 g. of KI in 300 ml. of distilled water).

Procedure. Cut fresh frozen sections, 20 to 30 t~ thick. Incubate them for 2 to 4 hours at 37 ° in a substrate solution made up by dissolving ~a T. Takeuchi and H. Kuriaki, J. Histochem. Cytochem. 8, 153 (1955).

[18]

HISTOCHEMICAL METHODS FOR ENZYMES

389

about 50 mg. of K-glucose-l-phosphate and 10 mg. of adenylic acid in 15 ml. of distilled water. Add 10 ml. of acetate buffer, a few drops of insulin, and about 5 rag. of glycogen (not strictly necessary). Immerse the sections for a few minutes in dilute Gram-Lugol's solution. Mount the sections in a mixture of 1 part of Gram-Lugol's solution and 4 parts of glycerol. Results. Enzymatically formed glycogen stains in shades of purplish or blackish; the background, in brown-yellow. The stain is not permanent. Specificity. The specificity is apparently absolute. ~-Esterase

24

Principle. Certain esterases hydrolyze a-naphthyl acetate. The naphthol liberated is coupled with a diazonium salt to form an intensely colored azo dye. Reagents a-Naphthyl acetate. 0.05 M phosphate buffer, pH 7.8 to 8.0. Diazo Red RC or Diazo Blue B Salt.

Procedure. Use paraffin sections of material fixed in cold acetone or cold neutral formalin. In the case of formalin fixation, the fixative must be washed out thoroughly before dehydration. Dissolve about 10 mg. of naphthyl acetate in 1 or 2 ml. of methanol; add 50 ml. of buffer; stir in about 50 mg. of either of the diazonium salts mentioned. Filter the mixture into a Coplin jar. Incubate the slides at room temperature (in the case of highly active tissues, cooling with ice may be advisable) until, under the microscope, the sites of activity appear in a rich red-brown (Red RC Salt) or black (Blue B Salt) shade (about 5 to 20 minutes). Counterstain as desired. Mount in glycerin jelly. A S E s t e r a s e 25

Principle. The principle is the same as that of ~-esterase. Reagents 1% solution of naphthol AS acetate 17 in acetone. Propylene glycol. 0.05 M phosphate buffer, pH 6.3 to 6.5. Diazo Garnet GBC Salt. ~* G. Gomori, J. Lab. Clin. Med. $§, 802 (1950). 25 G. Gomori, Intern. Rev. Cytol. 1~ 323 (1952).

390

TECHNIQUES FOR METABOLIC STUDIES

[18]

Procedure. Use material as for the a-naphthol method. Dissolve about l0 mg. of naphthol AS acetate in 1 or 2 ml. of methanol; add 10 ml. of propylene glycol and shake. Add, under constant stirring, 40 ml. of buffer. Stir in about 50 mg. of diazonium salt. Filter into a Coplin jar. Incubate at room temperature until, under the microscope, the sites of activity appear in a carmine shade (about 20 to 60 minutes). Counterstain as desired; mount in glycerin jelly. Specificity. The enzyme responsible for the hydrolysis of naphthol AS acetate appears to be, at least in some cases, different from a-esterase. C h o l i n e s t e r a s e 2e,27

Principle. Cholinesterase hydrolyzes acetylthiocholine. The thiocholine liberated is precipitated in the form of its copper mercaptide; the latter is transformed into dark brown cupric sulfide. Reagents Acetylthiocholine.28 Cupric sulfate (CuSO,'5H20). Glycine. Magnesium chloride. M acetate buffer, pH 5 to 5.3. Saturated solution of sodium sulfate. Procedure. Use frozen sections of fresh tissue or of tissue fixed briefly (4 to 12 hours) in cold neutral formalin. Some cholinesterases are quite resistant to formalin; others, especially those of lower species, are readily destroyed by any fixation. Unfixed sections must be stored in a saturated solution of sodium sulfate until they are incubated. Dissolve 0.3 g. of cupric sulfate, 0.375 g. of glycine, and 1 g. of magnesium chloride in a mixture of (1) 25 ml. of acetate buffer and 175 ml. of distilled water or (2) 25 ml. of acetate buffer and 175 ml. of saturated sodium sulfate. Solution 1 is for use with fixed tissue; solution 2 for unfixed tissue. Dissolve about 10 mg. of acetylthiocholine in a few drops of distilled water and add 5 to 10 ml. of solution 1 or 2. Incubate the sections at 37 ° for 30 to 60 minutes. Rinse in several changes of distilled water. Transfer into a dilute solution of light yellow ammonium sulfide (1 drop in 10 to 20 ml. of distilled water) for 2 minutes. Rinse in distilled water. Counterstain as desired. Dehydrate and mount. 28G. B. Koelle and J. S. Friedenwald, Proc. Soc. Exptl. Biol. Med. 70, 617 (1949). 27R. Couteaux, Arch. Intern. Physiol. 59, 526 (1951). 28Availablefrom LaWall and Harrison, 1921 Walnut St., Philadelphia 3, Pennsylvania

[19]

ELECTRON MICROSCOPY OF CELLULAR CONSTITUENTS

391

Results. The sites of activity are in a green-brown shade. Specificity. The method appears to be absolutely specific. Aminopeptidase 29,~0 Principle. Some aminopeptidases hydrolyze naphthylamides of amino acids. Naphthylamine liberated is coupled with a diazonium salt to form an intensely colored azo dye.

Reagents 1-Leucyl-~-naphthylamide chloride. ~7 0.2 M phosphate buffer, pH 6.0 to 6.2. Diazo Garnet GBC Salt.

Procedure. Use frozen sections of tissue fixed in cold neutralized formalin (10%), or cryostat sections or frozen-dried material. Dissolve 5 to 10 rag. of leucyl naphthylamide in a few drops of methanol; add about 40 ml. of distilled water and 10 ml. of buffer. Stir in about 25 mg. of diazonium salt. Filter into a staining jar. Incubate the sections at room temperature until, under the microscope, the sites of activity show up in a fiery orange-red shade (usually 5 to 15 minutes). Counterstain with hematoxylin; mount in glycerin ielly. The slides are not permanent; the azo dye tends, after a while, to undergo a rearrangement into coarse needle crystals, with loss of accurate localization. Specificity. The method appears to be entirely specific. ~9 G. Gomori, Proc. Soc. Exptl. Biol. Med. 87, 559 (1954). ~0 M. S. Burstone, J. Natl. Cancer Inst. 16, 1149 (1956).

[19] Electron Microscopy of Cellular Constituents

By

FRITIOF S. SJOSTRAND

Introduction For many years biochemists have used fractionation of cells as a preparatory technique and have been studying the biochemical properties of particles that have not been accessible to an acceptable direct morphologic analysis and identification through light or electron microscopy. In this situation the attitude of various biochemists has varied. To some, the relationship between the particles and the structure of cells as described by morphologists has been considered as of less importance when corn-

[19]

ELECTRON MICROSCOPY OF CELLULAR CONSTITUENTS

391

Results. The sites of activity are in a green-brown shade. Specificity. The method appears to be absolutely specific.

Aminopeptidase29,~0 Principle. Some aminopeptidases hydrolyze naphthylamides of amino acids. Naphthylamine liberated is coupled with a diazonium salt to form an intensely colored azo dye.

Reagents 1-Leucyl-~-naphthylamide chloride. ~7 0.2 M phosphate buffer, pH 6.0 to 6.2. Diazo Garnet GBC Salt.

Procedure. Use frozen sections of tissue fixed in cold neutralized formalin (10%), or cryostat sections or frozen-dried material. Dissolve 5 to 10 rag. of leucyl naphthylamide in a few drops of methanol; add about 40 ml. of distilled water and 10 ml. of buffer. Stir in about 25 mg. of diazonium salt. Filter into a staining jar. Incubate the sections at room temperature until, under the microscope, the sites of activity show up in a fiery orange-red shade (usually 5 to 15 minutes). Counterstain with hematoxylin; mount in glycerb~ jelly. The slides are not permanent; the azo dye tends, after a while, to undergo a rearrangement into coarse needle crystals, with loss of accurate localization. Specificity. The method appears to be entirely specific. 29 G. Gomori, Proc. Soc. Exptl. Biol. Med. 87, 559 (1954). a0 M. S. Burstone, J. Natl. Cancer Inst. 16, 1149 (1956).

[19] Electron Microscopy of Cellular Constituents By

FRITIOF S. SJOSTRAND

Introduction For many years biochemists have used fractionation of cells as a preparatory technique and have been studying the biochemical properties of particles that have not been accessible to an acceptable direct morphologic analysis and identification through light or electron microscopy. In this situation the attitude of various biochemists has varied. To some, the relationship between the particles and the structure of cells as described by morphologists has been considered as of less importance when com-

392

TECHNIQUES FOR METABOLIC STUDIES

[19]

pared with the fact that particles of various sizes may be isolated. The classification through sedimentation properties in the centrifuge has been considered a sufficient specific morphologic principle of classification. This means a purely chemical interest in these cell fractions, and frequently a satisfaction for descriptive biochemistry. To other biochemists and to the morphologists the biochemical study of various cell fractions has been especially interesting because it makes it possible to relate biochemical properties and structural organization of the cells. The breaking up of cells through fragmentation releases the various cell components, part of which will be undamaged and part of which will be more or less severely damaged. The size of particles isolated through differential centrifugation range all the way down to macromolecular dimensions. In the fractions containing the smaller particles there will be the greatest risks for contamination with fragments from damaged larger particles. A morphologic analysis of the cell fractions would furnish an important aid in identifying and estimating the particle population within the various fractions. These fractions will in fact never represent " p u r e " fractions. For such a morphologic control electron microscopy performed at a rather high resolution is necessary. When trying to relate the particles present in the fractions to the structural components of the cells, light microscopy has been of little help and has made possible a severe oversimplification of the problem. Electron microscopy of cells and tissues as worked out during the last years has created a new situation in this respect. The so-called ground substance of the cytoplasm has been demonstrated as structurally rather complicated. Membrane systems, particles of various sizes, vesicles, and fibrillar structures appear in this region. Furthermore, it has been observed that mitochondria and granules of about the same size may appear side by side. The cell membrane, the existence of which was earlier assumed on indirect evidence, appears now as a very obvious and concrete structural component. The various components of the cells are well characterized from a morphologic point of view through their structural geometry, through their dimensions, and frequently through their internal organization. We may conclude that a detailed morphologic analysis by means of electron microscopy is necessary for biochemical work on various cell fractions in order to evaluate the composition of the fractions, to check the fractionation technique used, and to correlate the isolated particles with structural elements observed in the intact cell. From a technical point of view it will be of interest to present the techniques that may be used when applying electron microscopy to bio-

[19]

ELECTRON MICROSCOPY OF CELLULAR CONSTITUENTS

393

chemical studies of cell fractions and to survey the most important structural components of the cells and our means of identifying these components.

Specimen Preparation There are two methods for making preparations of cell fractions. We may make a drop preparation--that is, we may let a drop of the fraction dry on a supporting film covering the grid used as specimen carrier in the electron microscope. The other method is to fix and then embed a sample in methacrylate to make possible a preparation of ultrathin sections through the sample. Drop Preparations. Drop preparations are of limited value. Larger particles may not be identified because they are too big to allow a sufficiently detailed image. The most severe disadvantages of this method are the artifacts that will be produced during the drying of the specimen when a liquid-air interface is passed through the sample and produces various deformations and aggregations. As has been calculated by Anderson,1 the forces acting on the particles of the specimen are tremendous under these conditions. The particles will be flattened to the supporting film, and the surface of the particles will be rounded off. When making a drop preparation a small drop of the sample is transferred by means of a fine-bore pipet to the collodion or Formvar-covered copper grid used as specimen carrier in the electron microscope. Before the drop specimen is prepared, the sample may be fixed with osmium tetroxide (see below). This fixation makes the particles less sensitive to the subsequent drying and washing of the specimen. The osmium tetroxide also acts as an efficient electron stain. After the drops have dried on the supporting film the specimens are washed in double-distilled water to remove the salts or the sucrose of the suspending medium. After drying, the specimens may be examined directly in the electron microscope (Fig. 1). To increase the contrast of the image the specimens may be shadowed at a 10° angle with some suitable metal, such as chromium, which is easy to use in this connection (Fig. 2). If the metal shadowing has been performed at a known angle of incidence of the metal atoms with respect to the specimen surface, the thickness of the particles may be determined from measurements of the length of the shadows. These measurements are necessary for determining the size of the particles in a suspension as they will flatten to the surface of the supporting film under the influence of strong surface tension forces. If a drop preparation has to be made from a sediment, a complete resuspension is necessary. The pellet formed during centrifugation may 1 T. F. Anderson, Am. Naturalist 86, 91 (1952).

394

TECHNIQUES FOR METABOLIC STUDIES

[19]

FXG. 1. Unshadowed drop preparation of mitochondria isolated through fragmentation of inner segments of retinal cones of the perch eye which consist almost entirely of mitochondria. The inner segments were first isolated from the retina before being fragmented. The illustration shows clusters of mitochondria appearing as e m p t y sacks. A comparison between Figs. 1 and 2 and Fig. 8 clearly demonstrates the difference in amount of information that drop preparations supply as compared with ultrathin sections. Magnification 41,000 X.

[19]

ELECTRON MICROSCOPY OF CELLULAR CONSTITUENTS

397

is chilled with liquid nitrogen. T h e time for sublimation is about 15 minutes. T h e sublimation tube is then placed in a water bath at 70 ° for 10 minutes. Air is then slowly admitted and the copper support removed from the tube and transferred to a shadowing unit for metal shadow casting.

- -

To

Uscuum

pump

U~CUL 9rea~-

FIG. 3. Vacuum sublimation tube slightly modified, after Williams2 For explanation, see text.

In order to transfer the collodion film to specimen grids the film m a y be pulled away from the copper surface by means of Scotch tape in which holes corresponding to the size of the specimen grids have been punched. A second piece of Scotch tape is fastened to the surface of a piece of glass with the adhesive side up, and specimen grids are placed at proper distances upon this strip of Scotch tape with a piece of paper about 1 mm. square between each grid and the tape. The first piece of tape is then placed with the collodion-covered side toward the second piece of tape in such a way that the holes are centered over the grids. By pressing the collodion film between the adhesive surfaces of the strips of Scotch tape the film will be stretched over the grids and a sufficiently firm contact between the film and the metal of the grids will be assured to make it possible to remove the grids, now covered with collodion film, with a pair

398

TECHNIQUES FOR METABOLIC STUDIES

[19]

of tweezers. The specimens are now ready for examination in the electron microscope. The conservation of the outer form and the size may be sufficient for identification of some particles, for instance virus particles, and the form is better retained, the smaller the particles. Larger particles such as bacteria, on the other hand, are not well preserved when this technique is applied. The spray drop technique, whether used with or without freezedrying preservation, serves as a quantitative method for estimating the concenti'ation and relative distribution of various particles in a sample2 With sufficiently dilute suspensions the particles within a spray drop may be counted, and if polystyrene latex particles at a known concentration have been added to the suspension, the number of latex particles in the drop will give the original volume of the droplet. When the spray drop technique is not used in connection with freeze-drying a high-velocity spray is necessary and the material has to be dispersed in a volatile medium. A high-velocity spray gun that makes it possible to obtain droplet patterns from 5 to 20 t~ in diameter has been devised by Backus and Williams. 4 This gun (Fig. 4) consists of an outer envelope, A, made of a 7-mm. glass tube. This tube has one air inlet, B, and a threaded Lucite union, C and D, which makes it possible to insert a second tube, E, into tube A. Tube E is a glass tube about 3 mm. wi~e with a finely drawn portion at the tip and a small rubbed sleeve, F. This sleeve and the threaded Lucite insert G secure an airtight and stable connection with tube A. The Lucite insert, G, has a hole, the diameter of which corresponds to the diameter of tube E. The airtight connection is secured by compressing F between the concave end of G and the base of A. The inner diameter of the tip of tube E should be about 0.1 mm. and the outer diameter about 0.3 ram. The inner diameter of the tip of tube A should be 0.75 ram. These dimensions are critical. The spraying pressure may be 20 to 30 p.s.i., and the gun is then held 30 to 70 cm. from the specimen grid covered with a supporting film. The spray gun has to be carefully aligned. About 0.2 ml. is sufficient for a satisfactory spray drop pattern. The sample is introduced into the gun by means of a small pipet, H, which is inserted into tube E so far that its end reaches the constriction of tube E. A number of such pipets should be used to prevent contamination. Full advantage of the spray drop technique could not be made until the introduction of volatile suspending media by Backus and Williams. 4 A 2 % solution of freshly made ammonium acetate at pH 7.0 is a satisfactory suspending medium for use with several biological materials. Ammonium carbonate at a similar ionic strength and pH is also useful. Ammonium acetate approaches a pH of 4.0 as it dries, and ammonium

[19]

ELECTRON MICROSCOPY OF CELLULAR CONSTITUENTS

I

399

mF

C E

L~

FIG. 4. High-velocity spray gun according to Backus and Williams. ~ This spray gun represents one of Backus and Williams' alternative constructions. For explanation, see text. carbonate a p H of 10.0. Backus and Williams therefore recommend the use of both media for comparison. This technique has proved extremely valuable in virus studies. 5 The drop specimens of cell fractions are disadvantageous because the morphology of dried, metal-shadowed particles is frequently so different from t h a t of the various components as seen in intact cells. Furthermore, the resolution t h a t m a y be attained on such specimens is less than 5 R. C. Williams, Advances in Virus Research 2, 183 (1954).

400

TECHNIQUES FOR METABOLIC STUDIES

[19]

what is possible on ultrathin sections. Therefore, m a n y of the structural details useful for characterization and identification are lost. Ultrathin Sectioning. The most satisfactory preparatory technique for a detailed control of cell fractions is t h a t of ultrathin sectioning. I t makes possible the s t u d y of the morphology of various particles irrespective of their size and analysis at a high resolution. This means that the particles m a y be morphologically well defined and differentiated. T h e preparation of ultrathin sections is possible only after dehydration and embedding in methacrylate. This means that the particles have to be fixed in order to stand this treatment. Preservation by means of osmium tetroxide fixation has proved most valuable in electron microscopy of tissue cells but is not an ideal or completely reliable technique. It is especially difficult to preserve the morphology of isolated cells and the particles in cell fractions by means of osmium tetroxide fixation. F r o m a theoretical point of view freeze-drying would represent the most satisfactory method of preservation, as it makes it possible to stop, in an efficient way, chemical reactions and structural changes. However, contrast conditions in the s t u d y of frozen-dried maberial are so unfavorable t h a t in most cases this technique has to be combined with a subsequent electron staining with osmium tetroxide or some other electron stain (SjSstrand~). For the time being the problem of fixation has not been solved in a conclusive way as far as cell structure is concerned. This is especially true in work on structural components of cells after isolation. The most obvious factor that we know about and which affects the quality of preservation is the time elapsing from the death of the experimental animal to the moment when a sufficiently high concentration of osmium tetroxide has been reached within the cell to exert a stabilizing action on the cell structure. This time should not exceed 5 minutes. 7,8 I t might be t h a t the isolation of various cell components produces severe structural damage comparable to the changes observed post m o r t e m in intact cells. The less favorable preservation of isolated cell components as compared to t h a t of the same components in the intact cell m a y depend on the techniques used for isolation and m a y be a result of mechanical mistreatment as well as inadequate dispersion media; it is ~F. S. SjSstrand, in "Physical Techniques in Biological Research" (Oster and PoUister, eds.), Vol. 3, p. 241. Academic Press, New York, 1956. J. Rhodin, "Correlation of Ultrastructural Organization and Function in Normal and Experimentally Changed Proximal Convoluted Tubule Ceils of the Mouse Kidney." Stockholm, 1954. s H. Zetterqvist, "The Ultrastructural Organization of the Columnar Absorbing Cells of the Mouse Jejunum." Stockholm, 1956.

402

TECHNIQUES FOR METABOLIC STUDIES Dispersed material

Fixation in 1% osmium tetroxide solution, pH about 7.4, adjusted tonicity, at 0° or at room temperature 10-30 rain. Washing in isotonic salt solution 5-15 min. Dehydration in ethyl alcohol: 70% 5-15 min. 95% 15-30 min. Absolute alcohol 30 min. Embedding in a methacrylate mixture consisting of 95 to 90% n-butylmethacrylate and 5 to 10% methylmethacrylate to which 0.1 to 0.5% benzyol peroxide has been added. The methacrylate should be changed two to four times. 30-60 min. The specimens are transferred to No. 2-4 gelatin capsules filled with the methacrylate mixture which may be prepolymerized to a sirupy consistency. Polymerization of the methacrylate at 40 to 45 ° or at room temperature or through ultraviolet light irradiation 24-48 hours

[19] Pellets 30-60 rain. 15 min. 30 min. 30 min. 1-2 hours

1-2 hours

24-48 hours

Within the pellet obtained either before or after fixation the distribution of particles is uneven owing to the f o r m a t i o n within the pellet of several more or less distinct layers corresponding to different particle sizes and densities. I t is therefore difficult to obtain reliable information regarding the statistical distribution of particles within the pellet as a whole as the field of view t h a t is available in the electron microscope is rather restricted and the thickness of the sections is v e r y minute. F o r this reason, the use of extremely m i n u t e pellets m a y be advantageous. I t is, however, i m p o r t a n t to work out a technique to simplify the q u a n t i t a t i v e evaluation of particle population. I t m a y be of value to centrifuge the particles onto a fiat surface at the b o t t o m of the centrifuge t u b e in order to f o r m a thin uniform layer. If the fiat surface is covered b y a plastic or gelatin disk it m a y be loosened from the centrifuge t u b e and dipped into the fixing agent. A thin plastic or agar film m a y be applied to cover the sample in order to p r e v e n t losses during this treatment. After fixation, washing, and embedding in the ordinary w a y sections m a y be cut perpendicularly to the layer of particles. If this layer is sufficiently thin it will be possible to obtain reliable counts representative for the particle population. T h e p r e p a r a t i o n of ultrathin sections is a simple routine task. T h e prerequisites are a good microtome designed for cutting sections of a b o u t 200-A. thickness or less, a good cutting edge, the right orientation of the edge with respect to the face of the block to be cut, and a small dimension of this face. Several useful microtomes h a v e been constructed in the last years t h a t fulfill the requirements (see Sj6strand 1~ and Porter and ~2F. S. SjSstrand, Experientia 9~ 114 (1953).

[19]

ELECTRONMICROSCOPY OF CELLULAR CONSTITUENTS

403

B l u m ~ ) . As cutting edge either an especially sharpened razor blade 1°,14 m a y be used or the fresh edge formed when plate glass is broken in two directions at a b o u t a 45 ° angle to each other (Latta and Hartmann15). T h e orientation of the edge with respect to the face of the m e t h a crylate block should be such t h a t the front facet of the edge m a k e s an

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FIG. 5. The conditions regarding rake angle and bevel angle that have to be fulfilled for ultrathin sectioning when the minimum thickness of the sections is aimed at. ° angle, the rake angle, of a b o u t 30 ° with the normal through this face. T h e angle between the two facets at the edge should be a b o u t 50 ° and the bevel angle 6 to 10 ° (Fig. 5). T h e block should be t r i m m e d down to form an obtuse square p y r a m i d with a sharp tip. T h e surface area of the face to be sectioned should be a b o u t 0.05 to 0.1 m m . square in order to obtain the thinnest possible 13K. R. Porter and J. Blum, Anat. Record 117, 685 (1953) 14F. S. SjSstrand, Nature 168, 646 (1951). 16H. Latta and J. F. Hartmann, Proc. Soc. Exptl. Biol. Med. 74, 436 (1950).

404

TECHNIQUES FOR METABOLIC STUDIES

[19]

sections. Larger areas may be cut, but the requirements regarding the quality of the cutting edge increase with the size of this area. The sections are collected during the sectioning on a liquid surface (for instance, 10 to 20% ethyl alcohol) and then transferred to copper grids covered with a thin Formvar or collodion film. In reflected light the sections must not show any interference colors. A gray tone due to reflected light indicates that the sections may be useful.

The Most Common Structural Components of Tissue Cells The study of ultrathin sections through tissue cells by means of electron microscopy has made it possible to define in a rather precise way the morphologic characteristics of the common structural components of the cytoplasm. The definition is based on the geometry of the elementary structural units, their dimensions, inner organization, and mutual relationship. Such a clear-cut definition is of fundamental importance for the identification of various components in cell fractions. However, the analysis has to be carefully performed at a high resolution to reveal the details necessary for identification. It seems especially important to emphasize the many membranous components present in the cytoplasm which may disintegrate into small fragments. A pure identification of a membrane fragment is therefore of little value. An analysis that makes a differentiation between membrane fragments of various kinds has to be demanded. For a more complete description of the electron microscopy of the various cell components the reader is referred to the survey of SjSstrand.L8 Mitochondria. The analysis of mitochondria in ultrathin tissue sections has revealed such a characteristic structural organization that its pattern may be made use of as a means for identification. The description presented here is that originally given by SjSstrand 1°,17,~8 and Sj6strand and Rhodin ~9 and differs in several fundamental respects from that originally published by Palade.20 The mitochondria are bounded by a continuous surface membrane. In the interior a system of inner membranes is present which in most cases are oriented mutually parallel and perpendicularly to the long axis of the mitochondrion (Figs. 6 and 8). These inner mitochondria memle F. S. SjSstrand, Intern. Rev. Cytol. 5, 455 (1956). 1~F. S. SjSstrand, J. Cellular Comp. Physiol. 42, 45 (1953). 18F. S. SjSstrand, Nature 171, 30 (1953). 19F. S. Sj6strand and J. Rhodin, Exptl. Cell Research 4~ 426 (1953). 20G. E. Palade, Anat. Record 114~427 (1952).

406

[19]

TECHNIQUES FOR METABOLIC STUDIES TABLE I DIMENSIONS OF MITOCHONDRIA MEMBRANES Outer m e m b r a n e s

Organ

Inner membranes

Total Thickness of Thickness of Total Thickness of Thickness of thick- osmiophilic osmiophobic thick- osmiophilic osmiophobic ness, layers, interspace, ness, layers, interspace, A. A. A. A. A. A.

Retinal rod inner segment of guinea pig eye a 150 Proximal convoluted tubule cell of mouse kidney b 170 Exocrine cells of mouse pancreas c 140 Skeletal muscle of mouse ~ 170 H e a r t muscle of guinea pig ~ 170 Intestinal epithelium of mouse • 170 Tracheal epithelium of rat: Ciliated cells: 170 Goblet cells/ 180 Basal cells: 160 "Brush c e l l s " : 175

55

40

160

60

40

50

70

190

55

75

35

70

170

40

80

50

70

220

70

80

60

60

210

70

70

60

55

210

70

70

60 55 45 60

55 70 65 50

200 190 170 190

60 60 50 65

70 70 70 60

a F. S. Sjbstrand, J. Cellular Comp. Physiol. 42, 45 (1953). b F. S. Sjbstrand a n d J. Rhodin, Exptl. Cell Research 4~ 426 (1953); J. Rhodin, " C o r r e l a t i o n of U l t r a s t r u c t u r a l Organization a n d F u n c t i o n in Normal and Experimentally Changed Proximal Convoluted Tubule Cells of the Mouse K i d n e y . " Stockholm, 1954. c F. S. Sjbstrand a n d V. Hanzon, Exptl. Cell Research 7~ 393 (1954). d E. Andersson, unpublished data. • H. Zetterqvist, " T h e U l t r a s t r u c t u r a l Organization of the Columnar Absorbing Cells of the Mouse J e j u n u m . " Stockholm, 1956. / J . Rhodin a n d T. D a l h a m n , Z. ZeUforsch. u. mikroskop. Anat. 44~ 345 (1956). surface m e m b r a n e , free opaque particles of varying diameters a n d in most cases arranged in groups. Magnification 60,000 X. ( U l t r a t h i n section prepared a n d electron micrograph t a k e n b y Mrs. A. Kajland, one of the a u t h o r ' s technical assistants.)

[19]

ELECTRON MICROSCOPY OF CELLUL.~R CONSTITUENTS

407

branes are embedded in a homogeneous ground substance in which specialized regions may occur, characterized through their osmiophilia. The outer and inner mitochondria membranes are layered structures with two osmiophilic layers separated by a less osmiophilic interspace. The dimensions of these layers are strikingly constant for the mitochondria of a certain cell type. The dimensions of the inner membranes may, however, vary considerably when mitochondria from different types of cells are compared (see Table I). The relationship between outer and inner membranes is very intimate, with the inner membranes in contact with the outer membrane along some distance of their rims. The form and the extension of the inner membranes may vary. They may constitute complete or almost complete septa extending across the whole or nearly the whole diameter of the mitochondrion as, for instance, in the tubular cells of the kidney, v,~s In other types of cells they may show more irregular outlines, appearing like pieces of a jigsaw puzzle as in the inner segments of retinal rods of the guinea pig eye. 17 The inner membranes may also be oriented parallel to the long axis of the mitochondrion or form a wavy pattern. In the spherical mitochondria of the adrenal cortex a still more complicated pattern is observed. In some invertebrate mitochondria a system of tubules has been reported instead of inner membranes. 21 It is important to take into account that the mitochondria are rather labile components that may be severely affected by the process of fixation and embedding. The possibilities that the observed pattern may result from a destruction of a more genuine pattern should always be considered. The structural organization of the mitochondria is extremely sensitive to post-mortem conditions. Structural changes may be observed in the mitochondria when the osmium tetroxide has diffused into the cells 5 minutes after the death of the experimental animal. 7,8 The mitochondria swell spontaneously, the inner membranes are partially destroyed, and the less osmiophilic layer of the membranes swells. Through the swelling a central space extending along the whole length of the mitochondrion is formed. Palade's 2°,22 description of such a space as normal seems to depend on studying mitochondria which had been structurally changed after the death of the animal. When using osmium tetroxide solution for fixation we have to take into account that the molecules of the medium in which osmium tetroxide is dissolved diffuse through the tissue cells considerably faster than do the osmium tetroxide molecules which are characterized through their 21 B. Ben Geren and F. O. Schrnitt, Proc. Natl. Acad. Sci. U.S. 40, 863 (1954). 22 G. E. Palade, J. Histochem. Cytochem. 1, 188 (1953).

[19]

ELECTRON MICROSCOPY OF CELLULAR CONSTITUENTS

409

FIG. 7. T h e homogenate o b t a i n e d from the same liver as in Figs. 6 a n d 10 after the nuclei have been separated from the homogenate. T h e homogenate consists in this part of the pellet of m i t o c h o n d r i a with more or less swollen inner m i t o c h o n d r i a mem~ branes, fragments of q-cytomembranes~ fragments of cell membranes, and vesicular structures of various kinds. Magnification 17,000×.

412

[19]

TECHNIQUES FOR METABOLIC STUDIES

A

B

0/oI101o110© D

I

"

i , i

-. .. n

i

:

I I

.

i

::H

"E ,illlli. ENiTili

°.

.-:

°. °.

II

o

I00,4

I

rZ

/oo ~

F"

F1G. 9. Schematic presentation of the structural organization of mitochondria. A and B, three-dimensional reconstruction of the arrangement of inner and outer mitochondria membranes in two types of mitochondria, A the one represented by, for instance, kidney mitochondria, and B representing retinal rod mitochondria. C and D show the typical appearance of mitochondria patterns in sections. E gives some rather typical figures for the dimensions of inner and outer mitochondria membranes. F shows a hypothetical interpretation of the structure of mitochondria membranes in terms of lipid and protein molecules,

[19]

ELECTRON MICROSCOPY OF CELLULAR CONSTITUENTS

413

F1G. 10. Region in the cytoplasm of a r a t liver cell in which a great n u m b e r of d - e y t o m e m b r a n e s are seen along the right side of the picture. Some mitochondria m a y be compared with an opaque large granule at the upper left corner. Numerous vesicles a n d small opaque particles are present in the ground substance of the cytoplasm. Magnification 43,000X.

[19]

ELECTRON MICROSCOPY OF CELLULAR CONSTITUENTS

415

FIG. 1 1. Golgi apparatus in exocrine pancreas cell showing cross sections of Golgi membranes, or -y-cytomembranes partly separated by vacuolar spaces. Granules of various sizes, forms, and opacities in the ground substance in the Golgi region. In the upper part of the picture, a mitochondrion and a-cytomembranes. Magnification 63,000X. (Sj6strand and Hazon. sS)

416

TECHNIQUES FOR METABOLIC STUDIES

[19]

t h e m together. I t might be t h a t this cementing layer is a nonosmiophilic c o m p o n e n t of the ~,-cytomembranes. T h e ~,-cytomembranes a p p e a r in groups of four to six m e m b r a n e pairs. There are no connections extending between adjacent pairs across the 60-A.-wide interpair space separating the osmiophilic layers. T a b l e I I presents the m e a s u r e m e n t s t h a t h a v e been performed on this t y p e of cytoplasmic m e m b r a n e s . Figure 12 shows in a schematic w a y the structural differences between these three kinds of m e m b r a n e s . TABLE II DIMENSIONS OF THE GOLGI MEMBRANES

Organ

Number Thickness of of single membrane membranes, pairs A.

Mouse pancreas ~ 2-5 Mouse Varies with epididymisb function Mouse kidney ~ 4-6 Mouse intestinal epithelium d 2-6

Width of interspace Width of interspace between the two conbetween membrane stituent single mornpairs, A. branes of a pair, A.

60 70

60 70

140

60

90

50-200

60

60

50-200

a F. S. Sjbstrand andV. Hanzon, Exptl. Cell Research 7, 415 (1954). b A. J. Dalton and M. D. Felix, Am. J. Anat. 49, 171 (1954). c j. Rhodin, "Correlation of Ultrastructural Organization and Function in Normal and Experimentally Changed Proximal Convoluted Tubule Cells of the Mouse Kidney. Stockholm, 1954. d H. Zetterqvist, The ultrastructural organization of the columnar absorbing cells of the mouse jejunum. Stockholm, 1956. T h e cell membrane (Fig. 6) consists in m o s t cases of a 60-A.-thick osmiophilic layer. W h e n two cells are in close contact the osmiophilic layers of the two cell m e m b r a n e s are separated b y a 120-A.-thick nonosmiophilic interspace the dimension of which is v e r y constant. This interspace has been i n t e r p r e t e d as representing two 60-A.-thick nonosmiophilic layers, one belonging to each cell m e m b r a n e . ~6 T h e total thickness of the cell m e m b r a n e would t h e n be 120 A. T h e structure of the cell m e m b r a n e varies in different t y p e s of cells, and it m a y v a r y in different p a r t s of the same cell. I n the absorbing cells of the intestinal epithelium the brush border projections are bounded b y a triple-layered cell m e m b r a n e 105 A. thick. Where the neighboring cells are in close contact the cell m e m b r a n e s are similar to the description

[19]

ELECTRON MICROSCOPY OF C E L L U L A R CONSTITUENTS

417

a b o v e with a single osmiophilic layer of 60-A. thickness and a nonosmiophilic layer of 60 A. T h e p a r t of the cell m e m b r a n e facing the b a s e m e n t m e m b r a n e and the intercellular spaces consists of a triple-layered structure with a total thickness of 70 A. ~

B

J.

~ T o" 2OO,9

.

~

i

,;

, 1~04

Fro. 12. Schematic drawing of the various kinds of membranes occurring in tissue cells. A, a-cytomembranes; B, Golgi membranes or -y-cytomembranes; C, $-cytomembranes and the cell membranes of two adjacent closely packed cells in the tubular epithelium of mouse kidney; D, the nuclear membrane according to Afzelius. 36

Membranes bounding cytoplasmic vesicles or granules occur with v a r y ing frequency in different t y p e s of cells. T h e thickness of these m e m branes is a b o u t 50 A., and t h e y m a y be triple layered. Some vesicles are formed through the extraction of granular material during fixation and embedding.

418

TECHNIQUES FOR METABOLIC STUDIES

[19]

Osmiophilic particles (Fig. 10) are very common in the cytoplasm, especially particles with a diameter of 150 A. which frequently appear in small groups. These particles were discussed in connection with the a-cytomembranes. Fibrillar structures are common in some types of epithelial cells, as for instance in the epidermis. They represent in most cases special differentiations of the cytoplasm in certain types of cells and do not appear as a regular constituent of the cytoplasm. The nucleus appears from a structural point of view as a rather poor component of the cell. It is bounded by a nuclear membrane consisting of two osmiophilic layers separated by a less osmiophilic interspace. In several types of cells the nuclear membrane shows round regions within which it consists of only a single osmiophilic layer. 36 At the rims of these regions osmiophilic material is concentrated to give a ring-shaped appearance in tangential sections through the nuclear membrane. These specialized regions have been interpreted as pores in the nuclear membrane by Watson. 37,38 The endoplasmic reticulum is a term frequently used by American electron microscopists and originally proposed by Porter and Kallman 39 to describe a system of tubules and vesicles observed in tissue-cultured cells. This term is now being used for many kinds of structure in the cytoplasm (for instance, a-, B-, ~,-cytomembranes, vesicles, and tubular structures). This term is based on the assumption that all these morphologically rather different components represent different °aspects of one and the same structural component. It seems unsuitable to use terms which are based on hypothetical interpretations.

The Electron Microscopic Image The image of biologic specimens that we obtain in the electron microscope is mainly one of osmium-fixed material. The effect of the osmium tetroxide is to preserve the cell structure as well as to act as an efficient electron stain of certain structural components. We do not know to what extent the structure of the cell is preserved. The poor appearance of the nuclei might well depend on a lack of preservation of the ultrastructural organization of the nucleus. It seems reasonable to assume that the osmium tetroxide preserves only to some extent the structure of the cytoplasm and that several cytoplasmic components are destroyed structurally and dissolved during the drastic treatment. The dominance a6 B. 37 M. 38 M. 3~ K.

A. L. L. R.

Afzelius, Exptl. Cell Research 8, 147 (1955). Watson, Biochim. el Biophys. Acta 15, 475 (1954). Watson, J. Biophys. and Biochem. Cytol. 1, 183 (1955). Porter a n d F. Kallman, Ann. N . Y . Acad. Sci. 54, 882 (1952).

[19]

ELECTRON MICROSCOPY OF CELLULAR CONSTITUENTS

421

instance, in whole cells fixed various times after the d e a t h of the experimental a n i m a l - - i s a disruption of cytoplasmic m e m b r a n e s as a- and ~,-cytomembranes into fragments. These f r a g m e n t s exhibit a striking t e n d e n c y to form vesicular or t u b u l a r structures. 25 T h e vesicles so formed m a y act as osmometers. T h e f r a g m e n t a t i o n also facilitates a loss of material t h r o u g h extraction before the fixation.

FIG. 14. Isolated small microsomes obtained from the same liver as is represented in Figs. 6, 7, 10, and 13. This fraction consists of opaque particles of very regular size with indications of an inner structure and of material which is less opaque and is irregularly arranged, partly forming a netlike structure. The very minute opaque particles scattered all over the picture represent contaminations, which in this case easily may be differentiated. Magnification 77,000X. T h e actual observations m a d e on the microsome fractions (Figs. 13 and 14) clearly show t h a t we are dealing with particles t h a t are not identical to the structural c o m p o n e n t s as described in the intact cells. These c o m p o n e n t s h a v e in f a c t been t r a n s f o r m e d into mainly vesicular structures of various dimensions and opacities. T h e t o p o g r a p h i c relationships which are so i m p o r t a n t for the identification in i n t a c t cells h a v e been spoiled. Therefore, m u c h work has to be done to find efficient m e a n s to identify the particles. ! T h e structural changes taking place during homogenization h a v e to be studied b y analyzing samples at various stages of f r a g m e n t a t i o n . T h e

422

TECHNIQUES FOR METABOLIC STUDIES

[19]

measuring of the thickness of the membranes appearing in fragments and the analysis of their internal organization seem at present to represent our most obvious aid for an identification. As the dimensions of the various membranes are rather similar, varying from 30 to 40 A. to 60 to 70 A. for the osmiophilic layers, it is obvious that the identification has to be based on high resolution electron microscopy. What seems obvious now is that the microsome fractions are mixtures of fragments from various cell components and that these fragments presumably are to a large extent derived from the various membrane structures observed in the intact cell. The earlier microsome concept according to which the microsomes were considered definite particulate components of the cytoplasm is not in agreement with our present knowledge of the ultrastructural organization of cells.

[20]

THE MEASUREMENT OF RADIOISOTOPES

425

[20] The Measurement of Radioisotopes B y DANIEL STEINBERG and SIDNEY UDENFRIEND

I. Introduction I n this chapter an a t t e m p t has been made to outline those aspects of the t h e o r y and practice of radioassay most likely to be useful to the enzymologist. Because of space limitations the present t r e a t m e n t of the subject m a t t e r cannot be complete. There are several excellent texts and monographs which m a y be consulted for more extensive and more detailed discussions. 1-8 Wherever pertinent, reference is made at the head of each section to recommended sources t h a t will supplement the material covered in t h a t section. Because radioisotope technique m a y be new to some users of these volumes, t h e o r y and b a c k g r o u n d material have been included, but only in the detail felt to be essential for good practice. Terminology peculiar to radiochemistry has been used, wherever possible, 9 in conformity with t h a t proposed by the National Research Council Conference on Nuclear Glossary.I° I t is hoped that, b y bringing together in s u m m a r y form information t h a t is sometimes difficult for the individual investigator to ferret out, 1 M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwich, "Isotopic Carbon." John Wiley and Sons, New York, 1949. M. D. Kamen, "Isotopic Tracers in Biology," 3rd ed. Academic Press, New York, 1957. 3 W. E. Siri, "Isotopic Tracers and Nuclear Radiations." McGraw-Hill, New York, 1949. 4 D. Taylor, "The Measurement of Radioisotopes." John Wiley and Sons, New York, 1951. 5 R. E. Lapp and H. L. Andrews, "Nuclear Radiation Physics," 2nd ed. PrenticeHall, New York, 1954. 6 R. D. Evans, Advances in Biol. and Med. Phys. 1, 151 (1948). W. W. Meinke, Anal. Chem. 25, 736 (1956). Reviews on nucleonics appear bienniaUy in this journal. s ,, Radioisotope Techniques," Vol. I. Her Majesty's Stationery Office, London, 1953. 9 It has been recently pointed out that the term "isotope" strictly applies only to families of atoms of identical atomic number and that a more generalized term for referring to unrelated atomic species may be desirable. The term nuclide is being used by physicists in this sense [T. P. Kohman, Am. J. Phys. 15, 356 (1947)]. Since this term has not yet found acceptance in the biochemical literature, "isotope" is used in this review to refer to both isotopes and nuclides. ~0A Glossary of Terms in Nuclear Science and Technology, National Research Council Conference on Nuclear Glossary, The American Society of Mechanical Engineers, New York, 1951.

426

T E C H N I Q U E S FOR ISOTOPE STUDIES

[20]

t h e c h a p t e r will be t i m e - s a v i n g a n d s t e p - s a v i n g . D i s c u s s i o n of specific counting problems has been limited to those peculiar to the isotopes s h o w n in T a b l e I, since t h e y a c c o u n t for m o s t r a d i o i s o t o p e w o r k in e n z y m o l o g y . ~1 TABLE I PROPERTIES OF COMMONLY USED RADIOISOATOPES a

Radioisotope

Half-life

Emax for ~ radiation

C 14 S 35 Ca 4~ p32 I TM

5600 years 87 days 164 days 14.3 days 8.05 days

0. 155 0. 165 0. 256 1.71 0.32 (15%) 0.60 (85%) plus ~ radiation

Data taken from Natl. Bureau Standards (U.S.) Circ. 499 (1950) and Supplements 1-3 (1951-1952), and from "Nuclear Level Schemes: Covering the Elements Ca-Zr," U.S. Atomic Energy Comm. TID-5$00 (1955).

II. Counting D e v i c e s A n a w a r e n e s s of t h e s c o p e a n d t h e l i m i t a t i o n s of c o u n t i n g d e v i c e s is e s s e n t i a l t o p r o p e r e x p e r i m e n t a l d e s i g n a n d i n t e r p r e t a t i o n . T h e followi n g d i s c u s s i o n is i n t e n d e d o n l y as a n i n t r o d u c t i o n a n d o u t l i n e . E x c e l l e n t d e t a i l e d t r e a t m e n t s a r e t o be f o u n d in a n u m b e r of g e n e r a l r e f e r e n c e sources. 12-1~

A. Ionization C h a m b e r s 16-18 T h e i o n i z a t i o n c h a m b e r m e a s u r e s t h e t o t a l c h a r g e of t h e i o n p a i r s p r o d u c e d in a g i v e n t i m e w i t h i n a closed c h a m b e r a n d c o l l e c t e d a t a v o l t a g e t h a t d o e s n o t l e a d to ion m u l t i p l i c a t i o n . T h e a c c u m u l a t e d c h a r g e 11 With the outstanding exceptions of oxygen and nitrogen, radioisotopes are available for virtually every element of importance in biology. A complete listing is found in: Nuclear Data, Natl. Bur. Standards (U.S.) Circ. 499 (1950), and Supplements 1-3 (1951-1952). 12 S. C. Curran and J. D. Craggs, "Counting Tubes." Academic Press, New York, 1949. 18 S. A. Korff, "Electron and Nuclear Counters," 2rid ed. D. Van Nostrand, New York, 1955. 14 D. H. Wilkinson, "Ionization Chambers and Counters." Cambridge University Press, Cambridge, 1950. 15 R. R. Wilson, D. R. Corson, and C. P. Baker, Preliminary Report No. 7, Nuclear Science Series, National Research Council, 1950. 1~ C. J. Borkowski, Anal. Chem. 21, 348 (1949). 17 H. Palevsky, R. K. Swank, and R. Grenchik, Rev. Sci. Instr. 18, 298 (1947). 18 G. L. Brownell and H. S. Lockhart, Nucleonics 10, No. 2, 26 (1952).

[9.0]

THE MEASUREMENT OF RADIOISOTOPES

427

can be measured in terms of mechanical displacement of a metal foil (electroscope) or as a change in the direct current across a vacuum tube. Most precise results are obtained when the ionization chamber is operated in conjunction with a vibrating reed electrometer. 17In this case the charge collector is made to vibrate rapidly in an electric field, producing an alternating current proportional to the charge, a real advantage in view of the greater stability of a.c. amplifiers. This latter type of instrument offers one of the most sensitive and most precise methods for assay of C 14 and H 3 when these are introduced directly into the chamber in gas form. 18Possibly because of the problems in constructior~ and maintenance, this counting technique is not widely used in general radioassay work.

B. Geiger-Miiller Counters The usual Geiger-MCtller (G-M) tube consists of a cylindrical cathode at ground potential and a fine, high-voltage central anode wire. This cylindrical chamber is filled at low pressure (5 to 40 cm. Hg) with one of a variety of ionizable gas mixtures and sealed at the end with a thin mica window. A charged particle entering the chamber may eject one or more electrons from the ionizable gas or from the wall. This initiates the series of events described below which results in a single voltage pulse, the height of which is independent of the nature of the entering particle and the number of ions initially produced. This pulse is registered as a single count by an appropriate amplification and registering system ("scaler") to which the tube is connected. Electrons produced by the ionization event are rapidly accelerated toward the central anode. As they approach it, their energies become sufficiently great to ionize additional gas molecules, producing additional electrons. In chain-reaction fashion these in turn cause further ionization. This rapid multiplication leads to the formation of as many as 108 to 101° electrons within about 1 ~sec. (Townsend avalanche). Collection of these on the central wire anode causes a negative pulse which is amplified and recorded by the scaler. The positive ions formed during the Townsend avalanche, being so much heavier than the electrons, drift at a considerably slower rate toward the cathode. For a time, then, there is a cloud of positive ions close enough to the central anode to decrease significantly the effective field strength. A second ionizing event occurring in the tube during this time will not generate a pulse sufficiently large to be registered. This is the basis of the resolving time of a G-]~¢I tube discussed further in the section on Coincidence Correction. If the G-1V[ tube contains only a simple gas such as argon or helium it is not self-quenching; i.e., the discharge following a single ionizing event is prolonged or actually continuous. Two types of events contribut,

428

TECHNIQUES FOR ISOTOPE STUDIES

[20]

to this continuing discharge. First, a positive ion on neutralization may emit a photon which in turn has a certain probability of initiating the discharge process all over again by ejecting a photoelectron from the counter wall. Second, the impact of the positive ions colliding with the cathode can eject new electrons. Since some 10 l° positive ions are involved, the discharge is likely to be continuous. These undesirable secondary events are prevented by adding a small amount of a polyatomic gas (quenching gas) to the tube. These quenching gases (e.g., methane, alcohol, amylacetate) absorb secondary photons by converting photon energy into vibrational or rotational energy. Instead of re-emitting a photon these gases have a strong tendency to dissipate the absorbed energy through decomposition. As a result of the quenching action each ionizing event produces one and only one voltage pulse. The useful life of a self-quenched G-M tube is determined in part by the amount of quenching gas and the amount of that gas destroyed per discharge. If approximately 101° molecules are destroyed per discharge and there are 1019 quenching molecules introduced into the tube when new, it would theoretically have a lifetime of 109 counts. When the partial pressure of the effective quenching material falls below a certain point, the counter performance becomes erratic and the plateau (see below) becomes shorter and less flat. The use of halogen gas quenchers considerably extends the lifetime of the tube and, in addition, decreases the operating voltages needed. Because the number of gas molecules decomposed per discharge increases with increasing operating voltage, it is best to use a voltage in the lower range of the plateau. Raising the operating voltage into the continuous discharge range even briefly will significantly shorten the useful life of the G-M tube. With a standard source in place, the count rate observed with a given G-1V[ tube is a function of the operating voltage as shown in Fig. 1. The voltage at which the system first responds is the starting potential, largely a function of the sensitivity of the detecting circuits. As the voltage is increased, the count rate increases for a time (proportional region or region of limited proportionality). Beyond a certain point (Geiger threshold voltage) it is seen that the count rate remains relatively constant and independent of the operating voltage (Geiger-Mi~ller region). Finally, at still higher voltages, the count rate increases at an accelerated rate and the tube goes into continuous discharge. Proper operating voltage for a G-~I tube is generally specified by the manufacturer, but this should be checked, particularly since it may change significantly as the tube ages. A source active ehough to give about 3000 to 5000 c.p.m, in the Geiger-Miiller region is placed under the tube. The operating voltage is raised in 25- to 50-volt increments, and

[20]

THE MEASUREMENTOF RADIOISOTOPES

429

1- or 2-minute counts are recorded. The plot of counts per minute versus voltage will resemble that of Fig. 1. (Care is taken to stop before the continuous discharge region is entered.) The fiat portion of the curve corresponding to the Geiger-Miiller region is the plateau. This should extend for 150 to 200 volts with a slope of less than 10% per 100 volts. The virtue of a long, fiat plateau is that precise voltage control becomes less crucial. The optimal operating voltage should be chosen about onethird of the way up the plateau from the Geiger threshold voltage.

CONTINUOUS DISCHARGE GEIGER THRESHOLD

l

/

VOLTAGE,,.~

i

/

STARTING/ OPERATING

VOLTAGE

FIG. 1. Typical curve showing operating characteristics of the G-M tube.

C. Proportional Counters If a tube is operated at a voltage below the Geiger-Miiller range, in the proportional region indicated in Fig. 1, the size of the voltage pulses generated will be in proportion to the number of original ionizing events triggered by the entering particle. At this lower operating voltage the electrons ejected by the entering particle are not so greatly accelerated as they are in the Geiger-Mtiller region, and consequently the secondary ionizing collisions do not spread throughout the tube. In other words, the chain reaction is more or less limited to a small region in the immediate neighborhood of the original ionizing event. The greater the number of ionizing events occurring as the particle traverses the tube, the greater will be the number of these localized avalanches and the larger the integrated pulse at the central wire. The end result is that the size of the

430

TECHNIQUES FOR ISOTOPE STUDIES

[20]

pulse is proportional to the ionizing power of the particle. Proportional counting is particularly useful in distinguishing different types of particles or particles of different energies in the same sample. Discriminating circuits can be introduced that will record voltage pulses only in a specified narrow range. However, even when only S-particles are being considered, there are definite advantages to proportional counting. First, operation in the proportional region plus the use of a threshold discriminator will significantly reduce the background. If, as discussed further under Statistics of Counting, this is not accompanied by a disproportionate decrease in counting efficiency, the net result is to shorten considerably the time needed to count weak samples to a given accuracy. Second, the resolving time is generally decreased to the point where it can be neglected in ordinary applications. Finally, the voltage pulses are smaller than in the case of G-M counters (1 to 100 my.), an important factor contributing to the much greater useful life of proportional counters even when polyatomic gases are used in the filling. D . W i n d o w l e s s F l o w - G a s C o u n t e r s 19,2°

In principle, flow-gas counters do not differ from ordinary endwindow counters and, like them, can be used either in the Geiger-Mtiller region or in the proportional region. Since the gas in these counters is at atmospheric pressure, higher operating voltages may be necessary (higher than available with scalers designed for ordinary G-iV[ counting). For counting soft t~-radiation they offer much higher efficiency (1) because absorption losses in the end window are completely eliminated and (2) because samples can be introduced directly into the sensitive volume of the counting chamber. For example, an average end-window counter with a thin mica window may give about 10% counting efficiency for C 14 or S 35, whereas a windowless flow-gas counter can give 40 to 50% efficiency. If to this greater efficiency is added the lower background obtainable by keeping the size of the chamber small and by operating in the proportional region with an appropriate discriminator, near-optimal conditions for counting small, low-activity samples of weak f~-emitters are obtained. An example of a counter design offering these advantages, which has been successfully used in a large number of laboratories, is the Robinson flow-gas counter. 19 When used in the proportional region, background count rates as low as 2 to 4 c.p.m, are obtainable with efficiencies for C 14 of about 40%. A continuous flow of gas at atmospheric pressure or slightly above keeps the chamber filled and compensates for the slow 19C. V. Robinson, Science 112, 197 (1950). so W. Bernstein and R. Ballentine, Rev. Sci. Instr. 20, 347 (1949).

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THE MEASUREMENT OF RADIOISOTOPES

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leakage around the sample and sample holder. The lifetime of the tube is virtually infinite. For certain types of sample materials, the absence of a window introduces special artifact problems (potential radioactive contamination of the chamber, bizarre electrostatic effects, vapor contamination) that may interfere with proper counter operation. These can be largely eliminated by using a thin end window which, if the counter is operated at near-atmospheric pressure with flow gas, can be so thin that only a very small absorption loss is occasioned. Efficiency is somewhat further decreased because the sample is no longer actually within the sensitive volume of the tube. These losses, however, may be less important than the potential artifacts listed above. E. G a s - P h a s e C o u n t e r s 21-25

By introducing the labeled material directly into the counter as an integral part of the counting gas, self-absorption losses and end-window absorption losses are eliminated and 100% counting efficiency is approached. Van Slyke and associates have established and described in detail elegant procedures for gas-phase counting of C'402.22 Carbon compounds have also been converted to acetylene for counting purposes. 23,24 The techniques involved are, however, rather complex and time-consuming. They should be necessary only when samples of very low specific activity but rather large size are to be assayed. Liquid scintillation counting (see below), although it is initially more expensive, appears to offer the same efficiency as gas-phase counting, with greater flexibility and simplicity. Because of its extremely low-energy radiation (0.017 Mev.) tritium is virtually impossible to deal with except by gas-phase counting or with liquid scintillation counting techniques. Several methods for preparing and counting tritium in the gas phase have been described. 2',25-2s Again, these appear to be as efficient as liquid scintillation counting but do not approach the latter in simplicity and flexibility. 21R. F. Glascock, "Isotopic Gas Analysis for Biochemists." Academic Press, New York, 1954. 22D. l). Van Slyke, R. Steele, and J. Plazin, J. Biol. Chem. 192, 769 (1951); F. M. Sine×, J. Plazin, D. Clareus, W. Bernstein, D. D. Van Slyke, and R. Chase, J. Biol. Chem. 213, 673 (1955). 23A. R. Crathorn, Nature 172, 632 (1953). 24H. E. Suess, Science 120, 5 (1954). ~5W. Bernstein and R. Ballentine, Rev. Sci. Instr. 9.1, 158 (1950). ~6C. V. Robinson, Rev. Sci. Instr. 22, 353 (1951). ~ C. V. Robinson, Nucleonics 13, No. 11, 90 (1955). 2sK. E. Wilzbach, A. R. Van Dyken, and L. Kaplan, Anal. Chem. 26, 880 (1954).

432

TECHNIQUES FOR ISOTOPE STUDIES

[9.0]

F. Immersion Counters ("Dipping Counters")~9-~3 A Geiger-Miiller tube can be constructed in the form of a thin-walled, finger-shaped glass envelope which can be immersed over all or most of its sensitive volume in a solution of the sample to be assayed. Contact between sample and counting tube can be achieved in a number of ways. The sample can be placed in a special tube with shape complementary to that of the counting tube "finger." With a rack and pinion arrangement, the sample tube is moved up under the counting tube and the liquid sample is forced up into the narrow space (1 to 2 mm.) between the two. Alternatively a sample jacket 1 to 3 mm. in diameter can be permanently sealed directly onto the G-M tube and fitted with inlet and outflow leads. Samples are pipetted or sucked in for counting, sucked out, and the chamber rinsed with appropriate solvents before the next sample is introduced. Still another type of apparatus utilizes a cylindrical, thinwalled glass vessel as sample holder which fits snugly around the counter tube. 34 The advantage of this arrangement is that the sample does not contaot the counting tube and contamination is less of a problem. The double layer of glass, of course, reduces the sensitivity. Because of the structural requirements, the walls of immersion counters cannot be made thinner than about 20 mg./cm. ~. Consequently this is a procedure limited to studies with pa2, Na24 K42, or other highenergy ~-emitters. The technique is particularly useful for studies in which the experimental material is already in solution and is highly radioactive. In these circumstances the advantages of the procedure stand out, namely: (1) the avoidance of the need for self-absorption correction, (2) the avoidance of any conversion and plating procedures, (3) highly reproducible and efficient geometry, and (4) the preservation of the material unchanged in form and concentration for use in further studies or for further characterization. Immersion counting is not intrinsically a sensitive counting technique because of the large losses by absorption in the solvent and in the tube walls. Even in the case of p82 (E~x. = 1.7 Mev.) a layer of material 1 mm. from the tube wall loses about 50 % of its radiation in penetrating to the counter wall and another 15 to 20% in getting through the counter wall. Greater sensitivity will almost always be obtained by taking an aliquot ~9 W. F. Bale, F. L. Haven, and M. L. LeFevre, Rev. Sci. Instr. 10, 193 (1939). 90j. C. Wang, J. F. Marvin, and K. W. Stenstrom, Rev. Sci. Instr. 13, 81 (1942). 31 R. B. Barnes and D. J. Salley, Ind. Eng. Chem. (Anal. Ed.) 1B, 4 (1943). 3~A. K. Solomon and H. D. Estes, Rev. Sci. Instr. 19, 47 (1948). 33N. Veall, Brit. J. Radiol. 21, 347 (1948). 34E. Chargaff, J. Biol. Chem. 128, 579 (1939).

[9.0]

THE MEASUREMENT OF RADIOISOTOPES

433

of the material to dryness and counting with an ordinary end-window counter. If the radioactive solute is present at a concentration of 1%, the dry weight of the solid sample prepared from 6 ml. will be 60 rag. When this is plated over an area of about 3 cm. 2 and counted with an efficiency of about 20 % (instead of about 5 % in the immersion counter) the over-all sensitivity is clearly greater. When, however, the a m o u n t of radioactivity is not limiting the advantages listed above m a y outweigh the disadvantages, since concentrating the solutions, plating, and making self-absorption corrections are time-consuming operations. G. Liquid Scintillation Counters 35-41

T h e liquid scintillation counter is based on the ability of certain organic materials to emit photons when excited by the passage of charged particles. The radioactive sample to be assayed is dissolved in a medium which contains in addition one or more organic phosphors. 42 Instead of a Geiger-Miiller tube, the detector device is now a photomultiplier tube which makes use of the photoelectric effect to amplify the energies of incident photons and convert t h e m into pulses which, as in the case of the G-IV[ tube, are amplified further and registered. Since the counting medium is almost completely transparent to the photons resulting from ~-particle excitation of the phosphors, one in effect substitutes nonabsorbed, easily detected photons for the strongly absorbed weak ~-particles so difficult to count efficiently b y other methods. When the chemical form of the sample will permit it, v e r y large amounts of materials of low specific activity can be introduced and counted with little or no decrease in efficiency. This is in strong contrast with ordinary counting procedures where self-absorption limits the effective size of sample to the "infinite thickness" level. One of the chief technical problems in liquid scintillation counting is the high intrinsic background contributed b y the " n o i s e " in the photomultiplier tubes. This is overcome by (1) use of low-temperature chambers to reduce thermionic emission and (2) use of paired photomultiplier tubes 35j. B. Birks, "Scintillation Counters." McGraw-Hill, New York, 1953. 36G. A. Morton, International Conference on Peaceful Uses of Alomic Energy, Vol. 14, p. 246, United Nations, New York, 1956. 37G. T. Reynolds, F. B. Harrison, and G. Salvini, Phys. Rev. 78, 488 (1950). 38F. N. Hayes, R. D. Hiebert, and R. L. Schuck, Science 116~ 140 (1952). 39F. N. Hayes and R. G. Gould, Science 117, 480 (1953). 40D. L. Williams anal A. R. Ronzio, J. Am. Chem. Soc. 72, 5787 (1950). 41F. N. Hayes, D. L. Williams, and B. Rogers, Phys. Rev. 92, 512 (1953). 42R. K. Swank, Nucleonics 12, No. 3, 15 (1954).

434

TECHNIQUES FOR ISOTOPE STUDIES

[20]

operating in coincidence. 43 In this way backgrounds as low as 20 c.p.m. can be achieved while still retaining 50% efficiency for C 14, and backgrounds as low as 75 c.p.m, with 8 % efficiency for H 3. The second maj or problem is t h a t of bringing the sample into solution with the organic phosphor. In the case of materials soluble in organic solvents, gram quantities can be dissolved together with the phosphor. This permits accurate counting of very low specific activity materials which it m a y not be possible to assay by any other method. Some materials, not themselves readily soluble in organic solvents, can be converted to chemical forms permitting solution of relatively large quantities in the phosphor-containing medium. For example, Passman et al. 44 have described a technique for complexing CO2 with an organic-soluble amine so t h a t up to 5 millimoles of C02 can be counted as a single sample. Recently it has been shown that, with reasonable precautions, fine s u s p e n s i o n s of insoluble materials can be efficiently and reproducibly counted. 45 Phosphor solutions have been developed t h a t will permit the inclusion of larger amounts of water, 46 and with further development greater and greater flexibility is to be anticipated. For example, Steinberg (unpublished data) has recently found t h a t tritium in intact, unprocessed proteins can be assayed. The d r y protein can be dissolved in the amine described b y Passman et al. 44 ( " H y a m i n e " ) or other q u a t e r n a r y amine hydroxides and, presumably on account of complex formation, will then stay in solution in phosphor-containing toluene. B y the use of appropriate pulse discriminators liquid scintillation counters can be used to count, sequentially or simultaneously, both C 14 and H 3 in the same sample, a real advantage in double-labeling studies.

H. Gamma-Ray Counters Ordinary G-M counters are inefficient for 7-ray photons because the probability t h a t these penetrating rays will produce an ion pair while traversing the sensitive volume of. the tube is small. G-M tubes specially constructed for ~,-ray sensitivity are available. 47 The increased ~-ray efficiency is accomplished by fabricating the tube with a heavy metal wall and in some cases by introducing a lining or meshwork of metal. In 4aThe passage of each E-particle causes the release of multiple photons. A count is passed to the "scaler" only when both photomultiplier tubes are excited simultaneously. In this way the background count rate due to thermionic emission, which is random, is markedly decreased. 44j. M. Passman, N. S. Radin, and J. A. D. Cooper, Anal. Chem. 28, 484 (1956). 45O. Bltih and F. Terentiuk, Nucleonics 10, No. 9, 48 (1952). 4eE. C. Farmer and I. A. Berstein, Science 115, 460 (1952). 47F. E. Hoecker and P. N. Wilkinson, Nucleonic8 11, No. 9, 64 (1953).

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THE MEASUREMENT OF RADIOISOTOPES

435

this way a higher percentage of the incident photons are absorbed and eject photoelectrons into the chamber. The Lauritsen electroscope, although it is not a very sensitive instrument, can be useful in standardization of relatively large amounts of v-emitting isotopes. Maximal efficiency is obtained by the use of a well-type scintillation counter. 48 The sample is lowered into a cavity within a large crystalline scintillator. (e.g., thallium-activated NaI). In this way a high percentage of the emitted v-rays are absorbed in the crystal and their energy is converted to lower energy photons. By suitable optical coupling these photons are directed to a phototube and the output of the phototube is amplified and registered. Operated at room temperature, the background is generally quite high (100 to 300 c.p.m.). Therefore, for low-activity samples, counting of the I TM /~-particles in a flow-gas counter is equally efficient. However, simplicity of sample preparation combined with the very high efficiency (about 50% for I TM) still makes scintillation counting the most desirable system for counting high-energy ~/-ray emitters such as Na ~4, Cr 51, and I~31.49

I. Auxiliary Equipment and Commercial Availability The rapid-fire output pulses from the primary detecting device, after appropriate amplification, are counted and recorded. Since mechanical devices are too sluggish to respond reliably at the high counting rates generally involved, a scaling circuit is interposed. This responds electronically and "counts" up to 64, 128, 256, or, in the case of the "decascalers," 100 or 1000 events, and then trips a mechanical register. If a scale of 128 is being used, for example, the final count is obtained by multiplying the "counts" on the mechanical register by 128 and adding to the product the sum of the numbers corresponding to the indicator' lamps lit at the end of the count. The decimal-oriented sealers operate similarly but can be read more directly. The counting-rate meter is a device that electronically integrates the output pulses from the detector and, within an accuracy indicated for the particular circuit used, expresses them as a voltage proportional to the average count rate. This device is readily coupled to an apparatus for graphic recording and is frequently used for scanning paper chromatograms or other extended sources for localization of radioactivity. Small, conveniently portable instruments are available for surveying for radioactive contamination and for roughly estimating sample activities. Automatic sample changers have been designed for use with end48 H. O. Anger, Rev. Sci. Instr. 9.9, 912 (1951). 49j . H. Weisburger and H. J. Lipner, Nucleonics 12~ No. 5, 21 (1954).

436

TECHNIQUES FOR ISOTOPE STUDIES

[20]

window counters and also with windowless flow-gas counters. R e c e n t l y a design for an a u t o m a t i c sample c h a m b e r to be used with a well-type scintillation counter has been d e s c r i b e d ) ° Virtually all the e q u i p m e n t discussed in the preceding and in the following sections is available commercially, and the guides to sources listed below m a y be useful. ~1,52

III. Correction of the Observed Counting Rate As will become clear f r o m the following discussion of the m a n y and complex variables involved in radioassay, it is necessary ill a sense to look upon each count determination as a separate experiment in itself. T h e experimenter m u s t be aware of all the potential variables, and although it is rarely necessary to evaluate explicitly all of t h e m in absolute terms, each variable m u s t be carefully controlled or corrected for. D e t e r m i n a t i o n of absolute radioactivity s~-56 is rarely needed in biological studies, and so the following discussion is limited to m e a s u r e m e n t s of relative radioactivity. N o w t h a t absolute reference standards are readily available f r o m the N a t i o n a l Bureau of Standards, 57 v e r y close approximations of absolute radioactivity can be m a d e b y direct comparisons with such s t a n d a r d s under identical counting conditions.

A. Background Correction E v e n in the absence of a radioactive sample all counters continue to register a low but often highly significant background counting rate. This is due to (1) cosmic radiation, (2) ambient radiation (radioactive materials stored nearby, radioactive contamination, X - r a y a p p a r a t u s , radon), (3) contamination with isotopes in the planchet-holding assembly so H. L. Demorest and J. H. Erickson, Nucleonics 12, No. 7, 68 (1954). sl Buyers' Guide Section, Nucleonics 13, D-1 (1955). This section, published annually, lists manufacturers but gives no descriptive material. 5~Radiation Instrument Catalog, No. 3, Technical Information Service, U. S. Atomic Energy Commission, Publ. RIB-8, 1952. s8 Conference on Absolute Beta Counting, Preliminary Report No. 8, Nuclear Science Series, National Research Council, 1950. 54 H. H. Seliger and A. Schwebel, Nucleonics 12, No. 7, 54 (1954). 6, Status Report on Standardization of Radionuclides in the United States, Prelilninary Report No. 13, Nuclear Science Series, National Academy of Sciences, Washington, D.C., 1953. S6L. F. Curtiss, Measurement of Radioactive Isotopes, Natl. Bur. Standards (U.S.) Circ. 475 (1948). ~7Absolute reference standards are already available for the following radioisotopes among others: H 8, C 14, Na 22, Na 24, p3~, ilsl, Co60 RaD ~- E. Standard Samples and Reference Standards Issued by the National Bureau of Standards, Natl. Bur. Standards (U.S.) Circ. 552 (1954).

438

TECHNIQUES FOR ISOTOPE STUDIES

[20]

Thus at high counting rates a fraction of the particles entering the counter will fail to be recorded, since they follow each other too closely, and a coincidence correction will be necessary. At low counting rates the number of these "coincidences" or "near-coincidences" will be negligible, making this correction unimportant. The need for coincidence correction can be avoided by taking appropriately small aliquots of sample material for counting. When this is not feasible, all samples can be counted at a greater sample-tube distance or with a standard thin absorber introduced between sample and counter. The relation between resolving time (tr), observed counting rate (n), and true counting rate (N) is given by g

~

n

1 -- ntr

(1)

Thus, if tr = 0.6 msec. or 10-5 minute, the observed counting rate will be within 1% of the true counting rate when n is below 1000 c.p.m, and within 5% when n is below 5000 c.p.m. The resolving time is generally included in the data provided with commercial G-M counters, and coincidence corrections can then be calculated from equation 1 or from the nomogram described by Andrews. 60 There are several methods for determining experimentally the appropriate coincidence correction factors for a given counting apparatus• A detailed discussion of some of these is to be found in Calvin et al. 1 The following procedure is relatively simple and provides correction factors sufficiently accurate for most studies. ~ A series of planchets is prepared by drying carefully measured aliquots of a solution of a high specific activity ~-emitter of high energy (e.g., carrier-free H3P3204). There must be no significant self-absorption losses even in the heaviest samples counted. The smaller aliquots should be chosen to give observed counting rates below 1000. For these, the observed counting rate, after background correction, should be exactly proportional to the size of the aliquot plated, since there is no significant coincidence loss. As the size of the aliquot is increased, the observed counting rate will fall progressively further and further below the expected counting rate calculated from the size aliquot plated. An empirical • •expected count rate~ to curve may be drawn relating the r a t m ~ , ~ c o u r / t ra~] the observed count rate. The observed counting rates for experimental samples are then multiplied by the corresponding ratio to derive the true counting rate. 60 H. L. Andrews, Rev. Sci. Instr• 211 191 (1950)•

[20]

THE MEASUREMENT OF RADIOISOTOPES

439

C. Corrections Due to Interactions of Radiation with Matter 1. General Characteristics. 5,8,81 The measurement of radioisotopes is essentially the measurement of radiation intensities, and the major practical problems in radioassay are related to the ways in which these radiation intensities are altered by interaction with matter. Although it is rarely necessary to determine absolute intensities in biochemical studies, it is nevertheless important to be aware of all the potential variables so that relative radioactivities can be properly evaluated and compared. Before discussing some of the specific corrections arising from interactions of radiation w.ith matter it may be useful to review briefly some of the outstanding characteristics of such interactions. GAMMA-RAYS.The absorption of ~,-radiation, like that of other electromagnetic radiation, is theoretically a strictly logarithmic function of the thickness of the absorbing layer: I = Ioe -~d

(2)

where I0 = intensity of incident radiation. I = intensity of transmitted radiation. d = thickness of the absorbing layer (cm.). # = linear absorption coefficient (cm.-1). Instead of expressing the thickness of an absorber in centimeters, it is more convenient, particularly in discussing/~-ray absorption, to express it in grams per square centimeter (g./cm. ~) or milligrams per square centimeter (mg./cm.2). This amounts to multiplying the density of the absorbing material (g./cm. 3) by the thickness (cm.) of the particular piece of it being used as an absorber. Essentially this is a way of describing the m a s s of the layer through which the radiation is penetrating independent of the compactness of the physical form of the absorbing material. When d is expressed in grams per square centimeter t~is replaced by pro, the mass absorption coefficient (cm.2/g.). The mass absorption coefficient is a measure of the "stopping power" of a material for a given ~,-ray. A more commonly used expression is the half-thickness, which is the thickness of absorber that will reduce the radiation intensity to one-half its original value. The exponential expression for absorption takes exactly the same form as the radioactive decay equation (see below), and the half-thickness (d~), in analogy with the half-life, is expressed by the equation d~ -

0.693 ttm

6~L. E. Glendenin, Nucleonics 2, No. 1, 12 (1948).

(3)

440

TECHNIQUES FOR ISOTOPE STUDIES

[20]

Although the theoretical aspects of v-ray absorption are well defined, the experimental aspects are less so. The d~ varies considerably from one absorbing material to another, decreasing with increasing average atomic number. For a given absorbing material, the d~ increases markedly as the energy of the v-ray photons increase. Very specific data are required, then, before it is possible to calculate the expected absorption in a given experimental situation. More important are the deviations from the theoretical encountered in real counting problems. Strict]y exponential absorption is predicted only for the narrowly collimated beam normal to the plane of the absorber. For example, the radiation that is scattered by the Compton effect, although it is effectively removed from the "straight-through" beam, may very well enter a counting device near the source or be scattered back toward the counter at an angle such that it is recorded. Furthermore the secondary electrons produced by the photoelectric effect, although they do not represent transmitted v-radiation, may contribute more or less significantly to the recorded count depending on the type of counter used. In practice, then, unless care is taken to collimate (or remove the source to a considerable distance from the counter) and to eliminate counting of secondary electrons, it is necessary to determine absorption losses experimentally for each counting problem encountered. BETA-RAYS. There is no good theoretical reason why the absorption of /~-radiation should be exponential in form, but it is, or very nearly so. Unlike v-ray photons,/~-particles (negative or positive electrons) are not absorbed in an "all-or-none" fashion. For the most part a/~-particle is stopped by a series of interactions with orbital electrons, each one of which robs it of a fraction of its energy which can vary from practically zero to 100%. At each collision the direction of the particle may be radically shifted, particularly when the energy of the particle is low, as it is near the end of its path. Consequently the f~-radiation actually reaching the detector is the resultant of highly complex absorption and scattering effects. Furthermore, the energy of f~-radiation emitted by a radioisotope is not homogeneous. The disintegration of any particular atom can give rise to a/~-particle with energy ranging anywhere from zero up to a well-defined maximum value, E . . . . 6~ Between these extremes the energy of emitted f~-particles covers a broad continuous spectrum (Fig. 2), but the shape of the curve varies considerably from isotope to isotope. Fortuitously, the complex and ill-defined absorption of f~-particles 6= Energy of 8-particles is expressed in multiples of the electron volt (ev.) which is the kinetic energy acquired b y a single electron when accelerated across a potential difference of 1 volt (1 key. = 1 kiloelectronvolt = 1000 ev.; 1 Mev. = 1,000,000 ev.).

442

TECHNIQUES FOR ISOTOPE STUDIES

[20]

For f~-particles with energies between 0.15 and 0.8 Mev. the relationship is 57 R = 0.407E L~s

(5)

From an experimental point of view the important facts to be kept in mind about B-ray absorption are that the absorption process is only approximately described by a simple exponential equation and that a number of auxiliary effects such as backscatter and self-scatter, discussed I

~

l

'

I

'

I

'

I

'

I

'

I

'

I

,

I

'

I

,

J

'

I

,

I

'

I

'

I

'

I

'

I

8OO ,.i

_z 600

I.-

z 400 U

200 I00 50 0

f I 2

6

J

I I0

,

I 14

,

I 18

,

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,

I 26

,

30

34

38

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,

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54

MILLIGRAMS

FIG. 3. Typical self-absorptioncurve. The data were obtained by counting increasing amounts of a large homogeneoussample of C 14-labeledrat liver protein in planchets of the same diameter. A thin end-window G-M tube was used. below in more detail, alter the observed effects still further. Unfortunately, the theory is inadequate as a substitute for direct experimental measurements under specified, controlled conditions of assay. 2. Self-Absorption Correction. Some of the radiation emitted by a sample of finite thickness will be absorbed within the sample itself (self-absorption), the fraction so absorbed increasing with increasing sample thickness. Thus when increasing amounts of a material of a given specific activity are plated, the observed counting rate, instead of increasing linearly with sample size, slopes off as shown in Fig. 3, finally reaching a plateau, Because of this variation in the fraction of radiation lost by self-absorption with samples of varying thickness, appropriate corrections must be made before the relative activities of a series of samples can be compared.

[20]

THE MEASUREMENT OF RADIOISOTOPES

443

There are several ways of dealing with the problem of self-absorption. INFINITE THINNESS COUNTING. Sample weight may be kept down to such a low level that the losses through self-absorption are negligible. This is feasible when dealing with samples of high specific activity or with isotopes having high-energy radiation, such as p3~. The penetrating power of the p32 radiation is so great that even samples as thick as 5 mg./cm. 2 will show less than 5% loss due to self-absorption. On the other hand, a C 14 sample of the same thickness will lose close to 40% of its radiation through self-absorption. Infinite thinness counting with weak f~-emitters like C 14, S 35, and Ca 45 is possible only when the samples are of such a high specific activity that only very minute amounts need be plated for assay. Because of the backscatter effects, discussed above, all samples must be plated in precisely the same manner and on exactly the same type of planchet material. A hidden source of significant error arises from the notoriously nonuniform patterns assumed by samples prepared by evaporating solutions. The solute tends to be concentrated around the perimeter of the planchet or around the perimeter of the drop or drops as transferred to the planchet. This is easily overlooked, since the films of dried samples when counting at infinite thinness are, of course, generally invisible. Because the geometric efficiency of counting varies considerably with the distance of the source from the axial center of the tube, nonreproducibility of dried material on the planchet from sample to sample can lead to large errors. CONSTANT SAMPLE WEIGHTS. If all samples can be prepared at precisely the same weight and are precisely identical in composition, the observed counting rates can be directly compared without the necessity for a self-absorption correction. This is rarely convenient. INFINITE THICKNESS COUNTING. Beyond a certain thickness (in mg./cm. 2) radiation from the bottom layers of a sample will be completely absorbed. The lower portion of the sample does not then contribute at all to the observed counting rate. In a sense this is equivalent to counting samples of identical weight, since only the same fixed thickness at the top of each sample is actually being counted. For material of a given specific activity all infinitely thick samples give the same count. The counting rate for infinitely thick samples of different specific activity is directly proportional to their specific activity. Provided enough material is avai!able for counting, this is a most convenient solution to the self-absorption problem. The observed counting rates of infinitely thick samples are frequently cited directly in "counts per minute" but discussed as though they represented specific activities, which they in fact do. Instead of counts per minute per milligram or counts per minute per

444

TECHNIQUES FOR ISOTOPE STUDIES

[20]

millimole, the data can be considered as expressing specific radioactivity in terms of "counts per minute per infinitely thick planchet." Values for minimum sample thickness required for infinitely thick counting can be estimated from the range of the emitted f~-particle. For example, the range of C 14 /~-particles is 28.5 mg./cm. ~, and so samples over 30 mg./cm. 2 qualify as "infinitely thick." Because of scattering effects and differences between sample materials it is best in any given application to make certain that the samples are in fact "infinitely thick." Adding more material of the same specific radioactivity to the planchet should not change the counting rate. SELF-ABSORPTION CORRECTION CURVES. 68-65 Self-absorption curves should be prepared for the specific material being studied and with the specific counting apparatus and geometry being used. Arbitrary application of curves obtained for other materials or for the same material but with different counting arrangements can introduce significant error. This is so because the so-called "self-absorption curve" depends not only on absorption within the mass of the sample but also on such additional variables as self-scattering, backscattering, energy spectrum of the E-particles emerging from the source, counting geometry, window thickness, and the type of detecting device. A series of standard planchets is made up containing increasing weights of labeled material taken from a single, large homogeneous sample. Optimally these planchets should give count rates in the range of 200 to 2000 c.p.m, so that accurate values may be quickly obtained. The observed counts, corrected for background (and, if necessary, for coincidence losses), when plotted against sample weight will give a curve like that shown in Fig. 3. If, now, the ordinate is redrawn with the count rate of the plateau ("infinitely thick" sample) assigned a value of 1.00, correction factors can be read directly from the curve. Dividing the observed count rate by the fraction read from the ordinate for the appropriate sample weight converts to the count that would have been observed for an "infinitely thick" planchet of material of the same specific activity. The corrected values obtained are then directly proportional to the specific activities of the unknown samples. Multiplying by the weight of the samples in milligrams gives values directly proportional to the total counts in the planchet. An alternative procedure is to plot counts per minute per milligram against sample weight in milligrams, the data being collected as in the 63 p. E. Yankwich, T. H. Norris, and J. Huston, Anal. Chem. 19, 439 (1947). e4 F. C. Henriques, Jr., G. B. Kistiakowsky, C. Margnetti, and W. G. Schneider, Ind. Eng. Chem., Anal. Ed. 18, 349 (1946). s6 W. F. Libby, Anal. Chem. 19, 2 (1947).

[9.0]

445

THE MEASUREMENT OF RADIOISOTOPES

preceding example. The specific activity of a sample near the middle of the range of sample weights to be normally encountered is arbitrarily assigned the value 1.00. Then the specific activity of the remaining standard planchets is expressed as a fraction (or multiple) of this value, and a new scale for the ordinate is constructed on this basis. Observed specific activities (corrected, of course, for background and coincidence) are then converted to a common basis by dividing by the ordinate fraction corresponding to the appropriate sample weight. An example of a curve obtained by this procedure is shown in Fig. 4. I

I

I

I

I

I

I

I

]

I

I

I

l

I

f

I

I

1

1

I

I

I

I

I

5

I0

15

20

25

30

]5

40

45

50

55

60

,,°I

too E o. 9O (,.) >I--

80

" o:

.

7O 6O 0

50 n."

40 U.

3O

UJ n U)

2O I0 0

MILLIGRAMS

Fro. 4. Replot of data from Fig. 3 as specific activity (c.p.m./mg.) versus milligrams of sample per planchet.

Because of the greater uncertainty attaching to the assay of very thin samples it is best not to use preparations of less than about 2 mg./cm. 2, especially for reference purposes, unless unavoidable. For the same reason, extrapolation to zero thickness may be deceptive and inapplicable to the experimental situation. As can be seen in Fig. 4, the scatter of experimental points becomes excessive at low sample weights. The empirical procedure described above will give a completely dependable basis for correction independent of theoretical considerations, provided the samples are uniformly and reproducibly plated and enough samples (15 to 25) are used to obtain a smooth curve covering the range of sample weights to be encountered. Samples must be dried to constant weight before counting. It has been found that the empirical self-absorption correction curve

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THE MEASUREMENT OF RADIOISOTOPES

447

about 78%, and on lead to almost 90%. 71 Infinitely thin plates are sometimes useful when dealing with very high specific activity materials, but it is obvious that absolutely identical support material must be used to permit valid comparisons of samples. As the weight of sample increases, the contribution of backscattering from the support decreases. More and more of the downward-directed radiation is now scattered from the deeper layers of the sample (self-scattering) rather than from the planchet material. In the case of BaC1403 plated on aluminum, the percentage of backscattered radiation actually increases with increasing sample thickness, since the C ~4/3-particles reflect more strongly from BaCO3 than they do from aluminum. As the thickness of the sample counted is increased, it is observed that a larger fraction of the radiation is counted than would be calculated on the basis of known factors such as geometry, self-absorption, and simple backscattering. This anomalous effect is attributed to a "focusing" effect of the sample tending to direct a larger than theoretical fraction of its emitted radiation in the vertical direction. The magnitude of the selfscattering effect can be large, and it varies from one source material to another. For example, S 35 counted as a thick layer of PbSO4 can give a 25% higher counting rate than the same amount of S 3~ counted as a thick layer of benzidine sulfate. 72 Because of the potential magnitude of backscattering and selfscattering, it is important to be aware of these effects and to avoid directly comparing assays on different materials or materials plated differently. Because of the difficulty in evaluating these effects it is inadvisable in routine work to attempt to make explicit corrections for them. Experimentally determined self-absorption curves, discussed below, include implicitly any corrections for backscattering and selfscattering in the particular cotmting system used and for the particular materials being assayed.

D. Counting Efficiency Correction Only a fraction of the total radiation from a given sample is actually counted by the G-M tube. This fraction, designated efficiency, is a function of the geometrical relation of sample to counting tube, absorption in intervening air and in the end window, operating voltage, fluctuations in ~1D. Christian, W. W. Dunning, and D. S. Martin, Jr., Nucleonics 10, No. 5, 41 (1952). 73D. W. Engelkeimer, J. A. Seller, E. P. Steinberg, and L. Winsberg, Radiochemical Studies: The Fission Products, Vol. 9, Div. IV, p. 56. National Nuclear Energy Series, McGraw-Hill, New York, 1951.

448

TECHNIQUES FOR ISOTOPE STUDIES

[9.0]

sensitivity of the counting apparatus, and other factors. In general, the over-all efficiency of a thin end-window counter will be in the neighborhood of 20% for high-energy f~-particles like those of p3~ and in the neighborhood of 5 to 10% for weak ~-particles such as those of C 14 and S 3~. Considerably greater efficiency is possible with windowless flow counters (40 to 50%) or with liquid scintillation counters (50 to 90%). The efficiency of ordinary G-M tubes for -y-rays is generally less than 5 % of their efficiency for f~-particles and special counting devices are required (see Gamma R a y Counters). 1. Geometry. The spatial relationship between the sample, the sample supporting apparatus, the intervening absorbers, and the detecting device (geometry) determines the fraction of emitted radiation that can enter the detecting device. Optimal geometry is, of course, obtained by suspending the sample within the counting device so that all the emitted radiation can potentially be counted (4~- geometry). For solid samples plated on ordinary planchets the maximum theoretical efficiency if the sample is placed within the counting device is 50% (2~ geometry). The term geometry can be used in referring in a general way to the sample-counter arrangement, or it can be used to mean specifically the fraction of the total solid angle about the sample subtended by the counter. In a given end-window counting arrangement the efficiency falls off very sharply as the sample is moved down away from the counter. If the losses due to absorption and to scattering effects are ignored and only the geometric efficiency is considered, it can be shown, for example, that a sample placed 2 em. from the window will be counted with an efficiency less than one-half that obtainable with the sample placed 0.5 cm. from the window (window radius, 2.46 cm. ; sample radius, 1.91 cm.). J Exact centering of the sample is equally important. A point source of radioactivity if moved out so that it lies just under the edge of the tube will give only one-half the count it will give when coaxially centered under the tube. In addition to these purely geometric factors the losses due to absorption in the air layer between the sample surface and the window become very important when dealing with weak/~-emitters. Even a 1-mm. layer of air will absorb about 5% of C 14 radiation. Finally, it must be recognized that, owing to the complex nature of the interactions of f~-rays with matter, scattering from the sample supports, lead housing, etc., can lead to very different observed counting rates independent of the other factors discussed above. Beta-particles in a closed counting chamber are like bullets in a canyon, ricocheting crazily from surface to surface in an unpredictable manner. Because of their

450

TECHNIQUES FOR ISOTOPE STUDIES

[20]

for example, to rely on a UX~ or R a D -~ E reference standard. The ideal reference standard is one made with the particular isotope to be assayed. BAC1403 STANDARD. An amount of BaC140~ that will give an observed counting rate of approximately 1000 is plated on a planchet identical with those to be used for assaying samples. To prevent mechanical losses and possible exchange reactions a thin coating should be layered over the standard, in which case the sample before sealing should have a somewhat higher count to allow for the absorption loss. Collodion or nail polish can be used, but a conducting material such as silver paint is less likely to give trouble due to electrostatic effects. Ordinarily C 14 is an adequate reference standard for work with C ~4, S 36 or Ca 4~. E. D e c a y C o r r e c t i o n

Radioactive decay is the prototype of a unimolecular reaction. Each unstable atom in a sample of a given radioactive material has an equal probability of disintegrating in a n y given time interval, and so the rate of disintegration, dN/dt, is directly proportional to the number of radioactive atoms, N, present at any moment:

dN

-- d--/ = k N

(7)

where k is the disintegration constant, the fraction of the atoms disintegrating per unit time. Rearranging and integrating we obtain: -(ln~ N2 - ln, N1) = h(t2 - t~)

(8)

If we let No = number of radioactive atoms at time zero, and N = number of radioactive atoms remaining at time t, we obtain the usual form of the decay equation: ln, - ~ = ),t

or

No 2.303 lOgl0 - ~ = ~t

(9)

When one-half the labeled atoms originally present (No) have disintegrated (N = N0/2), we have No lne N~-2 = lne 2 = Xt~

(10)

The time it takes for this decay to occur is called the half-life or t~ for the particular isotope. The natural logarithm of 2 is 0.693, so we have 0.693 t~ = ~, (11) The half-life (t~) and the disintegration constant (h) are simply alternative ways of expressing the decay rate of an isotope and can be

[@.0]

THE MEASUREMENT OF RADIOISOTOPES

451

readily interconverted by the above relationship. The decay rate of radioisotopes is for all practical purposes an immutable property, having a fixed value for each isotope independent of its chemical form or the physical and chemical stresses to which it may be subjected. In the above equations N has been defined as the absolute number of radioactive atoms present. This can be expressed in terms of the absolute number of disintegrations per minute (d.p.m.) in the sample, since that, is directly proportional to N (equation 7). Most commonly, however, one actually uses the observed counts per minute (c.p.m.) in a particular detecting system which is in turn proportional to the absolute disintegrations per minute (d.p.m.). Provided the values in observed counts per minute for No and N in equation 9 are obtained under identical conditions and are appropriately corrected for other factors as discussed below, they will be directly proportional to the number of radioactive atoms at time zero and time t. Since only the ratio of the two count rates enters the equation, the absolute values are not needed. Half-lives and some other properties of the more frequently used radioisotopes are given in Table I. Comprehensive compilations of data for all known isotopes are available in a number of reference sources.3.~ If an experiment extends over a time period significantly long relative to the t~ of the isotope used, it is necessary before comparing samples to correct the observed activities of all samples to the activity they would have had if counted at the same time. A convenient rule of thumb to use in deciding if a decay correction is necessary is that in a time period equal to one-tenth the half-life the activity of a sample will decrease by 6.5%. Exact corrections can be made by substituting the appropriate disintegration constant in equation 9 and expressing the time in corresponding units. Much more convenient is the use of a graph of correction factors which can be simply prepared on semi-logarithmic paper. A straight line is drawn from the point 1 on the ordinate at zero time through the point 0.5 on the ordinate at a time corresponding to t~. The line can be extended to cover as many half-lives as may be needed. The counts for samples assayed at any time, t, from the beginning of the experiment are corrected back to the initial time simply by dividing by the fraction read off the ordinate. Although convenient, the accuracy of this method is limited by the scale of the graph paper used. More precise corrections can be made with log tables or, more quickly, with a log-log slide rule. The simplest way of dealing with decay corrections, however, is to set, aside an aliquot of the initial labeled material for use as an internal standard in a given experiment and count this along with each batch of samples. By expressing all sample activities relative to the activity of this standard counted at the same time, the decay correction is made auto-

452

TECHNIQUES FOR ISOTOPE STUDIES

[20]

matically, thus eliminating tedious calculations. This direct comparison makes the decay correction independent of any errors in the published half-life value, and the repeated counting of the internal standard may, in addition, show up radioactive impurites of different half-life in the original material.

IV. Units of Radioactivity and the Reporting of Radioactivity Data The curie is that amount of any radioactive isotope in which 3.7 X 10 ~° atoms will disintegrate per second. More commonly used are the subunits, the millicurie (inc.), 3.7 X 107 disintegrations per second (d.p.s.) and the microcurie (~c.), 3.7 × 104 d.p.s. Since count rates are generally expressed in counts per minute (c.p.m.) it is useful to remember that 1 ~c. --- 2.22 × 10 s d.p.m. These units of radioactivity define a rate of disintegration and not an amount of material. However, for any particular isotope the rate of disintegration is, as discussed above, directly proportional to the number of radioactive atoms present and can be considered equivalent to an expression of the amount of labeled material. As can be seen from equation 7, the actual number of atoms of a radioactive isotope corresponding to 1 inc. of radioactivity will depend on the disintegration constant or the half-life (see equation 11). For example, 1 mc. of p3~ undiluted by stable phosphorus (carrier-free) would represent only about 3.5 × 10-6 mg. of phosphorus, whereas it would require 0.2 rag. of undiluted C ~4 to give the disintegration rate corresponding to 1 inc. Specific radioactivity is a measure of the amount of radioisotope per unit of sample, and there are many different ways of expressing it. When the absolute disintegration rate is known, it is most often expressed as millicuries per millimole (inc./raM.) or as millieuries per gram (mc./g.) or in subunits analogous to these. More commonly, when only the relative count rate is known in a given counting system, the millicurie is replaced by counts per minute (c.p.m.) or counts per second (c.p.s.) per unit weight of sample. In some cases, for example in studies of reaction pathways, it is more informative to express specific activity in terms of the atom being used as a tracer. Frequently C 14 results are expressed not per unit weight of compound but per unit weight of carbon in the compound. In some studies the specific activities of individual carbon atoms may need to be separately determined for comparative purposes. A host of expressions for specific activity have been proposed for application in particular studies, and a survey of some of these has been made by Schulman and Falkenheim. 74 74j . Schulman, Jr., and M. Falkenheim, Nucleonics 3, No. 4, 13 (1948).

[20]

THE MEASUREMENT OF RADIOISOTOPES

453

Unfortunately there is no accepted standard procedure for reporting radioactivity data in biological experiments. Because of the diversity both in the nature of experimental design and in the types of equipment used for radioassay, complete standardization is not possible. This being the case, every effort should be made to report such data in sufficient detail and in such a manner as to permit the reader, even though he may not be experienced in radioassay techniques, to evaluate the results properly. The following items are suggested as a minimum to be presented when reporting radioisotope experiments: i. Type of counting equipment. The kind of detecting device employed is particularly relevant in interpreting raw count data. 2. Background counting rate. 3. Observed counting rate--the actual range of counts per minute recorded for the samples as prepared for radioassay. 4. Statistical error of counting (see Section V). 5. Types of corrections made on the observed count, particularly the type of self-absorption correction made. Of course, there is no need to elaborate this basic information in detail along with the final tabulated data. It can easily be summarized in brief form along with the other data of the experimental section. Generally the radioactivity data will have to be presented in a derived form in the body of the paper. An attempt should be made, however, to avoid extrapolating too far from the observed counting data. For obvious reasons raw counting data obtained on microgram quantities should not, for example, be reported as counts per minute per milligram.

V. Statistics of Counting and Counting Artifacts A. Statistical Error of Counting Radioactive decay being a random process, the closeness with which the rate observed during a finite counting time approaches the " t r u e " rate increases with the total number of disintegrations observed. Since every determination involves a background correction, calculation of the uncertainty in the corrected count rate must take into consideration the uncertainty in this background rate. The standard deviation, S.D., of any measurement in which N random events are recorded is simply __+ ~ If a total of 400 counts is recorded, for example, S.D. = +_20 counts, and the chances are about 2 in 3 that the " t r u e " count lies between 380 and 420, and 19 in 20 that it lies between 360 and 440. Standard deviation expressed in counts per minute is +_ %/N/t, where t is the time in which N counts are recorded. What-

[20]

THE MEASUREMENT OF RADIOISOTOPES

o

f h-

,8 1111

455

!! 2

I

5

I

~

B(Ickgfound (cprn}

20

I0

20

30

40

60

80

I00

150

-

Must be counted

-

250

RATE FOR SAMPLE + BACKGROUND[(c.pm)obs]

Fie. 5. Nomogram for estimating counting time needed to reduce standard deviation to +__5 %. The necessary time over which the sample must be counted is deter-

mined from the observed or gross count rate (sample + background) during the first minute or two and the measured background rate (counted for at least 30 minutes). counting time to obtain 5 % accuracy is shown by the curves in Fig. 5 for a series of different background counting rates.

B. Counting Artifacts The application of the statistical evaluation above is predicated on the assumption t h a t the detecting device is completely stable in response and t h a t the only variations are due to the statistical randomness of the radioactive decay process. However, as in all measuring devices, the introduction of artifacts is almost inevitable. The most serious problems arise in connection with transient artifacts, since t h e y can easily go undetected. For example, intermittent operation of high-voltage equipment, leak detectors, X - r a y apparatus, and the like m a y lead to false high count rates for isolated samples. Similarly, operating defects in the detecting or scaling device m a y go undetected if t h e y are intermittent. Errors of this type m a y be disclosed only if several independent counts on the same sample are made at different times. Since reference standards are usually made so as to give several thousand counts per minute an artifact t h a t introduces only 10 to 20 spurious c.p.m, will not be detected in the count rate of the standard. However, if samples with less than 100 c.p.m, are being assayed very serious errors will be made. More frequent counting

456

TECHNIQUES FOR ISOTOPE STUDIES

[20]

of background would help under such circumstances but again only if the spurious count source continued at the same intensity during counting of both samples and background. In other words, artifacts may be present and go undetected, particularly with samples of low activity, until specifically checked for. Memory effect, contamination with radioactivity from previously counted samples, can be an important source of error, particularly in immersion and gas-phase counting and occasionally in windowless flowgas counting. Such an effect will, of course, show up in the background if the latter is checked between samples. Spurious counts may be occasioned by electrostatic effects. Plastic and paper are particularly apt to cause this type of artifact. The count rate may be high during the first few minutes and then stabilize subsequently. As mentioned above, counter discharges can be initiated by photons, and so with transparent window counters light artifacts are a potential source of error. Failure of a scaling stage or, more frequently, of a mechanical register may be overlooked particularly if intermittent. Introduction of a known 60-cycle pulse source, provided with the newer scalers, offers a simple check. Visual inspection of the scaling indicator bulbs and the register during a slow count will suffice to pick up simple failures.

VI. Preparation of Samples The manner in which samples are prepared for counting will be determined by the character of the radiation, the amount of radioactivity, and the properties of the isolated compounds.

A. Solid Samples The most frequently encountered form of C 14 in radioisotope work is C1402, either directly formed or derived from organic compounds by oxidation or by specific decarboxylation reactions. In the former case, such as collections of expired C140s or C1402 formed during in vitro incubations, the gas can be trapped directly in a solution of COs-free NaOH. Many procedures for trapping expired COs by bubbling the respiratory gases through towers of NaOH have been described. ~ C~402 formed during in vitro reactions can be trapped in the center-well alkali of a Warburg vessel or in an alkali-soaked fluted filter paper appropriately supported in an ordinary stoppered Erlenmeyer flask. For quantitative collection, of course, the incubation medium must be acidified (pH 5) and sufficient time allowed for complete absorption of CO2 from the gas phase

[20]

THE MEASUREMENT OF RADIOISOTOPES

457

at the end of an incubation. The alkali-soaked paper and washings from the center well are transferred to a 50-ml. glass-stoppered graduate cylinder which is made up to volume with water and shaken vigorously several times. Elution of carbonate from the paper is not instantaneous but is complete after 16 hours of standing at room temperature. When dealing with organic compounds, CO2 can be obtained by combustion procedures 75,7~ or by specific reactions such as ninhydrin decarboxylation. 7~ Frequently it will be necessary to modify published procedures for application to isotope experiments. For example, the net amount of carbon available in a tracer experiment may be less than the amounts for which a given method is designed. If only total radioactivity is to be determined, carrier can be added to ensure quantitative collection of CO2. Any conversion procedure should be checked with the specific material under investigation and with the quantities actually to be encountered. When small amounts of carbon must be handled and specific radioactivities are required it is obviously essential to avoid dilution with CO2 from room air during conversion procedures and during subsequent sample preparation. If the a m o u n t of sample is known or can be determined prior to radioassay (for example, manometric measurement of ninhydrin-labile CO2 or COs derived by wet combustion) subsequent dilution with unlabeled COs is no longer a problem. The specific radioactivity is calculated from the total counts in the sample and the amount determined before any unlabeled diluent is allowed access to the sample. In fact, it may be desirable, when the total radioactivity is not limiting, deliberately to add carbonate after measuring the amount of COs so that any losses during handling will represent a smaller percentage of the original amount and thus facilitate sample preparation. C1402 derived by any of the general methods discussed above is most commonly converted to BaC1403 for counting because the very low solubility of this compound permits quantitative recoveries and because its physical properties favor the preparation of uniform samples for counting. In some cases C~40~ can be directly trapped in Ba(OH)2 as, for example, in applying the ninhydrin amino acid decarboxylation method of Van Slyke. ~s Unless the COs is trapped in a centrifuge tube, however, such a procedure involves a transfer that is difficult to make 76D. D. Van Slyke and J. Folch, J. Biol. Chem. 136, 509 (1940). 76D. D. Van Slyke, J. Plazin, and J. R. Weisiger, J. Biol. Chem. 191, 299 (1951). 77D. D. Van Slyke, R. T. Dillon, D. A. MacFadyen, and P. Hamilton, J. Biol. Chem. 141, 627 (1941); see Vol. III [75]. 78D. D. Van Slyke, D. A. MacFadyen, and P. B. Hamilton, J. Biol. Chem. 141,671 (1941).

[20]

THE MEASUREMENT OF RADIOISOTOPES

459

The disk should be removed from the filter-funnel assembly while still slightly moist and dried slowly at room temperature or in a vacuum oven at low temperature. After weighing, the sample can be placed directly on a planchet for counting. If it will not lie flat it can be clamped in place with a ring that slips over the edge of the circle of paper and fits snugly around a holder, 64 or it can be pasted down to the planchet surface. Flaking, especially at the edges, tends to be a problem, and the samples must be gently handled to avoid cracking. The method of Armstrong and Schubert 81 is advantageous in this respect. Instead of removing the filter paper the entire filter cup with the precipitate in place is removed and placed under the counter. 3. A technique for packing washed precipitates onto the planchet by centrifugal force has been described. 82 In this case the planchet is incorporated as a part of the base of a centrifuge cup. 4. When the size of available sample permits, BaCO3 can be ground in a mortar and pestle, transferred as a dry powder to the planchet, and tamped down firmly to obtain a solid, fiat plate. A tamper milled to fit snugly within the walls of the planchet is convenient. After the powder is distributed uniformly over the bottom, the tamper is gently lowered into the planchet. Several sharp taps with a hammer give a firmly packed cake. When the products to be examined are volatile or difficult to precipitate or crystallize, conversion to C02 and BaC03 is virtually a necessity. On the other hand, many compounds lend themselves very well to precipitation and direct counting. In such cases the time-consuming conversion to BaCO3 is not at all necessary. As long as the isolated material is reasonably pure and plates well, a self-absorption curve can be prepared as described above and the samples can be counted as isolated. Moreover, in studies involving a series of homologous or closely related compounds the self-absorption correction curves frequently will be the same provided the average atomic numbers of the materials are not too different. For example, the average atomic numbers of phenylalanine, tyrosine, dihydroxyphenylalanine, and epinephrine are practically identical, and it is found that the self-absorption correction curves for these compounds measured in a windowless flow-gas counter are so close that one curve accurately serves for all of them. sa Karnovsky et al. 66 have published selfabsorption data on a number of organic compounds showing again that the self-absorption curves are very nearly identical. It should be noted, 81W. D. Armstrong and J. Schubert, Anal. Chem. 20, 270 (1948). 8~F. C. Larson, A. R. Maass, C. V. Robinson, and E. S. Gordon, Anal. Chem. 21, 1206 (1949). 8sS. Udenfriend, unpublished data.

460

TECHNIQUES FOR ISOTOPE STUDIES

[9.0]

however, that the introduction of a heavy atom into a derivative (as in conversion to BaCOn) may significantly alter the shape of the selfabsorption curve. The degree of disparity depends to some extent on the counting system employed. 66 Studies with S ~ usually involve conversion to sulfate and then precipitation as either BaS04 or benzidine sulfate. The radiation produced by S 35 is similar to that of C 14, and the same problems arise with respect to self-absorption and sample preparation. Actually most of the techniques described above for C 14 are equally applicable to S 85 studies. A discussion of sample preparation techniques with S 35 has been presented by Tarver. 84 Because of its penetrating radiation, pa~ presents no serious problems with respect to self-absorption, and so the physical form of the sample is far less critical than with C 14 or S 35. In most cases the product can be plated and counted in the form isolated. 1131 is most conveniently measured in solution by using a well scintillation counter to detect its ~,-radiation. However, it also emits an 0.6-1V[ev. ~-ray (87.2% of the disintegrations) and can be counted in solid form with ordinary G-]VI counters. Tritium/~-particles are so feeble (0.018 Mev.) as practically to exclude counting solid samples.

B. Liquid Samples Labeled compounds in solution can be assayed either as thin layers in ordinary planehets or in immersion-type counters as discussed above. The outstanding virtue of both techniques is the high degree of sample uniformity obtainable. With the latter method no self-absorption correction is needed, since all samples are counted in layers of identical thickness, determined by the dimensions of the apparatus. With the former method no self-absorption correction will be necessary if "infinitely thick" layers of solution are counted. When thinner layers are counted the sample is always absolutely flat 86 and the thickness can be determined volumetrically from the density of the solution and the dimensions of the planchet dish used. Because of these virtues, liquid sample plating has been applied even in the demanding area of absolute standardization of isotopes. ~4 The vapor from volatile solvents and even from water may be incompatible with stable counting behavior in windowless gas-flow counters. However, m a n y of these counters can be 84 H. Tarver, Advances in Biol. and Med. Phys. 2, 281 (1951). s5 Some error can be introduced b y " m e n i s c u s effects" a t the edges a n d b y "creeping." These can be minimized b y the use of a n appropriate surface active agent.

[20]

THE MEASUREMENT OF RADIOISOTOPES

401

fitted with an extraordinarily thin end window (0.2 to 0.8 mg./cm.2), which will prevent vapor effects and yet not decrease efficiency very much. Some solvents can be used directly in windowless counters because of their low vapor pressure (e.g., formamide), and when such solvents can be used the range of applications for liquid sample counting is greatly increased because of the gain in efficiency.88,87 Preparation of samples for liquid scintillation counting has been discussed above (Section II, G). Research in this area is increasing rapidly in intensity and in scope. Large strides can be safely predicted for the near future.

C. Gas Samples21 Materials to be counted in the gas phase must be converted to a gas that will not interfere with the operating characteristics of the counting tube or ionization chamber. The gases that have been successfully assayed by this technique include C1402,tS,76,s8 tritiomethane, 26-2m9 and C ~acetylene. 23.24 The preparation of the gaseous sample calls for a complex manifold system fitted with appropriate manometric equipment. The size of sample introduced is usually determined by measurement of its partial pressure in the system. In the case of many gases the amount of material that can be counted is limited, since at higher partial pressures they interfere with tube operation. For example, Robinson's system for counting tritium after conversion to methane permits the equivalent of 0.2 ml. of water to be introduced as methane. 2°,27 On the other hand, as much as 15 mg. of carbon in the form of C02 can be assayed by the procedure of Van Slyke et al. 22 The gas must be perfectly dry, since the introduction of even small amounts of water vapor will seriously alter the counting characteristics of the system. Although gas-phase counting offers several advantages, outstanding of which is the maximal efficiency obtained, it has not been widely used in biochemistry, no doubt because the procedures are complex and time-consuming.

VII. Detection of Radioactivity on Paper Chromatograms Radiochemical analysis utilizing paper chromatography permits detection and identification of minute amounts of material. The sensitivity and simplicity of the procedure make it most useful. 86A. Schwebel, H. S. Isbell, and J. V. Karabinos, Science 118, 465 (1951). 87D. L. Tabern and T. N. Lahr, Science 119, 739 (1954). 88K. Wilzbach and W. Y. Sykes, Science 120, 494 (1954). 8~K. E. Wilzbaeh, L. Kaplan, and W. G. Brown, Science 118, 522 (1953).

[20]

THE MEASUREMENT OF RADIOISOTOPES

463

components in a chromatographic strip may be close together and may in many cases smear into odd shapes, the use of a slit perpendicular to the strip will often give misleading results concerning separation. Improved resolution can be obtained only at the expense of reduced sensitivity. Furthermore, when isotopes with high-energy radiation are used (I TM, p~2, etc.) they may penetrate the masking piece so that an appreciable number of counts will be derived from portions of the chromatogram adjacent to that in front of the slit. 3. The autoradiographic procedure offers the maximum of sensitivity for visualization of radioactive chromatograms. The high resolving power of the chromatographic technique is excellently matched by photographic resolution. Exposure of a chromatogram to a photographic plate results in darkened areas exactly corresponding to the separated labeled components. It is then possible to trace these bands or spots from the photograph onto the original chromatogram and separate and elute each radioactive band no matter how close or misshapen the bands may be. 93 In addition, the photograph provides a permanent record of the chromatographic results. Although almost any type of film can be used for this purpose, it is best to select a fast, relatively coarse-grained emulsion, since the resolution even with such coarse-grained emulsions is adequate. Films which have been used successfully in many laboratories are Eastman No-Screen and Eastman Type K, Industrial. These can be obtained in many different sizes, making it possible to photograph iudividual strips or even large two-dimensional chromatograms. Before exposing the chromatogram to a photographic plate, all traces of solvents should be removed, since some solvents may damage the emulsion or produce an intolerably high background fogging. Guide marks may then be drawn on the chromatogram with "radioactive ink " ordinary ink to which some highly radioactive nonvolatile material has been added. These marks appear on the autoradiograph and later permit exact superposition on the chromatogram for definition of the radioactive areas. The next step is carried out in a dark room under the appropriate safelight for the film used. The marked chromatogram is placed in direct contact with the film and attached in such a manner that no movement of either will occur during exposure. This can best be achieved by use of commercially available lightproof cassettes. Weights placed on top of the cassette ensure the closest contact between film and chromatogram, which is essential for ~naximum resolution. The exposure time needed is determined by the type of film, the 93 S. Udenfriend and M. Gibbs, Science 110, 708 (1949).

464

TECH~IC~UES FOR ISOTOPE STUDIES

[20]

nature of isotope emission, the total number of counts, the thickness of the filter paper used for chromatography, and the area covered by the band. The times of exposure and amounts of radioactivity required for detection of a number of isotopes can be estimated from the data presented by Steinberg and Solomon24 For instance, with Type K Industrial film a spot with density 0.1 above background film density is obtained in 24 hours if the absolute B-flux is 900 d.p.m./cm. ~. With the average endwindow G-M counter this corresponds to an observed count rate of approximately 100 to 150 c.p.m, when the paper is placed 1 cm. below the tube for assay. In order to get this intensity of radiation emerging from Whatman No. 1 filter paper, three times this total amount of radioactivity must be contained within the paper. It should be emphasized that these values represent the minimum requirements and whenever possible more radioactivity should be used. When the amount of radioactivity is limiting, long exposure times can, of course, be used. The film density obtained is a simple function of the total number of f~-particles striking per square centimeter. Therefore, the density will, in the case of long-lived isotopes like C ~4, be approximately a linear function of exposure time. For S 35 and Ca 45 exposure requirements are very similar to those for C 14. In the case of pure H-emitters, the exposure time required when the emergent ~-flux is equal increases with increasing E ..... Losses by absorption in the filter paper will, of course, be less for the more energetic emitters. Gamma-ray emitters are extremely inefficient in producing photographic densities. Data for Ca 45, I TM, P~, and Zn ~5 have been published 94 and can serve as a rough guide in estimating required exposure times for a specific problem. After exposure the X-ray films are developed according to standard procedures. The dried film is placed on an X-ray viewer, and the chromatogram is again laid on top of it in exactly the same position as during exposure, with the "radioactive i n k " guide lines as coordinates. The dark bands can then be traced directly on the paper. Each one can then be cut out and eluted for further study. Historically, photographic methods were the earliest used to detect radioactivity, and this procedure is now widely employed to determine localization of labeled molecules in tissues. These histological techniques are adequately covered in a number of texts and will, therefore, not be discussed here25 ~4D. Steinberg and A. K. Solomon, Rev. Sci. Instr. 20, 655 (1949). 95G. A. Boyd, "Autoradiography in Biology and Medicine." Academic Press, New York, 1955.

[20]

THE MEASUREMENT OF RADIOISOTOPES

465

VIII. Multiple Labeling It is often desirable to determine the fate of more than one atomic species in a biological reaction. The two atoms may be independent, they may be part of the same compound, or they may combine to form a product. Instead of carrying out two separate experiments, each with a differently labeled material, double labeling makes it possible to make both observations in the same experiment, thereby eliminating biological variation. Studies have been carried out on the simultaneous incorporation of Clt-glycine and inorganic p32 into DNA and RNA 98 and on the simultaneous incorporation of Cl*-acetate and tritiated water into fatty acids2 7 Studies of this type yield information concerning relative rates and time sequences in a biosynthetic process which would be very difficult if not impossible to obtain any other way. Multiple labeling can also be used to determine time relations by introducing the same precursor labeled in different ways at different time intervals. Such a technique was applied by Green and Anker in determining the rates of antibody synthesis at different time intervals after the injection of antigen. 98 Double labeling used in conjunction with isotope dilution makes possible corrections for losses during isolation or chemical reaction. ,~ Another application of double labeling using S 35 and I TM has been made in the isotope derivative method, which is described below. This procedure has been applied to quantitative assay of amino acids and as a method for chemical identification at the microgram level. 100 The successful use of double labeling depends on methods for differential detection of the two isotopes. The methods for differential detection may depend on differences in rates of decay or in energy or quality of the radiation. Tait and Williams ~°~ have published an excellent paper concerning the assay of mixed radioisotopes in which each of the above methods for differential detection have been discussed. A simple technique for determining Na 24 and K 42 in a mixture by combining immersion counting and scintillation counting has been described by Robinson 9o p. M. Roll, tI. Weinfeld, E. Carroll, and G. B. Brown, J. Biol. Chem. 220, 439 (1956). 97j. H. Balmain, S. J. Folley, and R. F. Glascock, Nature 169, 447 (1952). 98 H. Green and H. S. Anker, Biochim. et Biophys. Acta 13, 365 (1954). 09 W. B. Stewart and H. H. Rossi, Nucleonics 11, No. 10, 66 (1953). 100 S.Udenfriend, J. Biol. Chem. 187, 65 (1950). 10~j . F. Tait and E. S. Williams, Nucleonics 10, No. 12, 47 (1952).

[9.0]

THE MEASUREMENT OF RADIOISOTOPES

467

per minute per mole (C~) gives the moles of amino acid in the original mixture: Moles of amino acid - Ts/S X I C~

IX. Isotope I~ffects The use of radioactive isotopes as tracers is predicated on the fundamental assumption that the labeled molecules will in all respects behave in a manner indistinguishable from the corresponding normal molecules in the system under investigation. In almost every biological application this assumption is fully justified, or at least the deviations are below the limit of experimental error in the study. However, it should be recognized that whereas the qualitative behavior of a radioisotope in chemical reactions will not differ from that of the stable isotope (the isotopes have identical electronic configurations), the quantitative aspects of their behavior (the isotopes have different masses) may be very different. Artifacts arising from such differences are termed isotope effects. Isotope effects are most likely when the percentage difference in mass between the isotopes is greatest, as in the case of hydrogen versus deuterium or, the extreme case, hydrogen versus tritium. When the labeled atom is a stably linked part of a large molecule and only the fate of that molecule as a whole is under investigation, then the effective mass difference is the difference in the formula weights of the unlabeled and labeled compound and becomes much less significant. For example, the mass difference between molecular hydrogen and molecular tritium would be 200 %, but the mass difference between stable valine and valine singly labeled with tritium would be less than 2 %. Even when the mass differences are very small, however, the possibility of isotope effects cannot be dismissed a priori, as will be seen in the examples below. Even though the differences in reaction rate for a single reaction may be all but undetectable, the amplification of such a small effect over a long series of sequential reactions can lead to highly significant isotope fractionation. After all, by patient protracted recycling U 23~ and U 238 can be separated on the basis of the minute differences in diffusion constant between their gaseous derivatives. Some examples of isotope effects that have been observed and their magnitudes may be useful. Weigl and Calvin ~°3 have shown that carbon dioxide uptake by algae from a C1202-C~40~ atmosphere results in considerable fractionation, the lighter form being taken up more rapidly. At the point at which 70% of the carbon dioxide had been fixed the relative specific activities of the carbon in the gas phase, in the liquid phase, and in the algae were 10.~j . W. Weigl and M. Calvin, J. Chem. Phys. 17, 210 (1949).

468

TECHNIQUES FOR ISOTOPE STUDIES

[20]

99, 106, and 81. Partition chromatography, as might be anticipated because of the repeated "adsorption-desorption" steps involved, has been found to effectively fractionate isotopically.labeled compounds. For example, Van Dyken TM has reported on the column fractionation of tritium-labeled formic acid from its stable analog. The original sample had a specific activity of 5.8 ~c./meq. but the specific activity of the eluted 1.5

GLYCINE i

"3"

ALANINE

,.o

,.~ 0.5

> . , ~ OI I3.0 V--z U ~JX 2.0 O. 0 ~ . 1

-

0.5 o:5. I J7s

r leo

I , las

t

t

I

I R f i oo

r

a

I t 19s

I

r

r

I ,, aoo

t

FRACTION NUMBER FIG. 6. Portion of the effluent curve from the chromatography of C14-]abeled amino acids on a 100 X 0.9-cm. column of Dowex 50: e, amino acid concentration as determined by ninhydrin; o, amino acid concentration as determined by C 14 count, 100% recovery assumed; and A, calculated specific activities.

material varied from 2.8 ~c./meq. at 10% elution up to 8.3 ~c./meq. at 90% elution. Similarly Piez and Eagle ~°5 have shown that the widely used procedure of Moore and Stein z°6 for separation of amino acids, using a 100-cm. column of Dowex 50, will separate CZ4-labeled amino acids from their normal analogs. Although the separation is not, of course, very great, it is enough to make the specific activity of the material in the first part of the eluted peak as little as one-fifth that of the last portion eluted (Fig. 6). On the basis of their studies Piez and Eagle suggest 104 A. R. Van Dyken, Abstracts of the 128th National Meeting of the American Chemical Society, Minneapolis, 1955, p. 280. z05 K. A. Piez and H. Eagle, Science 122, 968 (1955). ~08 S. Moore and W. H. Stein, J. Biol. Chem. 192, 663 (1951); 211, 893 (1954).

[9.0]

THE MEASUREMENT OF RADIOISOTOPES

469

that the magnitude of the isotope effect may depend on just which positions are occupied by the C 1~ as well as the total amount of C 14 in the molecules. Unless effects of this kind are explicitly ruled out it is obviously advisable to pool all portions of a chromatographic band before taking an aliquot for determination of specific activity. Isotope effects of this kind could not be demonstrated in one-dimensional paper chromatographic separations of amino acids. 1°7 The strength of the chemical bond is a function of the masses of the atoms involved, and the differences in bond stability can be calculated on theoretical grounds. Yankwich and Calvin found that in the thermal decarboxylation of malonic acid a C12--C ~2 bond was split more readily than a C12--C 14 bond, the difference being about 12%. In the case of bromomalonic acid the difference was as much as 40%. ~°s Perhaps the most dramatic example of isotope fractionation in biological systems is found in the studies of Catch ~°9 on algae grown from seed culture with C~402 as the sole carbon source. On analysis it was found that the specific activities of individual amino acids in the proteins of these supposedly "uniformly labeled" organisms varied considerably. The carbon in glycine, for example, had a specific activity of 0.65 mc./mM., whereas that in phenylalanine had a specific activity of only 0.42 mc./mM. The possibility that isotope fractionation during the isolation procedures contributed to the observed differences must be considered. Artifacts may also arise in biological systems when the radiation from the tracer material is sufficiently intense or prolonged to alter the properties of the system. ~1°-H2 The sensitivity of detection devices is such that this source of artifacts should not be a serious problem but it must be kept in mind at all times.

X. IsotopicaUy Labeled Compounds A large number of labeled compounds are now commercially available including almost all the biologically important amino acids, fatty acids, vitamins, and steroids. Information concerning commercial suppliers and their products may be obtained through the Atomic Energy Com107 D. Steinberg, unpublished observations. 108 p. E. Yankwich and M. Calvin, J. Chem. Phys. 17, 109 (1949). 109j. R. Catch, in "Radioisotope Conference 1954/' Vol. I, p. 258. Academic Press, New York, 1954. 110 H. J. Curtis, Advances in Biol. and Med. Phys. 2, 1 (1951). HI Biochemical Aspects of Basic Mechanisms in Radiobiology, National Academy of Sciences, National Research Council, Publication 367, Washington, D.C., 1954. ~12A. H. Sparrow and B. A. Rubin, Effects of Radiations on Biological Systems, Brookhaven National Laboratories, Publication BNL 97 (T-22), Upton, New York, 1951.

[9.0]

THE MEASUREMENT OF RADIOISOTOPES

471

and r a d i o a u t o g r a p h y is an excellent test of purity, particularly if several different solvent systems are used. However, even with these p r o c e d u r e , impurities can be overlooked if insufficient material is examined. If 20,000 c.p.m, of a c o m p o u n d were applied to W h a t m a n No. 1 paper, an i m p u r i t y appearing as a second spot representing over 1% of the activity would not be detectable after one d a y of exposure to an X - r a y film. Longer exposures and larger a m o u n t s of material would be needed to ensure detection of all impurities. Testing of purity should also be carried out periodically during the life of a labeled compound, even though the compound is originally pure and is k n o w n to be chemically stable. This frequent inspection m a y be needed to detect decomposition due to self-irradiation, ~3,H4 a problem which becomes more i m p o r t a n t as the specific activity of the compound increases. The problem of decomposition b y self-irradiation has been ably discussed b y L e m m o n . H5 Included in this article is a table listing rates of decomposition of several biologically i m p o r t a n t C ~4-1abeled compounds.

XI. Radiation Dosage and Protection Discussions of radiation hazards and methods of calculating radiation dosage are available in a n u m b e r of published texts. ~-3,H6 These and the m a n y excellent publications of the National Bureau of Standards H7 provide detailed information concerning the safe and proper handling of isotopes, radioactive waste disposal, monitoring techniques, and other aspects of radiation hazards. T h e isotopes of greatest interest to the biochemist (C ~4, S 35, H 3, and p32) emit only /3-radiation. T h e energy of the radiation from the first three is so low t h a t it is completely absorbed by the walls of the containers, and so the only hazard these present under ordinary circum~13B. M. Tolbert, P. T. Adams, E. L. Bennett, A. M. Hughes, M. R. Kirk, R. M. Lemmon, R. M. Noller, R. Ostwald, and M. Calvin, J. Am. Chem. Soc. 75, 1867 (1953). 1~4C. D. Wagner and V. P. Guinn, J. Am. Chem. Soc. 75, 4861 (1953). ~16R. M. Lemmon, Nucleonics 11, No. 10, 44 (1953). 116W. V. Mayneord and W. K. Sinclair, Advances in Biol. and Med. Phys. 3, 1 (1953). 117Handbooks of the National Bureau of Standards: No. 41, Safe Handling of Radioactive Isotopes; No. 47, Recommendations of the International Commission on Radiological Protection; No. 48, Control and Removal of Radioactive Contamination in Laboratories; No. 49, Recommendations for Waste Disposal of P~ and I T M for Medical Users; No. 51, Radiological Monitoring Methods and Instruments; No. 52, Maximum Permissible Amounts of Radioisotopes in the Human Body and Maximum Permissible Concentrations in Air and Water; No. 53, Recommendations for the Disposal of C 14 Wastes; No. 59, Permissible Dose from External Sources of Ionizing Radiation.

472

TECHNIQUES FOR ISOTOPE STUDIES

[20]

stances is through accidental ingestion or inhalation. Even radiation produced through ingestion of amounts up to 1 mc. of C14-1abeled compounds such as glycine is considered to be within permissible limits, l's Danger from mishandling of these isotopes is more frequently of concern because of possible contamination of the laboratory and counting equipment rather than because of threat to health. p32 is a f~-emitter of higher energy, and some radiation will penetrate the walls of the container. At preparative levels, with quantities of several millicuries, glass or plastic shielding should be used. In particular, safety goggles should be used to prevent radiation damage to the eyes. The ~-emitters with which the biochemist is mainly concerned are 1131, K 42, Na 22, Na 24, Cr 51, and Fe 59. Any large-scale studies with these v-emitters will require appropriate lead shielding and monitoring devices such as film badges and ionization meters. With these isotopes the problem of external radiation is a real one and should be given serious consideration. It should not be forgotten that ~/-emitters administered experimentally to animals or patients present potential hazards to the investigator also. The amounts of radiation emitted from experimental subjects given these isotopes are shown below (taken from the Radioisotope Guide of the N I H Radiation Safety Committee, February 23, 1954). Isotope 1in F e ~9 K .2 Na ~ N a 24

Approx. mr./hr, at 1 ft. from 1 mc. in subject 2.5 7.0 2.1 14.1 20.6

These quantites of radiation are appreciable. Thus, 2.5 hours of exposure at a distance of 1 foot to a subject who had received 1 mc. of Na ~4 would give 50 mr. which is the daily tolerance dose. Direct handling of millicurie amounts of ~/-emitting isotopes, as in a chemical experiment, should be avoided. Tongs and automatic pipets should be used.

Acknowledgment The authors are indebted to Dr. Charles V. Robinson for his generosityin reading the manuscript and making valuable suggestions and criticisms. 118A. M. Brues and D. L. Buchanan, Summary of Conference on the Toxicity of Carbon 14, Argonne National Laboratory Publication, ANL-4787, Chicago, 1925.

[21]

THE MEASUREMENT OF STABLE ISOTOPES

473

[21] T h e M e a s u r e m e n t of S t a b l e I s o t o p e s

By ANTHONY SAN PIETRO The use of stable isotopes for investigating biological problems, in which isotopes are employed as tracers for the constituent atoms of organic compounds, requires that a highly sensitive and accurate analytical procedure be available for determining the relative abundances of the isotopes. A number of methods which have been used for measuring isotope abundances in an element, such as the determination of atomic weights by chemical means or the analysis of optical spectra, are not applicable to biological investigations for one or more of the following reasons. First, the methods are not sensitive to small variations in the isotopic composition of the element under investigation. Second, large samples (of the order of 100 mg.) are required for analysis. In many biological experiments it is not feasible to obtain samples of this size. Third, the methods are too time-consuming to be useful for routine analysis. The only practical method for the precise determination of isotope abundance ratios of any stable element other than hydrogen involves the use of a mass spectrometer. For hydrogen the common procedures for determining isotope concentrations take advantage of the large difference in mass between hydrogen and its stable isotope, deuterium. In general, these methods are based on the variation of some physical property of water with its deuterium content. For example, the density of a purified water sample is a direct measure of its deuterium concentration. The more important of these methods have been described in detail in the excellent and authoritative book by Kirshenbaum 1 and will be discussed only briefly in a later section. The remainder of this article will, therefore, be devoted primarily to a discussion of the preparation of samples for mass spectrometric analysis of isotope abundance ratios.2 With the mass spectrometer it is possible to make accurate determinations of isotope abundance ratios on a routine basis and with a minimum of sample. In order to use this method, however, the element to be analyzed must first be converted into a convenient gaseous form. The gas should be such that the following requirements are fulfilled; (1) It should be easily preparable from the constituent atoms of organic com1I. Kirshenbaum, "Physical Properties and Analysis of Heavy Water." McGrawHill Book Company, New York, 1951. 2 See Vol. IV [37] for the preparation of gas samples of OTM for mass spectrometric analysis.

474

TECHNIQUES FOR ISOTOPE STUDIES

[21]

pounds. (2) Its structure should be as simple as possible from both the molecular and the isotopic viewpoint. (3) The gas should be readily p u m p e d out of the mass spectrometer; i.e., it should not be strongly adsorbed to the structural elements of the mass spectrometer.

The Mass Spectrometeff The mass spectrometer separates b y an appropriate combination of electrical and magnetic fields a beam of ions into a spectrum dependent on their masses. The relative abundances of particles of different masses is determined by collecting the ions on an insulated electrode (or electrodes). The ion current is a measure of the n u m b e r of positive ions falling on the collector electrode. A schematic diagram 4 of a mass spectrometer developed b y Nier is shown in Fig. 1. The gas sample is introduced into the gas-handling portion of the mass spectrometer and the pressure in this system adjusted b y means of the Toeppler pump. The sample is then continuously introduced into the mass spectrometer tube at such a rate t h a t the pressure in the ionizing region of the tube is maintained at approximately 10-4 mm. of mercury. The rate of flow is controlled b y means of a constriction between the gas-handling system and the mass spectrometer tube. In the ion source a heated tungsten filament emits electrons 5 which are accelerated and ionize the neutral gas molecules present in the mass spectrometer tube. Those gas molecules which are not ionized 6 are continuously removed from the mass spectrometer tube b y a high-speed diffusion pump. The positively charged ions are drawn out of the ionizing region and accelerated in the proper direction b y negative potentials on the electrodes indicated in the ion source. The ions are next subjected to the action of a uniform magnetic field. In this field t h e y travel in a eircu3 For a more detailed description of the mass spectrometer, see (a) A. O. C. Nier, in " A Symposium of the Use of Isotopes in Biology and Medicine," pp. 80-104. University of Wisconsin Press, 1948; (b) A. O. C. Nier, in "Preparation and Measurement of Isotopic Tracers" (D. W. Wilson, A. O. C. Nier, and S. P. Reimann, eds.), pp. 11-30. Edwards Bros., Ann Arbor, 1946. 4 Figure 1 appeared in an article by Nier ~ and is reproduced here through the kind courtesy and permission of the University of Wisconsin Press. 6The beam of electrons is collimated and held in a fixed position by the action of a weak magnetic field in the region of the ion source. This is not indicated in Fig. I. We are here concerned primarily with singly charged positive ions, that is, ions formed by the loss of one electron from the neutral gas molecule. It is not essential that the ionization of gas molecules be quantitative, since we are measuring the ratio of the relative abundances of ions of different masses rather than absolute amounts. The various isotopic molecules should, however, exhibit equal probability of ionization when bombarded by an electron beam of sufficient energy to cause ionization.

[9.1]

THE MEASUREMENT OF STABLE ISOTOPES

475

lar path. The radius of the orbit depends on the mass and energy of the ions. Heavier ions follow a path of greater radius than the lighter ions. In the usual situation where the isotope abundance ratio of a single element is being determined, the ion beam is thereby separated into two SAMPLE r~, CONSTRICTION GRIN[ TO ,"" PUMP

SOURCE

I

TOEPPLER PUMP

4i.,

M AG N ET--j~---~-* POLES MAGNET

LIGHTER

ION', HEAVIER IONS

NO.I

EXIT SLIT COLLECTOR NO.2

TO NO.I TO NO.2

VlERCURY DIFFUSION PUMP

PREAMPLIFIER

FIG. 1. Simplified schematic diagram of a mass spectrometer.

m a j o r ion b e a m s of different mass to charge ratio. These ion b e a m s are properly focused to fall upon the two collector plates, as indicated in Fig. 1, and the ion currents tO these plates measured. 7 T h e ratio of the intensities of the ion currents can be converted into a t o m per cent of the 7 I n Fig. 1, the mass spectrometer tube contains two collector electrodes for the simultaneous collection of ions of different masses. Alternatively, it is possible to employ a single collector a n d focus ions of different masses to fal! on the collector b y v a r y i n g either t h e accelerating voltage or the magnetic field, the other being held constant.

476

TECHNIQUES FOR ISOTOPE STUDIES

[21]

isotope under investigation b y appropriate formulas. These are presented in the later sections which deal with the individual elements. T h e p a t h of an ion in the mass spectrometer is given b y equation 1: B2r 2

me = 4.82 X 10-6- V where m e B r V

(1)

= = = = =

mass of the ion in atomic mass units. n u m b e r of electronic charges lost in ionization. magnetic field intensity in gauss. radius of curvature of the ion p a t h in centimeters. accelerating potential, t h a t is, the "potential through which the ion is accelerated, in volts. I t is evident from this equation that, if B and V are constant, the radius of curvature' of a given ion will be proportional to its mass to charge (m/e) ratio. 7 For reference, the stable isotopes of importance as tracers in biological systems are listed in the table together with their approximate natural abundances. NATURAL ABUNDANCES OF THE STABLE ISOTOPES OF BIOLOGICAL IMPORTANCEa

Element Hydrogen Carbon Nitrogen Oxygen

Isotope

Natural abundance, %

Hi H 2 (D) C1~ C 18 N 1~ N 15 Ole Ois

99.98 0.02

98.9 1.1 99.63 0.37 99.8 0.2

a Accurate values for the natural abundances of these stable isotopes have been obtained by Nier [Phys. Rev. 77, 789 (1950)]. Hydrogen 8 In order to use deuterium as a tracer for hydrogen in an organic compound, the isotope must be introduced into the compound in such a position t h a t it is nonexchangeable with the hydrogens of the medium. Thus, deuterium cannot be used to label the hydrogens of - - O H , - - N H ~ , - - S H , or COOH groups, since they undergo rapid exchange with the s H e a v y water can be purchased from the Stuart Oxygen Company, 211 Bay Street,

San Francisco, California.

[21]

THE MEASUREMENT OF STABLE ISOTOPES

477

hydrogens of water. 9 In general, any acidic proton will undergo exchange with the hydrogens of water; the extent of exchange will depend on the experimental conditions. For example, deuterium exchange under the influence of bases occurs on the a-carbon atom of ketones, esters, nitriles, etc. 10

Analysis by Mass Spectrometer ',~','2 Principle. Molecular hydrogen is the most suitable compound for the mass spectrometric analysis of deuterium. The procedure for determining the deuterium content of an organic compound involves the oxidation (or combustion) of the compound to water and the reduction of water to hydrogen gas. The oxidation is carried out in a standard combustion apparatus and involves no technical difficulties. The production of molecular hydrogen from water, however, must be accomplished in such a manner that no fractionation of the hydrogen isotopes occurs, that is, so that the isotope concentration of the hydrogen formed is the same as the water. Most of the reactions for liberating hydrogen from water cannot be employed, since they exhibit high fractionation factors (approximately 3 or greater) unless all the hydrogen of the water is quantitatively converted to molecular hydrogen. The method developed by Rittenberg T M and his co-workers, in which the reduction of water is carried out with zinc at about 400 °, has proved to be the method of preference. This procedure results in the quantitative production of molecular hydrogen which is equilibrated with respect to the reaction

H~ ~ D2 ~ 2HD

(2)

The equilibrium constant for this reaction has been calculated by Urey and Rittenberg 13 and is approximately 3.8 at 400 °. This calculated value has been verified experimentally. 14I t has further been demonstrated that the value of the equilibrium constant for this reaction does not vary greatly with temperature. '3.~4 The value of this constant is important in the derivation of equation 3. 9R. Schoenheimerand D. Rittenberg, J. Biol. Chem. 111, 163 (1935). 10For a detailed description of the structural requirements for an acidic proton, see L. P. Hammett, "Physical Organic Chemistry," pp. 243-245. McGraw-Hill Book Co., New York, 1940. 11j. Graft and D. Rittenberg, Anal. Chem. 24, 1 (1952). 1~D. B. Sprinson and D. Rittenberg, Supplement to the United States Naval Medical Bulletin, pp. 82-93, March-April, 1948. 13H. C. Urey and D. Rittenberg, J. Chem. Phys. 1, 137 (1933). 14D. Rittenberg, W. Bleakney, and H. C. Urey, J. Chem. Phys. 2, 48 (1934).

478

TECHNIQUES FOR ISOTOPE STUDIES

[21]

Apparatus and Procedure. The method developed and described by Graft and Rittenberg 11is the one most commonly used for the preparation of hydrogen samples for mass spectrometric analysis. A diagram of their apparatus is shown in Fig. 2.15 For the sake of simplicity of description, the apparatus m a y be considered to consist of two p o r t i o n s - - a combustion system and a system for the reduction of water and collection of hydrogen. TO Oil_ PUMP~,

Oz"-"~

IJ o 250CC( TO WATER

PUMP--"~

Fro. 2. Apparatus for the preparation of hydrogen samples for mass spectrometric analysis. 15 T h e combustion system consists essentially of a quartz or Vycor microcombustion tube, A, a tube filled with 5/[g(ClO~)2 for drying the oxygen, and a bubble counter for adjusting the rate of flow of oxygen during the combustion. A portion of the microcombustion tube is filled with cupric oxide or platinum wool which acts as the catalyst for the combustion. During the combustion process, the filled portion of the microcombustion tube is maintained at a t e m p e r a t u r e of 750 ° b y an electric furnace (which is not indicated in Fig. 2). The sample, which is introduced into the microcombustion tube in a platinum or porcelain boat, is cornbusted, and the resulting water is frozen out in trap C. The reduction of water takes place in the converter tube, B, which is filled with zinc granules. Approximately 200 samples of water can be reduced before the zinc filling has to be replaced. Reduction of water with a fresh zinc filling starts slightly above 360 °, although temperatures as high as 415 ° are required with progressive use. T h e time required for 15A detailed description of this apparatus has been published in Analytical Chemistry

by Graft and Rittenberg. ~1 Figure 2 is reprinted here from their original paper through the kind courtesy and permission of the authors and the journal.

[21]

THE MEASUREMENT OF STABLE ISOTOPES

479

the complete reduction of water to molecular hydrogen depends on the age of the converter tube but is usually about 5 to 8 minutes. The hydrogen formed is transferred to the sample bulb, 16 F, by means of the Toeppler pump, E. A detailed description of the operation of this apparatus has been published 11 and will not be repeated here. The advantages of this method are: (1) Small samples are required for analysis. In general, a sample of sufficient size to yield 3 to 5 rag. of water is used. In most biological problems, it is possible to obtain this amount of material. (2) The time required for analysis is short enough to be useful for routine analysis. Owing to the complexity of the apparatus, the time required for analysis will depend on the competence of the operator and will decrease with use. It is possible to carry out a complete analysis in duplicate, including the mass spectrometric measurements, in less than 1 hour. (3) A wide variety of compounds can be analyzed with this apparatus, for example, fatty acids, amino acids, sugars, and porphyrins. (4) Whereas the initial assembly of the apparatus may be time-consuming, the apparatus requires very little maintenance to keep it in operation once assembled. The major disadvantage of all apparatus of this type is that there is a large "memory effect" inherent in them. When, for example, an unlabeled compound is analyzed after one of high isotope content, the isotope concentration of the unlabeled sample will be greater than the expected value owing to holdup of water in the apparatus. The apparatus described previously suffers from this defect, as does the modified apparatus used by the author and all apparatus which Graft and Rittenberg 1~ investigated. Furthermore, the site of holdup does not appear to be restricted to any specific portion of the apparatus. It is interesting that the total amount of holdup of water, with both the original apparatus and the modification described below, is of the order of 50 to 100 ~ of water. With a sample sufficient to give 5 mg. of water, the holdup is only 1 to 2 %. It is evident that the effect of holdup can be neglected if the deuterium contents of successive samples are not too different. We have used a modification of the above method in which the reduction of water is carried out in individual conversion tubes which are discarded after use. T M The combustion apparatus is essentially that used le These sample bulbs m a y be o b t a i n e d from Eck a n d Krebs, Inc., 27-09 40th Avenue, Long Island City, New York. 1~ T h e a u t h o r wishes to express his gratitude to Drs. Theodore E n n s and Susanne yon Sehuching for their invaluable collaboration in the development of this modification a n d t h e assembly of t h e apparatus. 1~ F. P. C h i n a r d a n d T. Enns, Anal. Chem. 2§~ 1413 (1953).

480

TECHNIQUES FOR ISOTOPE STUDIES

[21]

by Graft and Rittenberg with the exception that the outlet end of the combustion train consists of a male ball joint. The receiver for the combustion water consists of a piece of 4-ram. tubing with a female ball joint (socket) at one end. A piece of 4-mm. tubing approximately 15 inches long will suffice for about three samples. These are held together with a clamp, and the ball joint is heated by shining an infrared lamp on it. This is done to prevent the combustion water from collecting in the area of the joint. Prior to burning a sample, the 4-mm. tubing is flamed, from the ball joint to the open end, with a microburner to remove any moisture. This is repeated several times. Then dry ice is placed around the 4-mm. tubing, about 3 inches from the open end, and the combustion is carried out. The combustion water collects in the 4-mm. tubing and is driven •

•

9"

2%

) ~\\

~'-ZINC DUST G L A 5 5 WOOL

-"

~

2"

:

Fro. 3. Sample tube for the reduction of water.

toward the dry ice by heating gently with a microburner. When all the combustion water has been collected and frozen, the 4-mm. tubing is sealed off with a hand torch. At this point, the combustion water is sealed in approximately a 3-inch length of 4-mm. tubing. (The sealed end of the 4-mm. tubing remaining on the combustion apparatus is cut off with a file. It is then flamed over its whole length as before to be sure that there is no water in it and used for the next sample.) The sealed 4-mm. tubing, which contains the combustion water, is scratched in the middle with a file to facilitate breaking it later and placed in a drying oven (80 °) until all the samples are combusted. The reduction of water is carried out in the individual reduction tubes of the type shown in Fig. 3. A piece of glass wool is placed next to the break seal in order to prevent contamination of the mass spectrometer with zinc dust. Approximately 2 g. of zinc dust is added to each reduction tube. The tubes are prepared prior to the combustions and left in the oven until needed. The zinc dust and the glass wool are kept in the oven at all times. The 4-mm. tubing containing the combustion water is placed in the zinc reduction tube which is then attached to a piece of pressure tubing and evacuated with a mechanical pump. A stopcock is present between the pressure tubing and the pump. During the evacuation (approximately 7 minutes), the reduction tube is cooled with dry ice to freeze the combustion water. After evacuation, the stopcock is closed and the reduction

[21]

THE MEASUREMENT OF STABLE ISOTOPES

481

tube tilted so that the 4-mm. tube slides into the pressure tubing. The 4-mm. tube is broken by bending the pressure tubing, and the two halves are allowed to fall back into the zinc reduction tube. The zinc reduction tube is cooled again with dry ice and the stopcock opened. (The 4-mm. tube can be broken quite rapidly so that the combustion water remains frozen.) The zinc reduction tube is then sealed off with a hand torch during evacuation. The reduction of water to molecular hydrogen is accomplished by heating the tubes for 4 hours at about 390 ° The hydrogen gas is released from the zinc reduction tube into the mass spectrometer by breaking the break-off seal with a piece of iron which is activated by an electromagnet. The method has been checked with samples of known deuterium content, and the expected values were obtained.~9 Since this is a modification rather than a new method, all the advantages of the original method described above apply here as well. There are two additional possible advantages of this modification: first, the simplicity of the apparatus required, and second, the fact that the sample for mass spectrometric analysis is in a sealed tube and can be stored indefinitely without fear of leakage. This latter fact is of considerable importance when a mass spectrometer is not immediately available--for example, when the samples have to be sent to another laboratory for mass spectrometric analysis. On the other hand, more time is required for the preparation of samples with this modified procedure. This is not a serious disadvantage unless the number of samples to be analyzed is very large. Calculation of Results. The deuterium content of a compound is expressed as atoms per cent excess of the isotope which is defined as the isotope concentration in excess of the normal abundance. This value is calculated from the mass spectrometric data by means of equation 3 in which R is the ratio of the intensities of the ion currents corresponding to the H-H and H-D molecules; i.e., R = (H-H)/(H-D). Atom per cent D -

100 2R+ 1

(3)

The term atom per cent D is defined as the ratio of the number of deuterium atoms in the sample to the total number of hydrogen and deuterium atoms times 100. The atom per cent excess D is obtained by subtracting the normal, abundance of deuterium, 0.02 atom %, from the value of atom per cent D calculated from equation 3. 19These samples were obtained through the kind generosityof Dr. David B. Sprinson.

482

TECHNIQUES FOR ISOTOPE STUDIES

[21]

Analysis by Density Methods A n u m b e r of density methods have been employed to determine the deuterium concentration of organic compounds. 1 These methods are based on the fact t h a t H~O and D20 differ in density b y approximately 105 parts per million. Since this difference is so large, the density of a sample of water is v e r y sensitive to its deuterium content. (A change of 1 part per million in the density corresponds to 0.001% deuterium.) The principal methods used for determining the density of water are the p y c n o m e t e r method, 1 the float method, 2° and the falling drop method. 21,22 Other methods such as the gradient tube method s3 have also been employed. T h e density can be determined conveniently to 2 to 4 parts per million (0.002 to 0.004 % deuterium) b y these techniques. T h e steps involved in these procedures are (1) oxidation of the compound to yield water, (2) purification of the water, and (3) the precise determination of the density of the purified water. T h e technical details of these procedures have been described elsewhere and will not be discussed here. I t should be noted, however, t h a t sufficient material to yield at least 100 mg. of water is required. Furthermore, the water must be extensively purified in order to obtain the precision indicated above; any slight i m p u r i t y will yield erroneous results. A satisfactory m e t h o d for the purification of small amounts of water has been described b y Keston et al. 2~ In addition, it is necessary to normalize the abundance of the oxygen isotopes if great precision is required, since the density of water depends not only on its deuterium content but also on the relative abundance of the oxygen isotopes. Finally, careful attention to details is required in the density determination in order to obtain reliable results.

Nitrogen 24 Principle. E l e m e n t a r y nitrogen is used for the mass spectrometric determination of the relative abundances of the isotopes of nitrogen. T h e procedure for determining the N 15 content of the nitrogen of an organic compound involves the conversion of the organic nitrogen to

20D. Rittenberg and R. Schoenheimer, J. Biol. Chem. 111, 169 (1935). 21A. S. Keston, D. Rittenberg, and R. Schoenheimer, J. Biol. Chem. 122, 227 (1937). ~ M. Cohn, in "Preparation and Measurement of Isotopic Tracers" (D. W. Wilson, A. O. C. Nier, and S. P. Reimann, eds.), pp. 51-59. Edwards Bros., Ann Arbor, 1946. 2s C. Anfinsen, in "Preparation and Measurement of Isotopic Tracers" (D. W. Wilson, A. O. C. Nier, and S. P. Reimann, eds.), pp. 61-65. Edwards Bros., Ann Arbor, 1946. 24N15 can be purchased from Distillation Products Industries, Rochester 3, New York. It is available as ammonium nitrate in which only the ammonium radical has been enriched, as potassium phthalimide, as nitric acid in approximately 2 M

[21]

THE MEASUREMENT

OF STABLE ISOTOPES

483

a m m o n i a and the oxidation of a m m o n i a to nitrogen gas. 12,25,26 The nitrogen is introduced into the mass spectrometer and the intensities of the ion currents corresponding to mass 28 (N14-N 14) and mass 29 (NI4-N la) are measured. The procedure employed for converting the organic nitrogen to a m m o n i a will depend on the structure of the compound being investigated and the nature of the information desired. The Kjeldahl procedure ~7,'-'8 (or one of its modifications) is the preferred method when the (~ompound contains only one nitrogen a t o m or where differentiation between constituent nitrogen atoms is unnecessary. It is often desirable to determine the isotope concentration of each nitrogen a t o m when the c o m p o u n d to be analyzed contains more than one nitrogen atom. I n such cases, a suitable method for the stepwise degradation of the compound must be devised. Unfortunately, there is no general degradative procedure which is applicable to all nitrogencontaining compounds. However, several reactions which have been used to liberate certain specifc nitrogen groupings as ammonia should be noted. The t r e a t m e n t of an a-amino acid with nit~hydrin results in the formation of ammonia, carbon dioxide, and an aldehyde having one less carbon atom. ~9 The a m m o n i a thus formed is suitable for the preparation of nitrogen for the mass spectrometer. The experimental procedure employed in this reaction has been described by ]V[acFayden.3° solution, and as potassium nitrate. All these are supplied in approximately 30 and 60 atoms % concentrations of N15; the ammonium nitrate is also available in 7 atom % concentration. Isotopic nitrogen enriched to more than 95% N Is is available from Isomet Corporation, P.O. Box 34, Palisades Park, New Jersey. It is available as nitric acid in approximately 5 to 9 M solution, as ammonium nitrate in which either the ammonium radical or the nitrate group or both are enriched, as potassium nitrate, as ammonium chloride, as potassium phthalimide, or as nitrogen gas. ~ D. B. Sprinson and D. Rittenberg, J. Biol. Chem. 180, 707 (1949). ~6D. Rittenberg, in "Preparation and Measurement of Isotopic Tracers" (D. W. Wilson, A. O. C. Nier, and S. P. Reimann, eds.), pp. 31-42. Edwards Bros., Ann Arbor, 1946. 27The Kjeldahl procedure has been described in detail by Ballentine; see Vol. I I I [145]. ~8If CuSO4 is used as the catalyst in the Kjeldahl procedure, a prolonged digestion (about 16 to 18 hours) is required for the complete conversion of the nitrogen of lysine, creatine, sarcosine, arginine, and ornithine. If a shorter digestion is used, the nitrogen gas, formed subsequently by the oxidation of ammonia, will be contaminated with an impurity which affects the measurement of the ratio of the intensities of mass 28 to mass 29 in the mass spectrometer, 1~ 29D. D. Van Slyke, D. A. MacFayden, and P. B. Hamilton, J. Biol. Chem. 150, 251 (1943). 30D. A. MacFadyen, J. Biol. Chem. 1§~, 507 (1944).

484

TECHNIQUES FOR ISOTOPE STUDIES

[21]

Ammonia can be liberated from amides and amidine derivatives by simple alkaline hydrolysis. For example, arginine is hydrolyzed to 2 moles of ammonia and 1 mole of ornithine by treatment with alkali. 3' The ammonia is derived from the two nitrogens of the amidine moiety of arginine. Enzymatic hydrolysis has also been used in certain instances. Thus, ammonia can be released from urea in the usual manner with urease. 32 TO AIR SUPPLY': (NH 3 FREE )

24/40

~ 19/58

l,~JELDAHL FLASK

125cc ERLENMEYER FLASK Fie. 4. Distillation apparatus for the preparation of ammonia samples for oxida-

tion with hypobromite. 3s Procedure. Enough material to yield 1 rag. of nitrogen is required for analysis. The organic nitrogen of the sample is converted to ammonia either by the Kjeldahl procedure or by a suitable degradative procedure. In the latter case, the ammonia formed is transferred by a stream of nitrogen into dilute HC1. The ammonia is liberated from the Kjeldahl digest, or the solution of the ammonia salt, with strong alkali and distilled in a stream of air (ammonia free) in the apparatus shown in Fig. 4 as follows: 33 The Kjeldahl digest is cooled, diluted with 25 ml. of water, and transferred to the standard taper Kjeldahl flask. Approximately 5 ml. of 40% NaOH is added through the addition tube, and the ammonia is distilled into

~, R. Schoenheimer,S. Ratner, and D. Rittenberg, J. Biol. Chem. 130, 703 ~1939). ~ P. B. Hawk and O. Bergheim, "Practical Physiological Chemistry," llth ed., p. 708. Blakiston Co., Philadelphia, 1944. 58Figures 4 and 5 appeared in an article by Sprinson and Rittenberg ~2and are reprinted here through the permission of the Department of the Navy.

[21]

THE MEASUREMENT OF STABLE ISOTOPES

485

5 ml. of 0.07 N H2S04. The distillation is continued until about 20 ml. of water has been collected in the receiver. Between successive distillations, the apparatus is disassembled and thoroughly washed to prevent cross-contamination of samples. ,o/3o The distillate is concentrated to a small volume (about 2 ml.) and transferred to one arm of the sample tube ~ shown in Fig. 5. 23 /40 Approximately 3 ml. of a strongly alkaline solution of sodium hypobromite 34 is placed in the other arm, and the vessel is evacuated to less than 0.01 ram. as indicated by a Pirani gage. A convenient vacuum system for evacuating four sample vessels simultaneously has been described by Sprinson and Rittenberg. s5 A diagram of their apparatus is shown in Fig. 6. 35 After evacuation, the vessels are closed by rotating the Y-tube and disconnected from the vacuum system. Immediately before analFIG. 5. Gas sample tube ysis, the sample is oxidized to nitrogen by mixing the hypobromite and the sample. When for oxidation of ammonium salts with hypobromite and the evolution of nitrogen has ceased, the liberation of carbon dioxide Y-tube portion of the sample vessel is im- from carbonates) s mersed in a dry ice-alcohol mixture to reduce the vapor pressure of water. The sample vessel is then attached to the gas-handling portion of the mass spectrometer by means of the 1°/~0 34 Alkaline hypobromite is prepared according to the procedure of Sprinson and Rittenberg. 26 "The hypobromite solution is prepared by the slow addition, with vigorous stirring, of 50 ml. of bromine to 150 ml. of 40 per cent (by weight) reagent quality N a O H held at 0 °. Since hypobromite decomposes less readily in strongly alkaline solutions, another 150 ml. of 40 per cent N a O H is added when the addition of bromine is completed. 1 ml. of this solution should oxidize from 10 to 12 rag. of NH~. W h e n stored in a refrigerator, it retains its activity for months. Freshly prepared hypobromite tends to liberate oxygen, but this effect generally disappears after the reagent has been allowed to stand for a few days. After this time the solution can be separated by decantation from the precipitated sodium salts. Occasionally a batch of hypobromite is obtained which continues to exhibit this decomposition. The cause is not known, but it can be demonstrated that the rate of evolution of oxygen is accelerated by cupric ions . . . . It is clear that contamination with copper of either the reagent or the ammonia sample must be avoided. For this reason HgS04 rather than CuS04 should be employed as a catalyst in the Kjeldahl procedure. Before use the hypobromite is diluted with an equal volume of water." 3s Figure 6 appeared in an article by Sprinson and Rittenberg ~5 and is reproduced here by permission of the authors.

486

TECHNIQUES FOR ISOTOPE STUDIES

[21]

joint and the sample introduced by rotating the Y-tube portion of the sample vessel. The sample vessels can be stored for several hours without leakage of air, provided the caps and the tubes are kept paired and not interchanged. If the mass spectrometric analysis cannot be carried out on the same day the samples are prepared, the vessels can be stored overnight in the refrigerator before mixing. They should be re-evacuated the next day prior to the liberation of nitrogen. This is necessary to prevent dilution of the

TO MERCURY _ 20 M M . ~ ~ PIRANI GAUGE .....I ~

© ]~40/'50 -15 MM. .40 MM. DRY ICE TRAP "30 MM.

F~. 6. Vacuum system for preparation of gas samples for mass spectrometric analysis, s5 nitrogen of the sample by the normal nitrogen of air which may have leaked into the sample vessels during storage. In spite of all these precautions, small amounts of air may contaminate the sample. The presence of air is indicated by the appearance of an oxygen peak at mass 32 and confirmed by a peak at mass 40 due to the argon content of air. The method of correcting for air leakage has been described by Sprinson and Rittenberg 12 and will not be repeated here. This correction should not be applied when the sample contains more than 3 % air. Calculation of Results. The atom per cent N ]~ is calculated from equation 4 in which R is the ratio of the intensities of the ion currents corresponding to masses 28 and 29; i.e., R = ( N 1 4 N I 4 ) / / ( N i 4 N ] 5 ) . 100 Atom per cent N 15 -- - 2R -t- 1

(4)

The atom per cent excess N ~6 is obtained by subtracting the normal

[21]

THE MEASUREMENT OF STABLE ISOTOPES

487

abundance of N 15, which is approximately 0.37 atom %, from the calculated value of atom per cent N 15. In the case of nitrogen the mass spectrometric analysis can be carried out with a precision of 0.003 atom % N 15 in the low concentration range. C a r b o n 3~

Within recent years, the radioactive isotope of carbon, C TM, has almost completely replaced the stable isotope, C 13, as a tracer for investigating the metabolic pathways of carbon. This is due to the increased availability and ease of analysis of the radioactive isotope. However, the stable isotope is still generally preferred in experiments with human subjects. Furthermore, it is often advantageous to employ multiple labeling of the compound under investigation. In such cases, the usual procedure is to label one carbon atom with the stable isotope and a different one with the radioactive isotope. Principle. The mass spectrometric determination of the C 13 concentration of an organic compound in general requires the preliminary conversion of the organic carbon to carbon dioxide. The carbon dioxide is introduced into the mass spectrometer, and the intensities of the ion currents corresponding to masses 44 and 45 are measured. 3~ A variety of methods have been employed for converting the carbon of an organic compound to carbon dioxide. The most common of these involves combustion of the compound in a microcombustion apparatus 26 or by a modification of the wet combustion method of Van Slyke and Folch. 38 The combustion procedure can be used only when all the carbon of the compound can be obtained in the form of carbon dioxide by combustion and when differentiation between constituent carbon atoms is unnecessary. The ninhydrin reaction can be used to liberate carbon dioxide from the carboxyl groups of amino acids. ~°,89 The reaction is carried out at pH 2.5 and the resulting carbon dioxide absorbed in Ba(OH)2 solution. at C13 can be purchased from Distillation Products Industries, Rochester 3, New York. I t is available as potassium cyanide a n d b a r i u m carbonate in approximately 60 atom % concentration of C 13. ~7 Although C 13 is most conveniently determined in the form of carbon dioxide, the analysis is complicated b y t h e contribution of C~201~O ~7 to mass 45. I n practice, however, where we are interested only in t h e excess above n o r m a l a n d not the absolute concentration of C la, t h e contribution of C1:0~"0 ~7 is eliminated when the normal a b u n d a n c e is s u b t r a c t e d from t h e absolute a b u n d a n c e (atom per cent value). as S. Weinhouse, in " P r e p a r a t i o n a n d M e a s u r e m e n t of Isotopic T r a c e r s " (D. W. Wilson, A. O. C. Nier, a n d S. P. R e i m a n n , eds.), pp. 43-49. Edwards Bros., Ann Arbor, 1946. a, This reaction c a n n o t be used for aspartic acid, since b o t h carboxyl groups are liberated as carbon dioxide.

488

TECHNIQUES FOR ISOTOPE STUDIES

[21]

Easterfield and Taylor 4° have described a useful reaction for the preparation of carbon dioxide from the carboxyl group of fatty acids. The decarboxylation is carried out in the presence of iron filings, and the carbon dioxide formed is absorbed in Ba(OH)~ solution. Procedure. The combustion system of the apparatus shown in Fig. 2 can be used to convert the carbon of an organic compound to carbon dioxide. 41 A 15-ml. centrifuge tube containing 5 ml. of a saturated solution of Ba(OH)2 is attached to stopcock 5. Stopcocks 1 and 2 are set so that the combustion gases pass through trap C, through stopcock 2, through stopcock 5 and into the tube containing the Ba(OH)2 solution. After the combustion has been completed and the system flushed, the centrifuge tube is removed and covered with a rubber cap. The precipitated BaCO3 is washed three times with water by centrifugation. The BaCO3 is suspended in about 1 ml. of water and transferred to one arm of the sample vessel shown in Fig. 5. A little dilute HC1 is placed in the other arm and the vessel evacuated. From this point, the procedure is identical to that described previously for the analysis of the nitrogen isotopes. Calculation of Results. The atom per cent C 13 is calculated from equation 5 in which R is the ratio of the ion intensities of mass 44 to that of mass 45; i.e., R = (44)/(45). 100 Atom per cent C 13 = - (5) R+I This formula ignores the contribution of the molecule C1~0~60~7 to mass 45. The atoms per cent excess C ~3 is obtained by subtracting the normal abundance of C ~, which is about 1.1 atom %, from the calculated value of atom per cent C ~a. 40 T. H. Easterfield a n d C. M. Taylor, J. Chem. Soc. 99~ 2298 (1911). 41 If the a p p a r a t u s shown in Fig. 2 is n o t available, a convenient combustion system has been described b y Rittenberg. ~6

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

489

[22] T h e S y n t h e s i s a n d D e g r a d a t i o n o f I s o t o p i c a l l y L a b e l e d Carbohydrates and Carbohydrate Intermediates B y S. ABRAHAM and W. Z. HASSlD

I. Synthesis of Labeled Carbohydrates A. Biological Methods

1. Photosynthetic a. Preparation of Uniformly Labeled Glucose-C 14, Fructose-C 14, and Sucrose-C 14. Principle. When green leaves are allowed to photosynthesize in the presence of C1402, randomly C14-1abeled glucose, fructose, and sucrose are produced. The sugars are extracted with aqueous alcohol, purified b y passing through ion exchange columns, and separated by the aid of paper chromatography. M ° Reagents

B a r i u m carbonate. 80 % lactic acid. Paraffin oil. 0.1 N sodium hydroxide solution, carbonate-free. 80% ethanol. Duolite ion exchange resins C-3 and A-3 (Chemical Process Co., Redwood City, California). Water-saturated phenol solution. Glucose, c.p. Fructose, c.p. Sucrose, c.p. n-Butanol (52.5%), ethanol (32.0%), water (15.5%) solution. 1E. W. Putman, W. Z. Hassid, G. Krotkov, and H. A. Barker, J. Biol. Chem. 175, 785 (1948). 2 E. W. Putman and W. Z. Hassid, J. Biol. Chem. 196, 749 (1952). 3 S. Udenfriend and M. Gibbs, Science 110, 708 (1949). 4 G. R. Noggle and R. A. Bolomey, Plant Physiol. 26, 174 (1951). s M. Gibbs, R. Dumrose, and F. Archer, Biochem. Preparations 2, 45 (1952). 6 A. P. Lamb and G. O. Burr, Federation Proc. 8, 217 (1949). 7 H. K. Porter and R. V. Martin, J. Exptl. Botany 3, 326 (1952). s A. N. Wick, M. E. Blackwell, and N. Hillyard, Arch. Biochem. and Biophys. 32, 274 (1951). 9 C. A. Dubbs, Science 109, 571 (1949). 10R. H. Burris, P. W. Wilson, and R. E. Stutz, Botan. Gaz. 111, 63 (1949).

490

TECHNIQUES FOR ISOTOPE STUDIES

[22]

Carboraffin (or any other decolorizing carbon with low sugar absorbency).

Apparatus. The photosynthesis chamber is made of a Pyrex glass tube (Fig. 1) 21 cm. in length and 5.5 cm. in inside diameter. The lower end consists of a drawn-down recurred entry tube fitted with a stopcock, a, and a 1°~ o tapered joint. The upper end bears a 6°/go joint, A1, to which a cover is fitted carrying an exit tube which is also fitted with a stopcock, b, and 10~o joint. The volume between stopcocks a and b is 500 ml.

b

B Fla. 1. Photosynthetic chamber, Erlenmeyer flask, and Florence flask.

When conducting an experiment, about 500 mg. of barium carbonate containing approximately 5.0 me. of C 14 is introduced into a 25-ml. Erlenmeyer flask, B, from which the neck has been removed. A cork disk, e, slightly smaller than the inside diameter of the chamber, A, is fitted to the top of the flask so that its position is fixed when it is placed in the chamber. The barium carbonate is then mixed with about 5 ml. of water, and a few drops of paraffin oil are added to prevent excessive foaming. One milliliter of 80% lactic acid is placed in a small test tube, f, cut off so that it rests at about a 45 ° angle when placed in flask B. This flask, containing the barium carbonate slurry and the test tube of lactic acid, is then placed in an upright position in the photosynthesis chamber, A, which was previously rinsed with water to assure a humid atmosphere. (Enough water should also be left in the recurring portion of the entry tube to serve as an indicator when the internal and external pressures are equalized.) A Florence flask, C, from which the neck has been removed is attached to a plywood disk, h, bored through with numerous holes. The flask is filled with water, and the petiole of the leaf, which has previously been kept in the dark for 24 hours, is inserted so that it reaches the bottom of the flask. The leaf and its container are placed in a vacuum desiccator

[22]

491

CARBOHYDRATE SYNTHESIS AND DEGRADATION

which is evacuated to about 20 cm. After the initial flow of gas bubbles from the petiole of the leaf ceases, the pressures are equalized and the water displaced in flask C is replaced. The leaf and container are then placed in the photosynthesis chamber, A, on top of the cork ring, e. A piece of moistened filter paper is placed over the plywood disk to prevent spattering of acid into the upper part of the vessel. The greased upperend ground taper, A 1, is fitted on, and with the entry tube, a, closed the chamber is partially evacuated through the exit tube which is then closed with stopcock b. With the chamber tilted about 30 ° the acid is dumped

=

[~..L.~z- --:

gz

:--',~:

I t]

"

line

i "-'~

gl

FXG. 2. Complete apparatus for photosynthesis.

into the barium carbonate slurry. (This should be done slowly so as to avoid excessive foaming.) When the reaction has subsided and all the barium carbonate has reacted, with liberation of the carbon dioxide, atmospheric pressure is restored by opening stopcock a. The chamber is then immersed in a cylindrical Pyrex water bath, 10 inches high and 18 inches in diameter, held in position by a condenser clamp on a heavy ring stand in the bath as shown in Fig. 2. Illumination is effected by two 100-watt bulbs in desk lamps gl and g2 placed opposite each other on the outside of the bath. A small fan (not shown in Fig. 2) is placed above the water bath so that a current of air passes over the surface of the bath. This maintains the bath temperature 3 to 4 ° above the prevailing room temperature. Illumination is continued for 18 to 24 hours. A relatively long period of illumination is used in order to ensure complete utilization of the available carbon dioxide and to increase the probability of obtaining uniformly labeled compounds. During the last hour of illumination a soda-lime tower, E, is attached to the entry tube of the photosynthesis chamber, and two carbon dioxide traps, F1 and F2, are attached in series to the exit tube. During this period about 5 1. of carbon dioxide-free air is pulled through the system by application of a vacuum at F2. Trap F1 consists of a 500-ml. jar fitted with

492

TECHNIQUES FOR ISOTOPE STUDIES

[22]

a sintered-glass aerator containing 335 ml. of 0.1 N sodium hydroxide; trap F~ consists of a 100-ml. test tube also equipped with an aerator and 65 ml. of 0.1 N sodium hydroxide. Titration of a 25-ml. aliquot of the alkali in traps F1 and F~ with 0.1 N hydrochloric acid after addition of 1 ml. of 10% barium chloride, with phenolphthalein as an indicator, usually gives the same value as the alkali control. Procedure. Two Canna leaves (Canna indica), each about 5.0 inches long and weighing approximately 5 g., are left in water for 24 hours in the dark to reduce the carbohydrate stores in the leaves. They are then allowed to photosynthesize for 22 hours in an atmosphere of radioactive carbon dioxide, obtained from 1.0 g. of barium carbonate containing C TM, in a chamber as shown in Fig. 1. Best results are obtained when photosynthesis is carried out at 17°, since it has been observed that the sucrose/ reducing sugar ratio is more favorable at that temperature than at 28 ° . (The ratio of sucrose to reducing sugars is 1.0 at 17 °, whereas it is 0.7 at 280.1 ) At the end of that period all the carbon dioxide is usually absorbed by the leaves. The leaves are removed from the chamber immediately, cut into small pieces, and immersed in boiling 80% ethanol. The ethanol used for killing the leaves is transferred to a small boiling flask of a Soxhlet extractor, the cut leaves placed in a Soxhlet extraction thimble, and the leaves extracted with 80 % ethanol during a period of 24 hours. The alcoholic extract containing the soluble sugars is concentrated under reduced pressure to a small volume to remove the alcohol, diluted with water, and partially purified by passing through two successive ion exchange columns containing Duolite C-3 and A-3. The combined neutral effluent and water washings are concentrated to a sirup under reduced pressure, transferred to a small beaker, and dried to constant weight in a vacuum oven at 40 °. The neutral residue, 217 mg., contains a small amount of pigmented chlorophyll decomposition products, is dispersed in 2.2 ml. of water, and usually amounts to 82% of the activity incorporated into the leaves. This solution is then placed in 0.01-ml. aliquots on eight 22]/~ X 181/~-inch Whatman No. 1 filter paper sheets. The aliquots are placed 1.5 cm. apart as a band 7 cm. from the long edge of the paper, leaving a margin on either edge of the paper. The beaker containing the sample is washed at least fourteen times with 0.2-ml. portions of water, and this water wash is also distributed on the eight papers, after the original spots have been dried in the manner previously described. After drying in air, the papers are placed in airtight cabinets, and the band chromatograms are developed for 24 hours by descending'chromatography with water-saturated phenol as the solvent. The chromatograms are then dried in a current of air at room temperature, placed in contact

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with 14 X 17-inch Eastman Kodak medical "No-Screen" X-ray films, and the latter developed after about I hour of exposure. The dark bands on the resulting radioautographs coincide in position with those of fructose and glucose. Sucrose, which is also present, does not show up on the chromatogram, as its RI value in water-saturated phenol is approximately the same as that of glucose. These dark areas of the film radioautographs are used as templates to locate the exact position of the bands of radioactive sugars on the filter paper chromatograms. This is done by placing the film on an illuminated cabinet with a transparent upper side and tracing the position of the bands on the superimposed filter paper. The radioactive areas may be checked through the use of any type of C 14 assay counter. The sections of the paper containing the sugar bands are cut out, one margin end of each resulting paper strip is cut to a pointed tip, and the strips are eluted in a small chromatography cabinet by capillary descent with water as the solvent. ~ The eluates, about 2 ml. from each strip, are collected in separate beakers and concentrated i n vacuo to a sirup. Each of the sirups is dissolved in water and again placed on filter papers as described above. The band chromatograms are then developed for 72 hours with a mixture of 52.5% n-butanol, 32.0% ethanol (95%), and 15.5% water, by volume. Before placing the filter papers in the cabinets, their lower edges are serrated so as to allow uniform dripping of the solvent. These chromatograms are air-dried, and radioautographs are prepared again by using X-ray films. The radioautographs obtained from the eluates containing the fructose usually show only one distinct band after 1 hour of exposure, whereas the radioautographs from the sucrose-glucose mixture are resolved into two bands, corresponding in positions to the location of authentic samples of sucrose and glucose. With the autographs as a guide, the positions of the sugars on the paper chromatograms are located, excised, and eluted with water as before. After the eluates are concentrated i n vacuo, sirups of glucose, sucrose, and fructose are obtained. The sirups are then rechromatographed, first with water-saturated phenol and then with the butanol-ethanol-water mixture. These sugars are eluted from the papers and the solutions clarified with a little decolorizing charcoal (carboraffin is preferred, since it adsorbs little sugar). After the charcoal is filtered off, the solutions are concentrated i n vacuo, and the following yield of sugars is obtained: glucose 72.4 mg., sucrose 70.2 mg., fructose 61.2 mg. On the basis of the original C1402 used, approximately 70 % of the radioactivity is incorporated into these three sugars. Of this amount 21%, 29%, and 20% is represented by glucose, sucrose, and fructose, respectively. 1~ j. R. Hawthorne, Nature 160, 714 (1947).

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TECHNIQUES FOR ISOTOPE STUDIES

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In order to establish the radiochemical purity of these sugars, small amounts of each of them should be subjected to two-dimensional paper chromatography and the papers exposed to X-ray films for several days until visible spots due to radioactive impurities can be observed. The purity of each sugar can be determined by counting the activities of the sugar and the contaminant spots on the chromatogram. The following results have been obtained by this procedure: 2 glucose 99.8% pure, sucrose 99.5% pure, and fructose 97.8% pure. The degree of purity of these sugars can be further increased by once more repeating the paper chromatographic procedure. Chemical degradation of the C14-1abeled glucose 12-~4 and fructose 16,16 reveals that they are evenly labeled with C TM in each of their carbon atoms. Bacterial degradation with L. casei of the inverted sucrose also showed that this sugar is uniformly labeled with C14.5 b. Preparation of Randomly Labeled D-Galactose-C TM and Glycerol-C 14. Principle. The marine red alga Iridea laminarioides (Iridophycus flaccidum) is allowed to photosynthesize in an atmosphere of C14-1abeled carbon dioxide and the galactoside, a-D-galactopyranosylglycerol, isolated chromatographically. Enzymatic hydrolysis of this galactoside followed by paper chromatographic separation of the products leads to the isolation of D-galactose and glycerol.~7

Reagents C~4-barium carbonate. Lactic acid, c.p. 80 % ethanol. 95% ethanol. Duolite ion exchange resins A-3 and C-3. Water-saturated phenol. n-Butanol-ethanol-water mixture. Petroleum ether (b.p. 30 to 60°). Liquid nitrogen.

Procedure. Approximately 1 g. of Iridea thallus, devoid of reproductive structures or nodules, is moistened with water and placed in the 50-ml. 1~ S. Abraham, I. L. Chaikoff, and W. Z. Hassid, J. Biol. Chem. 195, 567 (1952). 13 S. Abraham, E. W. Putman, and W. Z. Hassid, Arch. Biochem. and Biophys. 41,

61 (1952). 14 p. V. Vittorio, G. Krotkov, and G. B. Reed, Science 115, 567 (1952). in S. Abraham, P h . D . thesis, University of California, 1953. 16 M. Gibbs, J. Biol. Chem. 179, 499 (1949). 17 R. C. Bean, E. W. Putman, R. E. Trucco, and W. Z. Hassid, J. Biol. Chem. 204, 169 (1953).

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CARBOHYDRATE SYNTHESIS AND DEGRADATION

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flask, A, of the photosynthesis apparatus (Fig. 3); 40 mg. of BaC1403 is introduced into the C02-generating tube, B, and about 0.4 ml. of concentrated lactic acid pipetted into the side arm, C. This flask is maintained at a temperature of 15 to 20 ° by means of a water jacket. The apparatus is evacuated to approximately 200 mm. through stopcock a, and the C140~ generated by turning tube B and allowing the lactic acid from side arm C to drop slowly on the carbonate. After cessation of gas production, t4/20~

,o

A

Fro. 3. Photosynthesis apparatus for Iridea laminarioides. tube B is gently heated with a small microburner and the internal pressure of the apparatus equilibrated to about atmospheric pressure by allowing air to bubble in through the water manometer, D. (A slight negative pressure should be maintained to avoid the loss of CO2.) The photosynthetic chamber, A, is immediately illuminated for a period of 10 hours with a 150-watt reflector spot lamp (about 10,000 foot-candles). At the end of this photosynthetic period the alkali trap, E, containing 2 ml. of 1 N NaOH (carbonate-free) is attached to the manometer stopcock and the apparatus slowly evacuated. The apparatus may then be flushed out by allowing CO2-free air to enter through the manometer and the apparatus re-evacuated through the alkali trap. The residual C1402 is thereby trapped, precipitated as barium carbonate, and saved. About 99.5% of the C1402 is usually absorbed by the thallus, as judged by the residual activity obtained in this manner. The thallus is then

496

TECHNIQUES FOR ISOTOPE STUDIES

[22]

removed from the chamber, dropped into a 40-ml. conical centrifuge tube containing liquid nitrogen, and the frozen material finely ground with a pestle shaped to fit the bottom of the tube. The plant material is extracted twice with 15-ml. portions of boiling 80% ethanol, centrifuged, and the extracts decanted. The residue is thoroughly mixed with 1 ml. of water, 10 ml. of 95% ethanol added, and the mixture boiled on a steam bath. The solution is removed by centrifugation and decantation and the extracts combined. This latter procedure is repeated twice to ensure complete extraction. The combined extract is concentrated on a steam bath and dried i n vacuo at 40 °. The resulting residue, weighing about 66 mg., contains approximately 70% of the original activity supplied to the thallus as C14-barium carbonate. The dried extract is then taken up in water and transferred to a 12-ml. conical centrifuge tube and partitioned several times with petroleum ether. The solvent phases are separated by centrifugation, and the ether phase containing pigments and fats removed with a capillary pipet. The inorganic material is removed from this defatted extract by passage through 3 ml. of Duolite A-3 and C-4 ion exchange resin columns. The combined neutral solution and washings are concentrated i n vacuo. The residue weighs about 18 rag. and now contains about 48% of the original C14-barium carbonate activity. This residue is dissolved in 0.25 ml. of water and applied in 0.01-ml. aliquots as a band on Whatman No. I filter paper and tbe band developed by descending chromatography with water-saturated phenol. ~ The paper chromatogram is allowed to dry at room temperature and is placed in contact with an X-ray film and exposed for about 1 hour; the film is developed and used as a template to locate the band of galactosylglycerol on the paper. This band is cut out, washed with ether to remove nonvolatile impurities, and eluted by capillary descent with water. When the water eluate is concentrated i n vacuo a sirup is obtained weighing approximately 17 mg. and possessing about 42% of the original C 14 activity incorporated into the thallus. The galactoside sirup is then hydrolyzed by incubating it for 20 hours with 0.5 ml. of a 1.0 % solution of yeast invertase containing a-galactosidase. The hydrolyzate is concentrated, dissolved in 0.25 ml. of water, and placed on paper, as described before. The galactose is separated from the glycerol by chromatographing in water-saturated phenol. After the bands are located by radioautography, they are eluted and separately rechromatographed with a mixture of 52.5 % n-butanol, 32.0 % ethanol, and ]5.5% water. These paper chr matograms are then exposed to X-ray film for 2 days. This procedure allows the detection of radioactive impuri-

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ties of the order of 0.1%. Elution with water and filtration of the eluates through sintered-glass filters serves to remove any solid impurities. The galactose eluate, when concentrated m vacuo, yields a sirup weighing about 8.6 mg. and possessing approximately 25 % of the original C140~ activity incorporated into the plant. The yield of glycerol is about 5.0 mg. with an activity of approximately 12% of the original C1402 activity. c. Photosynthetic Preparation of C t4-Labeled Starch. Principle. When green leaves, such as Turkish tobacco leaves, are placed in the dark to exhaust the available reserve starch supply and then exposed to an atmosphere containing C140~, radioactive starch is re.adily synthesized. ~.7.~-2~ Reagents

Perchloric acid, 72%, 11.2 to 11.7 N, reagent grade. Iodine solution (7.5 g. of Is and 7.5 g. of KI ground with 150 ml. of water, diluted to 250 ml., and filtered through a Whatman No. 3 filter paper with suction). Alcoholic sodium chloride solution (350 ml. of ethanol, 80 ml. of water, and 50 ml. of 20 % sodium chloride diluted to 500 ml. with water). Alcoholic sodium hydroxide solution, 0.25 N (350 ml. of ethanol, 100 ml. of water, and 25 ml. of 5 N sodium hydroxide diluted to 500 ml. with water and filtered as above). 60% ethanol. 80% ethanol. 95% ethanol. Absolute ethanol. Absolute ether. 2 N sulfuric acid. Barium carbonate. Duolite ion exchange resins A-3 and C-3. Procedure. The Turkish tobacco plants are grown in Hoagland's solution 22 or sand culture. The leaves, weighing from 3.0 to 3.5 g. and from 15 to 18 cm. in length, are taken from the middle portion of the stem of plants about 2.5 feet high. Leaves of plants grown in culture 18j. p. Nielsen, Ind. Eng. Chem. Anal. Ed. 15, 176 (1943). 19 G. W. Pucher, C. S. Leavenworth, and H. B. Vickery, Ind. Eng. Chem. Anal. Ed. 20, 850 (1948). 20L. G. Livingstone and G. Medes, J. Gen. Physiol. 31~ 75 (1947). ~1 D. W. Dutton, Biochem. J. §6~ xlviii (1954). 22 D. R. Hoagland and D. I. Arnon~ Calif. Agr. Expt. Sta. Circ. 347 (1938).

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TECHNIQUES FOR ISOTOPE STUDIES

[22]

solution are usually harvested in the morning and placed in water in the dark until the following morning. Plants grown in pots of sand are placed with the container in the dark for a similar period of time, and the leaves are harvested just before the experiment. On a fresh-weight basis the leaves starved in this manner usually contain about 0.25% reducing sugars (glucose and fructose) and 0.15% sucrose. The residue after the extraction of these reducing sugars does not give the characteristic blue starch-iodine color, indicating the absence of starch. After 24 hours of illumination in the chamber (described above) in an atmosphere of carbon dioxide derived from 500 mg. of barium carbonate, about 0.85% reducing sugars, 0.65% sucrose, and 2.5% starch are obtained. Radioactive starch can be prepared by the following technique: A Turkish tobacco leaf is harvested in the morning and allowed to remain in the dark for 24 hours to exhaust its starch content. The leaf is then placed in the photosynthetic chamber in an atmosphere of C1402, as previously described, and illuminated for 24 hours. At the conclusion of the photosynthetic period and after the chamber has been swept out with carbon dioxide-free air, the leaf is immersed in boiling 80% alcohol to inactivate the enzymes. It is then cut into small pieces and placed in a Soxhlet extraction thimble. The alcohol used in killing the leaf is transferred to the boiling flask of the extractor. The Soxhlet apparatus is assembled and the extraction continued for 6 to 8 hours, after which the alcoholic extract containing the soluble sugars (glucose, fructose, and sucrose) is set aside for subsequent isolation. At this time the leaf residue is completely devoid of plant pigment and is white in color. After the 80% alcohol extraction, the residue remaining in the Soxhlet thimble is dried at 50 ° in a vacuum oven for a period of 18 to 24 hours (0.3 to 0.4 g. of dry material is obtained). The dried leaf residue is quantitatively transferred to a small mortar, which is contained in an ice bath, 4.0 ml. of water and 3.0 ml. of 72% perchloric acid added, and the plant material ground with a pestle. The slurry is transferred to a centrifuge tube with a minimum of water and the residue spun down. The aqueous starch solution is decanted into a 250-ml. centrifuge bottle (previously marked at 100 ml. volume) and the residue re-extracted two more times in a similar manner. The combined extracts are then diluted to the mark with water and 6.6 g. of solid sodium chloride added. After the salt has dissolved, 10 ml. of the KI-I~ solution is added and the blueblack precipitated starch-iodine complex allowed to form for 30 minutes at room temperature and then 15 minutes at 2 ° . The bottle with its contents is centrifuged and the supernatant liquid tested for additional starch content by adding several drops of KI-I~ solution. If precipitation is complete, the supernatant liquid is decanted and the starch-iodine com-

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

499

plex is washed twice with the alcoholic sodium chloride solution which removes the excess iodine. The complex is decomposed and the starch precipitated by the addition of 2 to 4 ml. of 0.25 N alcoholic sodium hydroxide solution. The crude starch is then centrifuged down and washed two times with 60 % ethanol. The crude starch is dissolved in about 5 ml. of water, filtered through paper into a 50-ml. centrifuge tube, and reprecipitated with 3 vol. of 95% ethanol. When precipitation is complete, the starch is centrifuged and washed with 60% ethanol, twice with 95% alcohol, twice with absolute alcohol, and finally with ether. After the ether has evaporated and the starch is ground to a powder, it is placed in a v a c u u m oven and dried at 50 ° for 24 hours. The yields of starch with this m e t h o d are approximately 20 to 25% calculated on the d r y basis of the alcohol-extracted plant material. C14-Glucose from Hydrolysis of Radioactive Starch. Drying of the starch is unnecessary if it is to be used immediately for the preparation of radioactive glucose. The starch can be immediately hydrolyzed with sulfuric acid after washing with 60% alcohol. T h e radioactive starch is dissolved in sufficient water to make a 0.2% solution, and an equal volume of 2 N sulfuric acid is added. The mixture is refluxed for 30 minutes on a hot plate and, after refluxing, the theoretical a m o u n t of powdered barium carbonate needed to neutralize the acid cautiously added. The precipitate is centrifuged and washed well with water. The combined supernatant liquid and water washings are then passed through Duolite ion exchange columns C-3 and A-3, having a bed volume of 25 ml. Each column is washed with 100 ml. of water. The deionized solution, about 300 ml., is concentrated to a small volume in vacuo at 50 °, transferred to a 25-ml. beaker, and taken to a sirup in a v a c u u m oven at 50 °. The C14-1abeled glucose obtained in this m a n n e r has been shown to be uniformly labeled with C14.2~ 2. Intact Animal

a. Preparation of D-Olucose-3~4-C TM from Olycogen. Principle. On administration of various Cl4-1abeled compounds (NaHC1403,~4-27 succinate-l-C~4, 2s palmitate-l-C TM~) to animals, glycogen or glucose pro23 p. V. Vittorio, G. Krotkov, and G. B. Reed, Proc. Soc. Exptl. Biol. Med. 74~ 775 (1950).

24H. G. Wood, N. Lifson, and V. Lorber, J. Biol. Chem. 159, 475 (1945). 25 D. B. Zilversmit, I. L. Chaikoff, D. D. Feller, and E. J. Masoro, J. Biol. Chem. 176, 389 (1948). ~e H. R. V. Arnstein and R. Bentley, Biochem. J. 54, 493 (1953). ~ A. K. Solomon, B. Vennesland, F. K. Klemperer, J. M. Buchanan, and A. B. Hastings, J. Biol. Chem. 140, 171 (1941). 2s S. Abraham and I. L. Chaikoff, Arch. Biochem. and Biophys. 41, 143 (1952).

500

TECHNIQUES FOR ISOTOPE STUDIES

[22]

duced by these animals has been shown to be labeled almost exclusively in the 3 and 4 positions.

Reagents 20 % glucose solution. 1 N sodium hydroxide. BaC140~. 0.5 M citric acid solution. Nembutal. 30 % potassium hydroxide solution. 95% ethanol. Ether, anhydrous. 2 N hydrochloric acid. 1 N sulfuric acid. Anion and cation exchange resins. Succinic acid-l-C 14. Physiological saline (0.9% sodium chloride solution). Saturated lead acetate solution. Saturated disodium phosphate solution. 97 % methanol. Acetic anhydride. Pyridine, anhydrous. 0.1 N sulfuric acid. Saturated barium hydroxide solution. Duolite cation exchange resin C-3 and anion exchange resin A-4. n-Butanol-acetic acid-water mixture 29 (500 ml. of n-butanol, 105 ml. of acetic acid, and 250 mh of water). Solution of phenol saturated with water.

Procedure. Isolation as glycogen. 26 Six rats (total body weight 1.5 kg.) are fasted for 24 hours, after which 6 ml. of a 20% glucose solution is administered to each animal by stomach tube. One hour later 2 ml. of an aqueous solution of NaHC1403 is injected into each animal. This solution is prepared by absorbing the C140~ liberated from 5 mc. of BaC1408 (200 mg.) in a slight excess of 1 N NaOH (1.5 ml.), diluting with water to 10 ml., and adjusting the pH to 7.5 with 0.5 M citric acid, making a final volume of 12 ml. After 2 hours the animals are anesthetized with Nembutal; the livers are extirpated and immediately placed in hot 30% K O H (80 ml.). After the solution has become homogeneous, 160 ml. of ethanol is added and the solution cooled at 0 ° overnight. The crude glycogen, amounting to 2 g., is centrifuged off, washed twice with ethanol, once with ether, and dried. The crude glycogen is then dissolved in 10 ml. of hot water and the :9 S. M. Partridge, Biochem. Soc. Symposium, Cambridge 3, 52 (1950).

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

501

insoluble residue removed by centrifugation. The supernatant solution is acidified to pH 4 with 2 N HC1, and 4 ml. of ethanol is added. The precipitate is centrifuged, and the pure glycogen is isolated from the supernatant by the addition of 4 ml. of ethanol. This procedure yields 1.1 g. of white product (12.7 gc.). The glycogen is then hydrolyzed for 3 hours at 100 ° with 1 N H~SO4 and the acid removed by passage of the solution through a column of Deacidite Ez ion exchange resin. Concentration of the neutral effluent to a thick sirup and crystallization with absolute ethanol yields D-glucose-3,4-C 14. b. Isolation of D-Glucose-3~4-C ~4 from Urine. 2s A dog weighing approximately 7 kg. is surgically depancreatized in the usual manner and, for a period of 3 months thereafter, injected twice daily with insulin and fed an adequate diet2 ° This period ensures complete healing of the wound and brings the animal into a good nutritional state. Insulin injections are discontinued, and about 30 hours later the dog is injected intravenously with a solution of 10.4 mg. of succinic acid labeled with succinic acid-l-C 14 which has been dissolved in 6.8 ml. of physiological saline. The urine is collected thereafter by catheterization. The dog is not fed after the injection of succinic acid-l-C TM but is allowed to have access to water. Isolation of Glucose. 12 The collected urine is clarified by the addition of 50 ml. of a saturated solution of neutral lead acetate, and the resulting precipitate is filtered through Celite. To remove excess lead, 30 ml. of saturated disodium phosphate is added and the precipitate once again filtered through Celite. Enough 95% ethanol is added to the filtrate to double its volume, and the resulting solution is concentrated in vacuo to a thick sirup at a bath temperature which should not exceed 60 °. This sirup is mixed with an equal volume of technical methanol (97%) and warmed on a steam bath until thoroughly mixed. The precipitate which forms is filtered and discarded. The clear filtrate is once again concentrated in vacuo to a thick sirup. The isolation of pure glucose from this sirup is accomplished by preparing the water-insoluble pentaacetyl-a-D-glucose and subsequent deacetylation as described below. Preparation of Pentaacetyl-a-D-glucose. L2 For every 5 g. of glucose in the sirup (as determined by reducing values), 23 ml. of acetic anhydride and 35 ml. of anhydrous pyridine are added. The reaction mixture is stored at 0 ° and shaken periodically for several days (usually 10 days) until the sirup is dissolved. The mixture is then poured into 125 ml. of ice water, with stirring. Within a short time crystals of pentaacetyl-a-D~0 M. L. Montgomery, C. Entenman, I. L. Chaikoff, and H. Feinberg, J. Biol. Chem. 185, 307 (1950).

502

TECHNIQUES FOR ISOTOPE STUDIES

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glucose are formed. The crystals are filtered and washed with ice water. The yield of pentaaeetate usually ranges from 60 to 70%, based on the reducing value of the urine. Deacetylation of Pentaacetyl-a-D-glucose. 12 For every gram of the pentaacetate, 10 ml. of 0.1 N H2SO4 is used for hydrolysis. The reaction mixture is put into a three-necked flask and heated on a steam bath under a reflux. The reaction is allowed to continue, with constant stirring, for 4 to 5 hours, by which time all the pentaacetate goes into solution. An amount of Ba(OH)2 solution sufficient to neutralize the sulfuric acid is added, and the resulting BaSO4 precipitate is filtered through Celite. The combined filtrate and washings are then passed successively over a Duolite C-3 cation exchange column and a Duolite A-4 anion exchange column (3 X 25 cm.), which serves to remove the inorganic impurities. The effluent and washings are then concentrated in vaeuo to a thick sirup, and the glucose is crystallized by the addition of absolute ethanol to the sirup. The yield of crystalline glucose is about 97%, based on the pentaacetate. The glucose thus obtained, when subjected to two-dimensional chromatographic analysis with butanol-acetic acid-water and phenolwater, 29should give a single radioactive spot occupying an area identical with that of the color spot that appears after the filter paper chromatogram is sprayed with a solution of aniline oxalate, or p-anisidine hydrochloride. Chemical degradation I~,2s showed that glucose obtained in this manner is labeled exclusively in the 3 and 4 positions and that these two carbon atoms contain an equal amount of C ~a activity. 3. Enzymatic a. Preparation of Sucrose Labeled with C TMin the Glucose or Fructose Component. Principle. An enzyme (sucrose phosphorylase) isolated from Pseudomonas saccharophila is capable of catalyzing the reversible phos-

phorolysis and transglycosidation reactions of sucrose according to the following equations :31-83 _+phosphate a-D-Glucose-l-phosphate + enzyme ~_ Glucose-enzyme ~ ±fructose Glucose-l-fructoside ~- enzyme (sucrose) 81 W. Z. Hassid, M. Doudoroff, and H. A. Barker, J. Am. Chem. Soc. 66, 1416 (1944).

32M. Doudoroff,H. A. Barker, and W. Z. Hassid, J. Biol. Chem. 168, 725 (1947). aa M. Doudoroff,J. Biol. Chem. 151, 351 (1943).

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C A R B O H Y D R A TSYNTHESIS E AND DEGRADATION

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Thus sucrose with C14-1abeled glucose can be prepared by allowing labeled a-D-glucose-l-phosphate to react with unlabeled fructose by the aid of P. saccharophila enzyme. Radioactive sucrose with " t a g g e d " fructose is prepared by allowing C14-1abeled fructose to exchange for fructose in the unlabeled sucrose molecule in the presence of the same enzyme. 34

Reagents Enzyme solution prepared from 4 g. of dried cells of P. saccharophila ~2,33 (see also Vol. I [28]). C14-1abeled glucose-l-PO4 35 prepared from C14-1abeled starch. 1 Citrate buffer, pH 6.64. Phosphate buffer, pH 6.64. Torula monosa, yeast. Duolite ion exchange resins C-3 and A-4. Absolute ethanol. Sucrose, c.p. C14-1abeled fructose2 Potato phosphorylase 35 (see also Vol. I [22]).

Procedure. Four grams of dry cells of Pseudomones saccharophila is extracted twice with M/30 SCrensen phosphate buffer at pH 6.64, ~2,33the extraction being made in 1-g. lots. The cells are well dispersed in about 10 ml. of buffer per 1 g. of bacteria, either by grinding in a mortar or by agitation in a Waring blendor, and centrifuged after 20 minutes at room temperature. The combined supernatant liquids are then treated with ammonium sulfate at 0.8 saturation and kept at +_5 ° for several hours. The precipitate is collected by centrifugation, resuspended in the original volume of buffer, and treated for 1 hour at 5° with 0.3 saturated ammonium sulfate. The precipitate is discarded, and the supernatant is brought to 0.63 saturation of ammonium sulfate and stored at 5° for 3 days or longer. During storage the interfering enzymes (invertase, etc.) are inactivated while the precipitate retains most of the phosphorylase activity. Enzyme solutions can be prepared by suspending the precipitate in phosphate, citrate, or bicarbonate-carbonate buffers at pH 6.4 to 7.0. If the preparations are not clear, the fractionation with ammonium sulfate can be repeated and the material that is insoluble in the buffer can be removed by centrifugation. If the presence of phosphate is not desired in the final preparation, the enzyme can be reprecipitated several 34 H. Wolochow, E. W. Putman, M. Doudoroff, W. Z. Hassid, and H. A. Barker, J. Biol. Chem. 180, 1237 (1949). 35 R. M. McCready and W. Z. Hassid, J. Am. Chem. Soc. 66, 560 (1944); see also Vol. I I I [16B].

504

TECHNIQUES FOR ISOTOPE STUDIES

[22]

times from M / 3 0 citrate adjusted to pH 6.64. Enzyme preparations can be dialyzed against tap water for as long as 18 hours with little loss of activity, but they are inactivated by dialysis against distilled water. C14-Labeled a-D-glucose-l-phosphate is prepared according to McCready and Hassid 3~from a sample of radioactive starch, with the use of potato phosphorylase. The potato phosphorylase solution can be prepared in the following manner. 36,~7 A sufficient number of potatoes to furnish i 1. of juice are peeled and sliced and kept under water until used, preferably within 1 hour. The slices are then drained quickly and pulped in a Waring blendor. The minced material is filtered through cheesecloth, rapidly heated to 50 °, and maintained at that temperature for 5 minutes. The heating serves to inactivate the ~-amylase present in the juice. While the coagulated suspension is still warm, solid ammonium sulfate is added to bring the specific gravity of the solution to 1.085. The precipitate is centrifuged within half an hour and discarded. The supernatant liquid is adjusted to a specific gravity of 1.152 with solid ammonium sulfate and then centrifuged. The precipitate, which is not entirely soluble in water, is suspended in 250 ml. of water, and fractionation is repeated in a specific gravity range of 1.095 to 1.145. The precipitated enzyme is dissolved in 100 ml. of water for two subsequent fractionations in the specific gravity ranges of 1.100 to 1.140 and 1.100 to 1.35, respectively. The pH is determined at frequent intervals through the preparation and is kept between 6.0 and 6.5 by the addition of dilute ammonium hydroxide. Rapid inactivation of the enzyme occurs below pH 5.8. The final enzyme precipitate is dissolved in 25 ml. of water for each liter of crude potato juice used and stored at I to 2 °. At that temperature the enzyme can be kept from 4 to 6 weeks with little change of activity. One of the important precautions in the concentration of the enzyme is a speedy separation of the freshly expressed juice from the pulp, as inactivation is particularly rapid at this stage. All the other steps in the procedure should be carried out as quickly as possible. Sucrose with C~4-Labeled Fructose. 34 An extract from 2 g. of dried cells from P. saccharophila is mixed with 2.5 g. of sucrose acid and 0.2 g. of C14-1abeled fructose in a total volume of 30 ml. of citrate buffer at pH 6.64 and incubated for 6 hours at 30 °. At the end of that time, the enzymatic digest is inactivated by heating on a steam bath and the remaining fructose fermented with a washed cell suspension of the yeast, Torula monosa. This enzyme ferments glucose, fructose, and mannose but does not attack sucrose or other disaccharides. The organism is grown 8, D. E. Green and P. K. Stumpf, J. Biol. Chem. 142, 355 (1942). ~7p. H. Hidy and H. G. Day, J. Biol. Chem. 160, 273 (1945).

[9.2]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

505

aerobically for 24 hours at 28 ° on agar plates containing 0.5% yeast extract and 1% glucose. The ceils are washed twice by centrifugation and resuspended in 0.033 M phosphate buffer, pH 5. A yeast suspension prepared in this way will decompose approximately 15 rag. of glucose per hour per 100 rag. of dry yeast under anaerobic conditions at 37 °. The initial glucose concentration should not be above 3 %. The yeast is removed by centrifugation and the electrolytes by the use of ion exchange columns Duolite C-3 and A-3. The neutral solution is evaporated to 10 ml., 2.5 vol. of ethanol is added, and the alcoholic solution filtered. After evaporation of the solution and treatment of the sirup with absolute alcohol, the sucrose is obtained in crystalline form. The crude product should be recrystallized twice from ethyl alcohol. On the basis of the activity of the C14-1abeled fructose originally added, the radioactivity of the sucrose is usually found to be 76% of the possible theoretical activity of equilibrium. Sucrose with C~4-Labeled Glucose. 34 The reaction mixture consisting of 0.5 g. of a-D-glucose-l-phosphate containing C t4, 0.2 g. of inactive fructose, and 2.5 g. of inactive sucrose (carrier) is mixed with an extract from 2 g. of dry cells from P. saccharophila. This mixture is made up in a total volume of 30 ml. of citrate buffer at pH 6.64 and allowed to react for 6 hours at 30 °. The isolation of the sucrose is carried out in the same manner as previously described. The radioactivity of the sucrose is usually found to be about 72% of the theoretical activity at equilibrium. b. Preparation of Maltose Labeled with C ~4 in Either the Reducing or Nonreducing Glucose Component. Principle. A phosphorylase extract obtained from Neisseria meningitidis is capable of catalyzing the reversible reaction maltose phosphorylase Maltose + inorganic phosphate ÷ 13-D-Glucose-l-PO4 A- D-glucose When C14-1abeled ~-D-glucose-I-PO4 and nonlabeled D-glucose are employed in the presence of this enzyme, maltose is obtained with C l~labeled in the nonreducing unit. Nonlabeled f~-ester and C~4-1abeled D-glucose produce maltose labeled with C ~4 in the reducing unit. 3s

Reagents Uniformly labeled C14-maltose (from C14-1abeled starch) page 507). 3s

C. Fitting and E. W. Putman, J. Biol. Chem. 199, 573 (1952).

(see

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

507

starch) is incubated for 6 hours at 37 ° with the maltose phosphorylase extract in the presence of 20 tLN[. of sodium phosphate in a total of 1 ml. The reaction mixture is then chromatographed, as described above, with water-saturated phenol as a solvent. The radioactive bands corresponding to maltose, glucose, and hexose monophosphate are located by reference to X-ray film radioautography. The hexose monophosphate band is cut from the paper, eluted with water, and rechromatographed in a propanolammonia-water solvent, 4[ with inactive f3-D-glucose-l-phosphate as a side marker. After excision and elution of this band, a product is obtained which is presumably a mixture of sodium and ammonium ~-D-glucose-1phosphate. This hexose phosphate is then incubated with 30 ~M. of inactive glucose in the presence of the enzyme in a volume of 0.5 ml. for 4 hours at 37 °. The reaction mixture is chromatographed with water-saturated phenol, and the band corresponding to maltose is excised and eluted. A glucose impurity amounting to about 4% can be eliminated by a second chromatographic development in the butanol-acetic acid-water solvent.2 Following this procedure, a yield of 2.4 nlg. of maltose sirup is obtained which may be crystallized by the addition of inactive maltose, dissolved in water, concentrated, and ethanol added. c. Preparation of Randomly Labeled Maltose-C ~4. Principle. C 14labeled starch is hydrolyzed with a-amylase and the resulting C ~4maltose isolated by a paper chromatographic procedure. 3s Reagents C14-1abeled starch (see page 497). Human saliva, prepared by diluting with 1 vol. of distilled water and filtering through .Celite with the aid of suction. 4"~ Sodium chloride, c.p. 0.2 M phosphate buffer, pH 5.8. Water-saturated phenol. n-Butanol-acetic acid-water mixture. Procedure. A sample of C14-1abeled starch (10 rag.) is mixed with 5 rag. of solid sodium chloride and dissolved in 2.0 ml. of water. After the addition of 0.1 ml. of phosphate buffer and 1 ml. of diluted saliva, the mixture is incubated for 2 hours at 40 °. The salivary amylase is then inactivated by heating the mixture on the steam bath and the enzymatic digest concentrated in dryness in vacuo. The residue is dissolved in 0.25 ml. of water and chromatographed as a band on Whatman No. 1 filter paper 42 W. Z. Hassid, R. M. ]V[cCready, and R. S. Rosenfels, Ind. Eng. Chem. Anal. Ed. 12, 142 (1940).

508

TECHNIQUES FOR ISOTOPE STUDIES

[22]

with water-saturated phenol as solvent. The chromatogram is allowed to develop for 24 hours. T h e areas on the paper containing the separated sugars are located b y the radioautographic procedure, cut from the chromatogram sheet, eluted with water, and again chromatographed as a band with butanol-acetic acid-water as solvent. This chromatogram is developed for about 48 hours to ensure complete separation of the products of the enzymatic hydrolysis of the C14-1abeled starch. These products which are located b y the radioautographic technique are found to be maltose, isomaltose, and glucose. T h e maltose band is cut from the paper, eluted with water, and the solution concentrated i n vacuo. A yield of about 6 mg. of maltose h y d r a t e is obtained. T h e randomly labeled maltose contains approximately the same specific activity as the starting C~-labeled starch. B. Chemical Methods

1. Cyanohydrin

a. Preparation of Cyanide-C ~4. The cyanohydrin reaction has proved to be v e r y successful for the synthesis of m a n y carbon-labeled sugars and sugar derivatives and has been used extensively. Thus the preparation of cyanide-C 14 from barium carbonate-C ~4 is of considerable importance. I n order for such a conversion to be practical, it should meet the following standards. 48 T h e synthesis should (1) give high and reproducible yields, (2) utilize a minimum of steps, (3) require simple apparatus, and (4) not be time-consuming. P r i n c i p l e . Five general types of procedures have been used to prepare cyanide, involving the following reactions: 1. Carbon dioxide or carbonate with a metal and ammonia 44-~° 2. Carbonate with azido 51-55 48F. L. J. Sixma, H. Hendriks, K. Helle, U. Hollstein, and R. van Ling, Rec. tray. chim. 73, 161 (1954). 44R. D. Cramer and G. B. Kistiakowsky, J. Biol. Chem. 187, 549 (1941). 45R. B. Loftfield, Nucleonics 1, No. 1, 54 (1947). 46p. Olynyk, D. B. Camp, A. M. Griffith, S. Woislowski, and R. N. Helmkamp, J. Org. Chem. 13, 465 (1948). 4~R. Abrams, J. Am. Chem. Soc. 71, 3835 (1949). 4sj. A. McCarter, J. Am. Chem. Soc. 73, 483 (1951). 49j. A. Bos, Experientia 7, 258 (1951). 50j. K. Jeanes, Science 118, 719 (1953). 51A. W. Adamson, J. Am. Chem. Soc. 69, 2564 (1947). 52G. 0. Henneberry and B. E. Baker, Can. J. Research B 28, 345 (1950) 53B. G. van den Bos and A. H. W. Aten, Jr., Rec. tray. chim. 70, 495 (1951). 54V. I. Maimind, B. V. Tokarev, and M. M. Shemyakin, Doklady Akad. Nauk S.S.S.R. 81, 195 (1951). 55A. G. MacDiarmid and N. F. Hall, J. Am. Chem. Soc. 75, 4850 (1953).

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

509

3. Sodium amide with sodium formate 56 4. Carbon dioxide with triphenylmethylsodium 57 5. Barium carbonate with potassium in the presence of ammonium chloride 43 Only the first (1) and the last (5) appear to satisfy the above-mentioned conditions. Of the various procedures reported for the preparation of cyanide-C ~4, three methods have been described which give high yields. In the first method, C14-barium carbonate is heated in the presence of zinc dust and metallic sodium in a stream of anhydrous ammonia which has passed over hot iron. The C ~4 cyanide thus produced is distilled into potussium or sodium hydroxide. The yield of cyanide obtained by this one-step process is reported to be quantitative. 5° However, the results are not consistent. In the second procedure, reported by McCarter, 48 a mixture of potassium-carbonate-C 14 and zinc dust is heated in a stream of dry ammonia gas which has passed over hot iron before passing over the mixture of alkali carbonate and zinc. Consistent yields of 90% have been obtained by this method. However, in this method conversion of the barium carbonate-C ~4to potassium carbonate-C ~4is required. An excellent review of the methods of cyanide synthesis is presented by Sixma et al. 43 These workers also describe a third procedure which makes use of the reaction between barium carbonate, ammonium chloride, and potassium. With this method, consistent yields of 94 % were reported. This third procedure is likely to produce the best results. Reagents for Method 1 ~o

C14-barium carbonate. Zinc dust. Metallic sodium. Anhydrous ammonia gas. Iron wire or powdered iron. 2 N sulfuric acid solution. 1 N sodium or potassium hydroxide solution. Procedure for Method 1. An intimate mixture of 1.0 mM. of C14-barium carbonate, 1.0 g. of zinc dust, and 0.2 g. of metallic sodium cut in small pieces is placed in a porcelain combustion boat of suitable size. A 5.0-g. ball of iron wire or 5.0 g. of powdered iron distributed throughout two plugs of Pyrex glass wool is placed in the mid-portion of a Vycor or quartz combustion tube (600 mm. in length and 19 mm. inside diameter). ~e j . W. Spyker a n d A. C. Neish, Can. J. Chem. $0, 461 (1952). 5T B. Belleau a n d R. D. H. Heard, J. Am. Che~n. Soc. 72, 4268 (1950).

510

TECHNIQUES FOR ISOTOPE STUDIES

[22]

The boat is then placed in the tube and pushed until it touches or nearly touches the iron plug. The end of the tube nearest the iron is connected to a cylinder of anhydrous ammonia, and a stream of gas is allowed to flow through a gas bubbler at a rate of 3 bubbles per second in and through the tube containing the mixture. That portion of the tube containing the boat and the iron plug is brought to 650 ° and maintained at approximately that temperature with an electric muffle furnace for a period of 4 hours. At the end of that time, the tube is cooled to room temperature, the flow of ammonia being continued until the tube is completely cooled. The boat and the contents of the tube, except the iron ball, are then washed with water into a 250-ml. flask which can be attached to a distillation head for subsequent distillation of the hydrogen cyanide. The solution in the flask is acidified with 2 N sulfuric acid, and 20 to 30 ml. of distillate is collected in a receiver containing a 20% excess of 1 N sodium or potassium hydroxide. Analysis by the argentimetric method 58 for cyanide, together with the data obtained from analysis of C ~4 content, showed that the yield is quantitative and that the specific activity of the labeled cyanide is the same as that of the starting C~4-barium carbonate. Reagents for Method 2 48

Potassium carbonate-C ~4. Zinc dust (reagent grade). Iron wire (0.01 inch in diameter "for standardizing"). Anhydrous ammonia gas. 1 N sodium or potassium hydroxide solution. 2 N sulfuric acid solution. 4 N potassium hydroxide. Procedure for Method ~. C14-Labeled potassium carbonate is readily obtained by passing the C1402, produced from barium carbonate-C 1~, into a slight excess of 4 N potassium hydroxide and evaporating the solution to dryness. The powdered anhydrous potassium carbonate-C ~4 is thoroughly mixed with about 1 g. of zinc dust and the intimate mixture transferred to a porcelain combustion boat (Coors, Size 2). The l~oat is then placed in a Vycor combustion tube (750 ram. in length and 19 ram. in inside diameter) containing from 3 to 4 g. of iron wire in the form of a loose ball occupying the mid-portion of the Vyeor tube. The boat is pushed into the tube until it touches the iron wire. ~s I. M. Kolthoff and E. B. Sandell, "Textbook of Quantitative Inorganic Analysis," rev, ed.~ p, 574. The Macmillan Co., New York, 1943.

512

[22]

TECHNIQUES FOR ISOTOPE STUDIES

by vigorous shaking. The tube is then placed in an electric furnace which is kept at 640 ° for 1 hour. After cooling, the tube is carefully opened and the excess potassium is destroyed by the slow addition of ethanol. The resulting solution is then transferred to a 250-ml. round-bottomed flask which can be attached to a distillation head for subsequent distillation of the hydrogen cyanide. With boiling water, the last traces of radioactive cyanide can be removed from the Supremax reaction tube. The solution in the distillation flask is acidified with 2 N sulfuric acid, and 20 to 30 ml. of the distillate is collected in a receiver containing a 20% excess of 1 N sodium or potassium hydroxide. With this procedure the yields obtained from eleven experiments averaged about 99%. A blank value of approximately 5 % was observed when no barium carbonate was present, probably due to a slight absorption of carbon dioxide on the surface of the potassium, so that the net yield was therefore about 94%. 48 b. Preparation of D-Glucose-l-C TM and D-Mannose-l-C TM. Principle. The condensation of D-arabinose with C~4-1abeled cyanide leads to the production of D-glucose-l-C TM and D-mannose-l-C TM. In the presence of sodium bicarbonate and carbon dioxide, the ratio of the epimeric nitriles is about 3 parts mannonic nitrile to 1 part gluconic nitrile, but in the presence of sodium carbonate this proportion is approximately reversed. 59 This procedure is based on that of Isbell et al. ~9 Production of D-Mannose-l-C TM

D-Arabinose -~ NaC14N -~ NaHCO3 Jr COs-~ D-Mannono-a-lactone-l-C TM-~ barium-D-gluconate-l-C TM (20%) (67%)

$

D-Mannose-l-C i~ (50% over*all)

Production of D-Glucose-l-C 14

D-Arabinose -~ NaC~4N ~- Na~CO3-* Barium D-gluconate-l-C TM~ D-mannono-~-lactone-l-C TM (61%)

(30%)

(D_Glucono_~_lactone_l_C,4) --~ D_Glucose_l_C,4 (45% over-all) Reagents

Sodium cyanide-C '4. 59 H. S. Isbell, J. V. Karabinos, H. L. Frush, N. B. Holt, A. Schwebel, and T. T. Galkowski, J. Research Natl. Bur. Standards 48, 163 (1952).

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

513

Sodium bicarbonate. Sodium carbonate. Sodium hydroxide. Sodium oxalate. 5 % sodium amalgam pellets. (The 5 % amalgam is prepared in the form of small pellets by pouring it in molten condition through a heated alundum thimble, having a small hole in the bottom, into a 70-cm. "shot tower" of mineral oil. The pellets are stored under mineral oil. They are blotted dry just before use, weighed, and rinsed with benzene to ensure removal of any residual mineral oil.) Carbon dioxide, gas and solid. o-Arabinose. D-Glucose. D-~V[annose. D-Mannono-8-1actone. Barium gluconate trihydrate. D-Glucone-~-lactone. Acetone, c.p. Ether, anhydrous c.p. Ethanol, c.p. Methanol, c.p. Isopropanol, c.p. Chloroform, c.p. 5~[ethyl Cellosolve. Oxalic acid dihydrate. Benzoic acid. Barium hydroxide octahydrate. Phenolphthalein indicator. Celite (diatomaceous earth). Decolorizing carbon (carboraffin). Amberlite IR-100, analytical grade (cation exchange resin, regenerated, thoroughly washed with water; immediately before use the resin is washed successively with methanol and water, in order to inhibit microbiological action) (Resinous Products Division of Rohm and Haas Co., Philadelphia, Pennsylvania). Duolite anion exchange resin A-4.

Preparation Favoring the Synthesis of D-Mannose-l-C 14. A solution containing 2 mM. of sodium cyanide-C 14 and 2.24 mM. of sodium hydroxide in a total volume of 5 ml. is placed in a flask and the solution frozen in a mixture of dry ice (solid CO2) and acetone. To the frozen mixture are added 1 g. of solid carbon dioxide and 20 ml. of a solution

514

TECHNIQUES FOR ISOTOPE STUDIES

[22]

containing 2 raM. of D-arabinose. The mixture is allowed to thaw, and the flask is stoppered and stored in a refrigerator overnight. After standing at room temperature for 2 days the solution is hydrolyzed by heating for 4 hours at 50 ° under reduced pressure, and then for I hour on a steam bath. The cations are removed by passage of the solution through a column (1.4 X 24 cm.) containing Amberlite IR-100, analytical grade, and the combined solution and washings are lyophilized. The residue is moistened with methanol, seeded with D-mannono-~-lactone, and allowed to stand for 3 days. During this time a considerable amount of the lactone usually crystallizes out. The entire residue is then dissolved in 0.5 ml. of Methyl Cellosolve. The solution is treated with anhydrous ether to incipient turbidity and is again seeded with D-mannono-~-lactone. The D-mannono-~-lactone-l-C 14 that crystallizes out after several days is separated, washed with ethanol and ether, and dried. The yield is about 192 mg. (54%), m.p. 151 to 152 °, [a]~° = ~-53 °. The combined mother liquor and washings are then concentrated to a sirup, 180 rag. of D-mannono-~-lactone carrier is added, and the mixture is dissolved in 1 ml. of Methyl Cellosolve. Ether is then added to incipient turbidity, and the lactone is allowed to crystallize in the course of several days. This crystallization yields about 187 mg. containing about 13 % of the radiochemical yield. The total recovery of radioactivity as D-mannono-~-lactone-l-C 14 is thus about 67% .... In order to isolate the gluconic acid-l-C 14 which is also produced by the cyanohydrin synthesis, the mother liquor and washings from the crystallization of the mannonic lactone carrier are combined and evaporated to a sirup. After the addition of 1 mM. of barium hydroxide octahydrate (315 mg.) and 3 ml. of water, the mixture is digested on a steam bath for 1 hour. Carbon dioxide gas is then bubbled through the solution until it is acid to phenolphthalein, and the mixture filtered by suction on a funnel precoated vcith Celite and decolorizing carbon. The combined filtrate and washings are lyophilized and the residue dissolved in 0.5 ml. of warm water. Methanol is added until the solution is turbid and then seeded with barium gluconate trihydrate. After several days the crystalline barium gluconate-l-C 14 trihydrate is separated and dried. The yield usually amounts to about 42 mg. (7%). An additional yield of barium gluconate containing approximately 12% radioactivity can be obtained from the mother liquor and washings by the addition of carrier. Thus the radiochemical yield of barium gluconate-l-C ~4 amounts to about 19%, and the total radiochemical recovery from the sodium eyanide-C ~4 is approximately 86 %. Reduction of D-Mannose-~-lactone-l-C ~4. The D-mannono-~-lactone is reduced as follows: One millimole (178 mg.) of the material is dissolved

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

515

in 20 ml. of ice water and transferred into the apparatus shown in Fig. 4. Benzoic acid (1.5 g.) is added and stirred into the solution only long enough to effect dispersion; 4.6 g. of 5% sodium amalgam pellets is added, and the entire contents of the tube stirred for 1.5 hours at 0 °. At the end of this time, the mercury which forms is removed with a pipet, 1.2 g. of benzoic acid and 4.6 g. of sodium amalgam pellets are added, and the stirring is continued for an additional 2 hours or until all the amalgam

1/4" stainless steel stirrer ....

I|

li

tl

II II I I II

I I

T1 g

I

Stainless steel stopper g 24/40 fitted with greaseless bearings

I

i I i;i I ~, I.F-44

,,I I

,!,

Q,

i|

Heavy-walled glass tube

FIG. 4. Apparatus for reduction of sugar lactones by sodium amalgam.

is spent. The mercury is again removed as before, 1.26 g. of oxalic acid dihydrate is added, and the resulting benzoic acid is removed by extraction with chloroform. The aqueous solution is then mixed with 2 vol. of methanol and 2 vol. of ethanol which serve to precipitate out the sodium salts. The sodium salts are removed by filtration, washed with methanol, and set aside for recovery of any unreacted mannonic acid. The combined filtrate and washings are concentrated at reduced pressure to about 10 ml. and again diluted with 2 vol. each of methanol and ethanol. The salts are separated, ancl the solution is once again concentrated at reduced pressure to about 5 ml. and passed through a column (1.4 X 10 cm.) containing an equal mixture of Amberlite IR-100, analytical grade, and

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

517

dried. The yield of D-mannono-~-lactone-l-C 14 crystals amounts to about 0.11 g. Addition of carrier (0.2 g.) to the mother liquors and washings followed by crystallization yields another crop of about 0.18 g. of radioactive lactone. The total mannono-~-lactone-l-C 14 corresponds to a 30% radiochemical yield. The over-all radiochemical yield of the two hexonic acids by this procedure is about 94 %. Conversion of Barium Gluconate-l-C ~4 to o~Glucono-~-lactone. An aqueous solution containing 0.581 g. of barium gluconate (milliequivalents) is passed over a column (1 X 30 cm.) containing Amberlite IR-100, analytical grade, cation exchange resin, and the column is immediately washed with water. The effluent is lyophilized at once, and the residue taken up in a small amount of Cellosolve. (In the event that D-glucose1-C 14 is the desired final product, the Methyl Cellosolve solution of gluconic acid can be evaporated and lactonized in the reduction tube (see Fig. 4), thus avoiding transfer and possible loss of material.) The solution is seeded with D-glucono-~-lactone crystals, and the solvent is removed by evaporation in a stream of dry air. In the course of about 1 day, the solvent is almost removed, leaving a partially crystalline residue. This residue is then moistened with Methyl Cellosolve, and evaporation is continued until the material appears to be completely converted to the characteristic chunky crystals of the ~-lactone. This material, corresponding to about 90% of the theoretical yield, may be used directly for the preparation of D-glucose-l-C 14 by reduction with sodium amalgam. Reduction of D-Glucone-~-lactone-l-C ~4 with Sodium Amalgam and Separation of Crystalline a-D-Glucose-l-C ~. In order to reduce the D-glucono-~-lactone-l-C ~4, 0.7 g. of oxalic acid, 0.8 g. of sodium oxalate, and 20 ml. of ice-water are placed in the reduction tube containing 1 raM. (178 rag.) of the lactone. Immediately afterward, 2.3 g. of 5% sodium amalgam pellets is added, the mixture cooled in an ice bath, the tube closed, and the mixture vigorously stirred until the amalgam is spent. After removal of the mercury, 3 vol. of methanol is added, and the precipitated crystalline salts are removed by filtration, washed with methanol, and set aside for recovery of any unreacted gluconic acid. The alcoholic solution is then neutralized with aqueous sodium hydroxide and finally evaporated almost to dryness under reduced pressure. The residue is extracted with several small portions of methanol (total volume, about 10 ml.), and the extract filtered and diluted with water. The solution is then passed through a column (1.4 X 10 cm.) containing equal parts of cation and anion exchange resins (Amberlite and Duolite). The combined solution and washings are evaporated under reduced pressure to remove the methanol, and the concentrate is finally lyophilized to a sirup, weighing about 184 rag. The sirup is dissolved in a small

518

TECHNIQUES FOR ISOTOPE STUDIES

[22]

amount of methanol, transferred to a tared tube (total volume with washings, about 4 ml.), isopropyl alcohol added to incipient turbidity, and the solution seeded with crystals of D-glucose. Crystallization, which usually begins immediately, is allowed to take place over a period of several days. On decantation of the mother liquor and drying of the crystals a yield of 144 mg. of D-glucose is obtained. Specific rotation at equilibrium [a]~2 = +51o; m.p., 147 to 149 °. The yield of D-glucose-l-C TM from the reduction of the ~-lactone is thus about 80%. On addition of D-glucose carrier to the mother liquors and concentration of the solution the yield of radiochemical glucose is increased to about 45%. Frush and Isbell 6° have recently described two macroprocedures for the efficient reduction of aldonic acid lactones through the use of sodium amalgam in the presence of an oxalate buffer. The first employs a stainless steel flask equipped with a stainless steel stirring rod fitted with a curved vane that conforms in shape to the flask and sweeps the bottom. The second method makes use of a high-speed blendor. Either procedure is suitable for the preparation of rare sugars from their respective lactones in relatively large amounts. With these methods, L-glucose was prepared from L-glucono-8-1actone (10.0 g.) in 88% yields. c. Preparation of D-Glucose-6-C TM and D-Glucuronolactone-6-C TM. Principle. 5-Aldo-l,2-isopropylidene-D-xylofuranose prepared from the sodium periodate oxidation of 1,2-isopropylidene-D-glucofuranose is reacted with sodium cyanide-C TM. The product is hydrolyzed to the corresponding 1,2-isopropylidene-D-glucuronic acid-6-C 14, which is then lactonized 81 reduced with lithium aluminum hydride, and hydrolyzed to D-glucose-6-C14. 62 The procedure as outlined by Sowden 6~ gave yields of about 12%, and the method described by Shafizadeh and Wolfrom 63 gave yields of about 15%. A modification of these procedures has recently been reported by Schaffer and Isbell ~4 which increases the yield of D-glucose-6-C TM to 48% based on the starting sodium cyanide-C ~4.

Reagents Sodium metaperiodate. 1,2-Isopropylidene-D-glucofuranose. 6~.e so H. L. Frush and H. S. Isbell, J. Research Natl. Bur. Standards 54, 267 (1955). 61 j. C. Sowden, J. Am. Chem. Soc. 74, 4377 (1952). s~ S. Roseman, J. Am. Chem. Soc. 74, 4467 (1952). sa F. Shafizadeh and M. L. Wolfrom, J. Am. Chem. Soc. 77, 2568 (1955). 84 R. Schaffer and H. S. Isbell, U.S.A.E.C. Rept. No. NBS 4048 (1955); J. Research Natl. Bur. Standards in'press (1956). 86 D. J. Bell, J. Chem. Soc. 1935, 1874. ~6 C. L. Mehltretter, B. H. Alexander, R. L. Mellies, and C. E. Rist, J. Am. Chem. ~oc. 73, 2424 (1951).

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

519

Ethylene glycol. Chloroform, c.p. Acetone and dry ice. Benzene, c.p. Sodium cyanide-C 14 (see page 508). Sodium hydroxide. 1.0 N acetic acid. Sodium sulfite. Sodium carbonate. Amberlite IR-120 H. Barium hydroxide • 8H20. Carbon dioxide gas. Methanol, c.p. Toluene, c.p. Decolorizing carbon (Norit or Carboraffin). Anhydrous diethyl ether, c.p. (stored over sodium wire). Lithium aluminum hydride solution in ether. 67 Anhydrous ethanol. Glacial acetic acid. Sulfuric acid. Duolite ion exchange resin A-4. 2-Propanol. Methyl Cellosolve.

1,2-O-Isopropylidine-D-xylodialdopentofuranose. To a stirred solution of 50 g. of sodium metaperiodate and 400 ml. of water in a 1-1. flask surrounded by an ice bath was added during 30 minutes 50 g. of 1,2JOisopropylidine-D-glucofuranose.65,e6 The stirring was continued for an additional 20 minutes and the excess periodate then decomposed by the addition of ethylene glycol. The water was removed by freeze-drying, and the residue was extracted with four 100-ml. portions of chloroform. The combined chloroform extract was clarified by filtration through carbon and concentrated to a heavy sirup under reduced pressure. The sirup was dissolved in 100 ml. of water and once more concentrated under vacuum to remove all traces of chloroform. The concentrate was again dissolved in 50 ml. of water, filtered, and stored in the refrigerator for several weeks. The crystals that formed were separated, washed with water, and then recrystallized from water to give 29.9 g. of 1,2-O-isopropylidine-D-xylodialdopentofuranoside hydrate. The specific rotation of the hydrate is [~]~D° = 25.6 ° (in water). 67W. G. Brown, Org. Reactions 6, 484 (1951).

[9.~]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

521

glucuronolactone-6-C ~4 mixed with some brown residue. The product was used without further purification for the preparation of D-glucose-6-C 14. D-Glucose-6-C 14. The reaction vessel consisted of a 300-ml. threenecked Grignard reaction flask into which were inserted a condenser and a pressure-equalized dropping funnel, both protected with drying tubes. Through the third neck, stirring with a magnetic stirrer, 30 ml. of a diethyl ether solution of 13.2 mM. of lithium aluminum hydride 67 was added. The crude C14-1abeled lactone was dissolved in 70 ml. of diethyl ether and then added at a dropwise rate from the funnel over a period of 30 minutes to the stirred lithium aluminum hydride solution. The residual labeled material was rinsed into the reduction flask with ether, and the reaction mixture was refluxed for 30 minutes in a water bath at 50 to 60 ° while the solution was stirred. The mixture was then allowed to cool to room temperature after which 25 ml. of anhydrous ethanol was added dropwise, with continued stirring, followed by 130 ml. of water containing 6 ml. of glacial acetic acid to destroy the excess lithium aluminum hydride. After the supernatant ether was evaporated by a current of air an aqueous solution remained, which was then passed through 100 ml. of cation exchange resin (Amberlite IR-120 H), and washed with water until 725 ml. of eluant was collected. The solution was concentrated at reduced pressure, and finally 10 mN[. of aqueous sulfuric acid was added to make 200 ml. of 0.1 N sulfuric acid solution. The solution was heated for 2 hours in a boiling-water bath (steam bath) and, after cooling to room temperature, was passed through 40 ml. of anion exchange resin (Duolite A-4). A total of 550 ml. of eluant was collected, concentrated i n vacuo to about 50 ml., filtered through a bed of decolorizing carbon, and lyophilized. The residue was dissolved in methanol and a small amount of insoluble material removed by filtration. On conoentration of the filtrate to about 5 ml., addition of 2-propanol to incipient turbidity, seeding with crystalline D-glucose and, as crystallization occurred, with periodic additions of 2-propanol, a crop of crystalline D-glucose-6-C 14 was obtained. The crystals were separated from the mother liquor and washed with a mixture of methanol and 2-propanol. Recrystallization yielded 485 mg. of D-glucose-6-C ~4 of a specific activity of 9.2 ~c./mg. Through the use of 2.0 g. of carrier D-glucose, an additional 993 ~c. of labeled glucose was obtained. The over-all radiochemical yield based on the sodium cyanideC 14 was 48%. S o d i u m D-Glucuronate-6-C ~4 Monohydrate. In a 100-ml. flask equipped with a magnetic stirrer, an electrical heater, and a reflux condenser, a mixture of 896 rag. of barium 1,2-O-isopropylidine-D-glucuronate-6-C~4 (590 t~c.), 10 ml. of Amberlite IR-120 H, and 40 ml. of water was heated to reflux temperature for 50 minutes. The cooled mixture was filtered

522

TECHNIQUES FOR ISOTOPE STUDIES

[22]

through a small bed of resin and washed until 175 ml. of filtrate was collected. The clear filtrate was neutralized with sodium hydroxide, concentrated under reduced pressure, treated with decolorizing carbon, and filtered. T h e concentrated filtrate was then treated with a few drops of ethanol and seeded with crystalline sodium D-glucuronate monohydrate. 6s The resulting crystals were separated and washed with aqueous ethanol. Recrystallization from aqueous ethanol yielded 595 rag. of the monoh y d r a t e with a total radioactivity of 509 ~c. B y using carrier, an additional 34 ~c. of sodium D-glucuronate-6-C 14 m o n o h y d r a t e was obtained from the mother liquor, giving a 92 % radiochemical yield. D-Glucurone-6-C 14. A solution of 927 rag. of barium 1,2-O-isopropylidine-D-glucuronate-6-C ~ (165 ~c.) in 50 m l . . o f water and 10 ml. of Amberlite IR-120 H resin was treated as described above for the preparation of sodium D-glucuronate-6-C 14. After removal of the resin, the solution was concentrated at reduced pressure, passed through a filter coated with decolorizing carbon, and then lyophilized. The residue was dissolved in 5/[ethyl Cellosolve, seeded with o-glucurone, and allowed to crystallize. Recrystallization produced 438 rag. of D-glucurone-6-C 14 having a total activity of 133.5 ~c. B y use of carrier an additional 19.3 #c. of labeled glucurone was obtained, making the total radiochemical yield 93 %.

2. Nitromethane a. Preparation of Glucose-1-C TM and M a n n o s e - l - C 14. Principle. As developed b y Sowden, 69,70the procedure consists in converting methanolC ~4to methyl iodide by the m e t h o d of Norris 7~and then to nitromethane. 7~ Condensation of the nitromethane-C TM with D-arabinose according to Sowden and Fischer 78 yields 1-nitro-l-deoxysorbitol and 1-nitro-l-deoxyD-mannitol and subsequent conversion of the two nitro alcohols to the respective hexoses b y the Nef reaction.74 Reagents

C14H~0H. Hydriodic acid. Silver nitrite. D r y sand. 88W. Hach and D. G. Benjamin, J. Am. Chem. Soc. 76, 917 (1954). B9j. C. Sowden, Science 109, 229 (1949). 70j. C. Sowden, J. Biol. Chem. 180, 55 (1949). 71j. F. Norris, J. Am. Chem. Soc. $8, 639 (1907). 7~V. Meyer, Ann. 171, 23 (1874). 7~j. C. Sowden and H. O. L. Fischer, J. Am. Chem. Soc. 68, 1312 (1944); 67, 1713 (1945); 68, 1511 (1946); 69, 1048, 1963 (1947). ~4j. V. Nef, Ann. 280, 263 (1894).

[9.~]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

523

Solid C02-chloroform mixture. Calcium sulfate. D-Arabinose. Absolute methanol. Sodium methoxide solution. Ether, c.p. Petroleum ether (b.p. 30 to 60°). Amberlite IR-100, analytical grade, and I)uolite A-4 ioi~ exchange resins. Absolute ethanol. Anhydrous acetone. 2 N sodium hydroxide. Concentrated sulfuric acid. Sodium carbonate. Phenylhydrazine. Acetic acid. Benzaldehyde. Congo red indicator. Barium carbonate. D-Glucose. Procedure. C14-Methanol (16 g., containing 4 me. of C 14) is converted to methyl iodide by distillation with hydriodic acid. A yield of approximately 59 g., or 83%, is obtained. 69,7°,7~ To an intimate mixture of 120 g. of silver nitrite and 250 g. of dry sand the methyl iodide is added dropwise under a cold finger maintained at - 4 0 ° by means of dry ice and chloroform. After the addition of the methyl iodide, the mixture is heated for 2 hours on the steam bath. The condenser is warmed to - 5°, and the gaseous methyl nitrite is flushed out of the system with a slow current of dry air into a re('eiver at - 7 0 °. The yield of methyl nitrite is approximately 4 g. (l 6 %). The nitromethane is distilled from the reaction flask with a slow stream of dry air into 20 g. of silver nitrite and calcium sulfate. About 18 g. of nitromethane (71%) is obtained. A suspension of 44 g. of D-arabinose in 50 ml. of absolute methanol is added to the 17.9 g. of nitromethane, and the mixture is treated with a solution of sodium methoxide (8.8 g. of sodium in 350 ml. of absolute methanol). The mixture is stirred for 5 hours at room temperature in a closed system, and the precipitated sodium nitro alcohol filtered and washed in rapid succession with cold methanol (-20°), ether, and petroleum ether. The mixed nitro alcohols are dissolved in 350 ml. of 75 j . D. Cox, H. S. Turner, and R. J. Warne, J. Chem. Soc. p. 3167 (1950).

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

525

converted to methyl-D-glucopyranoside. The methyl derivative is oxidized to a dialdehyde which on hydrogenation produces the corresponding dialcohol. This dialcohol is hydrolyzed, and the resulting glycerol is separated from the glyceraldehyde by removal of the latter as the 2,4dinitrophenylhydrazone. The identity of the glycerol obtained by this method was established from its tribenzoate derivative and from paper chromatographic data. 7s Several methods for preparation of glycerol labeled in a single position are described in the literature. ~7-79 Reagents

Uniformly labeled glucose-C 14 (see page 489). 0.083 N hydrochloric acid in anhydrous methanol. Benedict's solution. 0.37 M sodium periodate solution. 0.1 N sodium hydroxide solution. ~[ethyl red indicator. Barium chloride. Raney nickel. Hydrogen gas. Concentrated sulfuric acid. 2,4-Dinitrophenylhydrazine solution. 35 to 38% formaldehyde solution. Barium carbonate. Anhydrous acetone, c.p. Water-saturated phenol mixture, s° Butanol-acetic acid-water mixture, s° Saturated solution of lead tetraacetate in benzene (anhydrous). Pyridine, c.p. Be~lzoyl chloride. Ether. Saturated sodium bicarbonate. Anhydrous ethanol. Procedure. Uniformly labeled C14-glucose with a high specific activity caa be prepared photosynthetically according to the method of Putman el al. ~ (see p. 489). The methyl glucoside is prepared by dissolving the

76S. Abraham, J. Am. Chem. Soc. 74, 6098 (1952). 77A. P. Doerschuk, J. Am. Chem. Soc. 73, 821 (1951). 78H. Schlenk and B. W. DeHaas, J. Am. Chem. Soc. 73, 2921 (1951). 7~M. L. Karnovsky and L. I. Gidez, Federation Proc. 10, 205 (1951). s0 S. M. Partridge, Biochem. Soc. Symposia, Cambridge 3, 52 (1950).

526

TECHNIQUES FOR ISOTOPE STUDIES

[22]

sugar (1.0 g.) in absolute methyl alcohol containing dry hydrogen chloride (0.083 N HC1) according to the method of Fischer sl as described in the section on the chemical degradation of glucose. The yield of the mixed isomers is approximately 84 %. A small sample of this material should give a negative reaction with Benedict's solution. The methyl glucoside is then dissolved in 35 ml. of water, treated with 35 ml. of 0.37 M sodium periodate solution, and the mixture allowed to remain at 2 ° for 24 hours. The formic acid formed is titrated with 0.1 N sodium hydroxide to the methyl red end point and is usually found to be 100% of theoretical. A small aliquot of the solution at this point gives a positive Benedict's test. Solid barium chloride (1.6 g.) is added to the neutralized solution, and the resulting precipitate is filtered and washed with water. The filtrate is concentrated under reduced pressure at a bath temperature of less than 60 ° (the formic acid is recovered in the distillate), and the additional precipitate formed during this time is filtered and washed with water. The dialdehyde in aqueous solution'is then hydrogenated at 2700 p.s.i. and 140 ° for 18 hours, with Raney nickel as the catalyst. An aliquot of this solution gives a negative Benedict's test. The filtered solution is acidified with 3.5 mh of concentrated H2SO4 and is hydrolyzed under reflux for 3 hours. At the end of that time the solution turns slightly yellow in color. Addition of 42 ml. of 2,4-dinitrophenylhydrazine solution (4 g. of 2,4-dinitrophenylhydrazine -{- 20 ml. of concentrated sulfuric acid -~ 30 ml. of water ~ 100 ml. of 95% ethanol) precipitates the glycolaldehyde as its hydrazone. This is filtered, washed, and dried with a resulting yield of 1.0 g. (approximately 90%). Addition of 0.7 ml. of 35 to 38% solution of formaldehyde removes most of the excess 2,4-dinitrophenolhydrazine as the hydrazone, which is filtered and discarded. The H2S04 is neutralized with a large excess of solid BaCO3, and the resulting precipitate filtered and washed with distilled water. The combined filtrate and washings are treated with charcoal and filtered. The resulting slightly yellow solution is concentrated in vacuo, at 40 °, to a thick viscous mass. Extraction with boiling, anhydrous acetone, and final evaporation of the acetone at room temperature yields the glycerol-C 14. The recovered weight is about 0.4 g. (64 % based on glucose corrected for 78% glycerol in water as judged by its refractive index). The refractive index of this glycerol is 1.4398 at 25 °. This represents 77.6% glycerol in water. 82 The specific activity of the resulting glycerol is the same as the original glucose. Two-dimensional ,paper chromatography with water-saturated phenol 81 E. Fischer, Bet. 28, 1145 (1895). s~ "International Critical Tables," Vol. 7, p. 68. McGraw-Hill Book Co., New York, 1930.

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

527

and butanol-acetic acid-water s° reveals only one radioactive spot, which is identical with the color spot obtained with inactive glycerol, with a solution of lead tetraacetate in benzene as the color spray. Experiments with inactive glucose by the same method yielded glycerol (65%) which was identified (1) by its refractive index, as given above, and (2) by its crystalline glycerol tribenzoate derivative. Preparation of Glycerol Tribenzoate. To 0.5 g. of the synthesized glycerol is added 5 ml. of pyridine, and the solution is cooled on an ice bath for 10 to 15 minutes. Two milliliters of benzoyl chloride are added to this acid mixture, and the resulting mass is allowed to stand at room temperature overnight. The solution is then poured into 150 ml. of ice water (acidified with 5 N H~SO4) and allowed to stand for 2 hours. A clear oil separates out and is extracted with ether. The ether solution is washed several times with a saturated solution of NaHCOa and finally with cold water. The ether is evaporated, and the resulting oil is dissolved in a minimum of absolute ethyl alcohol. After the alcoholic solution is allowed to stand at 0 °, the glycerol tribenzoate crystallizes. The product is recrystallized from ethanol, m.p. 74-75 ° (lit. 75°). A mixed melting point with the tribenzoate prepared from c.p. glycerol is not depressed. Degradation of Glyeerol-C ~4. A small aliquot of the glycerol-C ~ is dissolved in absolute ethanol, and inactive glycerol is added. The ethyl alcohol is removed in vacuo at room temperature, and the resulting diluted glycerol-C ~4may be degraded according to the procedure outlined by Kritchevsky and Abraham.83 b. Preparation of D-Xylose-l-C 14 from D-Glucose-l-C ~4 by Means of Periodate. Principle. D-Glucose-l-C ~4 is acetonated to 1,2,5,6-diisopropylidine-D-glucofuranose-l-C14. 84 Controlled hydrolysis of this derivative yields 1,2-isopropylidine-D-glucofuranose-l-C ~4, which on treatment with periodate leads to the formation of the substituted dialdehyde 5-aldo-l,2-isopropylidine-D-xylofuranose. When reduced with Raney nickel followed by mild acid hydrolysis this dialdehyde gives D-xylose1-C I4 in 55 to 60% yield. 85

Reagents D-Glucose-l-C 14. Sulfuric acid, c.p. Acetone, c.p. 83 D. Kritchevsky and S. Abraham, Arch. Biochem. and Biophys. 39, 305 (1952). s4 C. L. Mehltretter, R. L. Mellies, J. C. Rankin, and C. E. Rist, J. Am. Chem. Soc. 73, 2424 (1951). ~ J. C. Sowden, J. Am. Chem. Soc. 73, 5494 (1951).

528

TECHNIQUES FOR ISOTOPE STUDIES

[22]

50% NaOH solution. HC1, c.p. Benzene, c.p. Methyl red indicator. Sodium metaperiodate. Starch-iodide paper. Glycerol, c.p., or ethylene glycol, c.p. Absolute ethanol. Anhydrous sodium sulfate. Chloroform, c.p. Hydrogen gas. Raney nickel. Duolite ion exchange resin A-4.

Preparation. To a suspension of 5.0 g. of powdered D-glucose-l-C TM in 100 ml. of acetone at 0 °, 4 ml. of concentrated sulfuric acid is added dropwise. The mixture is stirred with a magnetic stirrer in a closed flask for 4 hours at room temperature. About 140 mg. of unreacted glucose is then separated from the solution by filtration. The clear filtrate is made just alkaline with a 50% sodium hydroxide solution at 15 to 20 °. The resulting sodium sulfate is filtered and washed thoroughly with fresh acetone. The combined filtrate and washings are concentrated at reduced pressure, water is added, and the concentration of the solution continued to a volume of 50 to 70 ml. The solution is adjusted to pH 2.0 with concentrated HC1 and stirred for 4 hours at 40 °. The solution is then adjusted to pH 8 to 8.5 with a sodium hydroxide solution and extracted with an equal volume (100 to 125 ml.) of benzene which serves to remove the unchanged diacetone glucose. Methyl red indicator is added, and the aqueous layer is stirred with portions of powdered sodium metaperiodate, keeping the pH as near as possible to the end point of the indicator by the dropwise addition of aqueous sodium hydroxide. After the addition of 7.3 g. of periodate, the presence of excess oxidant can be tested for by using starch iodide paper. The excess periodate is destroyed by the addition of a few drops of glycerol or ethylene glycol. The solution is then concentrated at reduced pressure to a thick sirup. Anhydrous sodium sulfate is added to the sirup, and the acetonate dialdehyde is extracted with five 40-ml. portions of chloroform. Concentration of the chloroform extract at reduced pressure yields about 5 g. of crude acetonated dialdehyde. The product is mechanically shaken in 65 ml. of 95 % ethanol with hydrogen in the presence of 5 g. of Raney nickel for 90 hours at room temperature and atmospheric pressure. The Raney nickel is then filtered and washed with 95% ethanol, and the combined filtrate and washings

530

TECHNIQUES FOR ISOTOPE STUDIES

[22]

After removal of the catalyst by filtration, the solution is evaporated to dryness i n vacuo, and the residue extracted with hot pyridine. On cooling of the pyridine solution, the sorbitol crystallizes as its pyridine complex. After two crops of the complex are thus isolated, 2 g. of sorbitol is added to the mother liquor as carrier, and two additional crops of this complex are isolated. The combined crops of the sorbitol-pyridine complex are recrystallized from pyridine two times and after drying i n vacuo over H2SO4 at room temperature. The product weighs approximately 9.2 g. The total yield of anhydrous sorbitol-C 14 is about 6.4 g. after quantitative removal of the pyridine by heating the complex at 80 ° and 1 ram. of pressure over H2SO, for 48 hours. Another method for the preparation of sorbitol by using sodium borohydride with nonisotopic glucose results in an approximate yield of 80%. 89 The method employed by Wick et al2 ° involves the use of Raney nickel 9~ as the catalytic reducing agent on glucose-C 14.

II. Degradation of Labeled Carbohydrates A . Biological Methods 1. Lactobacillus casei

See Vol. IV [23].

2. Baker's Yeast The method employed by Barnet and Wick 92 is based on the principle that when glucose is fermented by baker's yeast to ethyl alcohol and carbon dioxide, the ethyl alcohol represents carbons 1, 2, 5, and 6, and the COz represents carbons 3 and 4 of the fermented glucose. The ethyl alcohol can then be oxidized to acetic acid 9~ and further degraded by the Schmidt (azide) reaction described below. 3. Leuconostoc mesenteroides

See Vol. IV [23] for degradation to lactate and acetate. a. Degradation of C14-Labeled Lactate and Acetate (see also Vol. IV [23]). P r i n c i p l e . The following reactions are involved in the complete 89M. Abdel-Akher,J. K. Hamilton, and F. Smith, J. Am. Chem. Soc. 73, 4691 (1951). 90A. N. Wick, M. C. Almen, and L. Joseph, J. Am. Pharm. Assoc. Sci. Ed. 40, 542 (1951). 91S. D. Borisoglebskii, J. Appl. Chem. (U.S.S.R.) 13, 571 (1940). 92H. N. Barnet and A. N. Wick, J. Biol. Chem. 185, 657 (1950). 9sA. C. Neish, "Analytical Methods for Bacterial Metabolism," National Research Council of Canada Report No. 46-8-3 (rev.), pp. 21-22, Saskatoon, Sask., 1950.

[22]

CARBOHYDRATE SYNTHESIS

AND D E G R A D A T I O N

531

degradation and isotopic assay of the individual carbon atoms of lactate and acetate :94,95 KMnO4,H +

CH3CHOHCOOH * CH3COOH + C02 (3) (2) (1) (3) (2) (1) CH3COOH NaN3,H~SO~ CH3NH,. + CO~ (3) (2) (3) (2) KMnO4,0HCH~NH2 + CO.., (3) (3)

(a) (b) (c)

Although m a n y procedures for the degradation of these compounds have been reported 24,95-99 which involve elaborate equipment, the method described here permits the complete degradation to be performed in the combined combustion-diffusion vessel described below24,1°° A p p a r a t u s and Reagents

Reaction flask. Ordinary 50-ml. narrow-mouthed Erlenmeyer flasks provided with a center well for CO~ absorption are used. A center well similar to t h a t used in a conventional Warburg flask is sealed to the bottom of the vessel. The exact dimensions of the well are not critical; about 12 mm. in diameter and approximately 3 cm. high will be found convenient (Fig. 5A). Serum bottle stoppers, sleeve type. Catalog No. 16201, Braun, Knecht and Heimann Co., San Francisco; catalog No. 2319, A. H. Thomas Co., Philadelphia, 1950 (Fig. 5B). Siphon for carbonate sample transfer. A siphon which facilitates the quantitative transfer of the carbonate solution from the center well of the combustion flask is shown in Fig. 5C. 5 % potassium permanganate solution in 2 N sulfuric acid. Carbonate-free NaOH solution, approximately 1.0 N. Carbonate-free N a O H solution, approximately 0.1 N. Saturated barium chloride solution. Phenolphthalein indicator. Phenol red indicator. 100% sulfuric acid (3 parts of concentrated H~SO4 and 1 part of fuming H2SO4 9~). 94j. Katz, S. Abraham, and I. L. Chaikoff, Anal. Chem. 27, 155 (1955). 95E. F. Phares, Arch. Biochem. and Biophys. 33, 173 (1951). 56H. A. Barker and M. D. Kamen, Proc. Natl. Acad. Sci. U. S. 31, 219 (1945). 97S. Aronoff, H. A. Barker, and M. Calvin, J. Biol. Chem. 169, 459 (1947). 98S. Roseman, J. Am. Chem. ~oc. 75, 3854 (1953). 99L. Daus, M. Meinke, and M. Calvin, J. Biol. Chem. 196, 77 (1952). ~0oj. Katz, S. Abraham, and N. Baker, Anal. Chem. 26, 1503 (1954).

532

TECHNIQUES FOR ISOTOPE STUDIES

[22]

2 N sulfuric acid containing 1 to 2 % H20~ (freshly prepared). 30% sodium hydroxide solution. H~S04 solution, approximately 0.10 N. 5 % potassium permanganate solution. Sodium azide (twice recrystallized from water and alcohol and stored in a desiccator over calcium chloride). 2 N sodium hydroxide solution (carbonate-free). To Vocuum

To Vacuum

9 B

A

FIG. 5. Combined combustion--diffusion vessel and siphon. The Oxidation of Lactate (Equation a). Two hundred micromoles of lactate is introduced into the vessel, and 2 ml. of 5 % K M n 0 4 dissolved in 2 N H~SO4 is added. T h e total volume can be in excess of 10 ml. ; however, for subsequent ease in distillation of the resulting acetate a small volume is preferable. One milliliter of standardized carbonate-free base (1.0 N) is placed in the center well. The flask is immediately capped and evacuated by inserting through the rubber cap a 20- or 22-gage hypodermic needle which is connected to a v a c u u m line. T h e flask is heated for 30 minutes in an 80 ° water bath and allowed to cool. The carbonate solution in the center well is collected by means of the siphon, barium chloride is added, and the excess base is titrated to the phenolphthalein end point. The BaCO3 is mounted and assayed for C14.1°1 T h e acetate is recovered b y steam distillation of the permanganate solution in a small M a r k h a m still, I°~ 20 vol. of distillate is collected, and the acetic acid 101C. Entenman, S. R. Lerner, I. L. Chaikoff, and W. G. Dauben, Proc. Soc. Exptl. Biol. Med. 70~ 364 (1949). 102R. Markham~ Biochem. J. 36, 790 (1942).

[22]

CARBOHYDRATE S Y N T H E S I S AND DEGRADATION

533

titrated with standard base. Typical results of such experiments with variously labeled lactates are presented in Table I. 94 TABLE I a DEGRADATION OF LACTATE-C 14

Products of reaction Per cent recovery Compound Lactic Lactic Lactic Lactic Lactic Lactic

Reaction

acid-l-C 14 KMnO4oxidation acid-l-C 14 K2S208oxidation acid-2-C TM KMn04 oxidation acid-2-C TM K~S20~oxidation acid-3-C TM KMnO~oxidation acid-3-C '4 K2S~0~oxidation

CO.~ Acetate

COs

97 100 99 92 95 92

24.6 8.5 2.4 35.8 1.3 18.1

98 -96 -96 --

Per cent activity in carboxyl carbon of Acetate lactate

Specific activity~

0 ------

96.5 -2.3 -2.4 --

a j. Katz, S. Abraham, and I. L. Chaikoff, Anal. Chem. 27, 155 (1955). b Counts per minute of BaC14Os assayed with an end-window Geiger-Mueller tube [C. Entenman, S. R. Lerner, I. L. Chaikoff, and W. G. Dauben, Proc. Soc. Exptl. Biol. Med. 70, 364 (1949)].

The Degradation of Acetate (Equation b). The sodium acetate solution is evaporated to dryness on the steam b a t h and the residue dissolved in a small volume of water (1 or 2 ml.). Aliquots are taken for determination of total activity by persulfate combustion, described in this section, 1°° and for the azide (Schmidt) reaction. The combustion with persulfate yields the average specific activity of acetate, which represents the 2 and 3 carbons of lactate. The aliquot used for the Schmidt reaction is put into a shell vial which conveniently fits into the center well of the reaction flask, and the solution is evaporated to dryness in a v a c u u m oven. A b o u t 0.2 ml. of 100% H.,SO495 followed b y about 40 mg. of recrystallized sodium azide is added cautiously and the contents mixed with a small, fine stirring rod which is left in the vial. The vial is placed in the center well and the flask capped, evacuated as described before, and placed in a water bath at about 35 °. The temperature is brought to 70 to 80 ° within 15 to 20 minutes, and maintained for half an hour. The vessel is cooled, and about 2 ml. of C02-free alkali (1.0 N N a O H ) is carefully injected into the main c o m p a r t m e n t by means of a syringe and a 3-inch No. 20 hypodermic needle. After about half an hour for CO2 adsorption, the v a c u u m is released b y inserting a hypo-

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

535

standard, and CO2-free base is put in the center well. The flask is again capped, evacuated, and the COs liberated by injecting excess dilute sulfuric acid into the alkaline permanganate solution. The methylamine can also be combusted with a large excess of persulfate (page 549). As seen from Table II, the yields of both CO2 and methylamine range from 90 to 95 %. B. Chemical Method with Lead Tetraacetate I. Determination of C 14 Activity of Individual Carbon Atoms of the Glucose Chain (see Vol. IV [23] for Biological Procedures)

Principle. The activity of the individual carbon atoms in the glucose chain is obtained from the combined data of the degradation of glucose and several of its derivatives. Their oxidation with specific oxidizing agents yields C02 either from a single carbon atom or from groups of carbon atoms. The general scheme is outlined in Fig. 6. I~ Degradation of glucose-C ~4 from various biological sources has been performed by this method.12.1s.2s.~03 2. Preparation of Glucose Derivatives

Reagents for the Preparation of Glucose Derivatives Methyl-D-glucoside. D-Glucose. Methanol, anhydrous. Hydrochloric acid, dry gas. Liquid nitrogen. Ethanol, anhydrous. Decolorizing carbon. D-Glucose phenylosazone. Phenylhydrazine hydrochloride, recrystallized. Sodium acetate. Ether. D-Glucose phenylosotriazole. Copper sulfate pentahydrate. n-Butanol. 2-Phenyl-4-formylosatriazole. Sodium metaperiodate. Methyl red indicator. Sodium hydroxide solution, approximately 0.1 N. ~0s F. W. Minor, G. A. Greathouse, H. G. Shirk, A. M. Schwartz, and M. Harris, J. Am. Chem. Soc. 76, 1658 (1954).

536

[22]

TECHNIQUES FOR ISOTOPE STUDIES

I

CHO

COOH

I

I

CHOH

CH2

I

I

HOCH

CHs

I

HBr I

I

J

+ HCOOH ~(o) cos (C=I)

CHOH ----* CO CHOH

CH~

I

CHsOH CHO

II

CHO

r I

I CHOH [

CHO

I

CHO

[

I

Pb(OAc)~

\ /

CH20 III H OCH3

H

\/

t

I

HOCH

\/

OCH3

C

C CHOH

/

CHsO + 2 COs (C-1, C-2)

CHOH CHCH~

I

\

CHOH CHCH3

HOCH

,

J

CHO

I Pb(OAc)4--* O

0

+ COs (C-3)

CHO [ CH

l

CHOH

I

l

CHsOH

CH

I

IV H

CH2OH OCH~

H

\/

C

C

HOCH

I

OCH3

\/

O

Pb(OAc)(

)

CHO

CHOH

CHO

CHOH

CHs

J

O _ + COs

(c-4)

I

CH2-FIG. 6. General scheme for degradation of glucose.

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

V

HC~N

HC~N

1 b )NC6H5 C-=N HOCH

+ HCHO

I

(C-6)

(C-l, C-2, C-3)

+2C0~

1

CHOH

i

)NC6H.~ C---N CHO

Pb(OAc),

. . . . . . --~

(C-4, C-5)

CHOH

t

VI

CH20H CHO

I

CHOH

I

HOCH

f I

CItOH

NaI(h

5 HCOOH + HCHO

1(o)

CHOH

CO2 (C-6)

I

CH:OH Fro. 6. (Continued)

Calcium gluconate. Barium iodide. Iodine. 0.4 N barium hydroxide solution. Sulfuric acid, concentrated. Lead carbonate. Silver sulfate. Hydrogen sulfide. Duolite cation exchange resin C-3. Calcium carbonate. D-Arabinose. Barium acetate. Ferric sulfate. Ascarite tower (for CO2-free air). 0.5 N sodium hydroxide solution. 30% hydrogen peroxide (Superoxol). Duolite anion exchange resin A-4. Barium chloride monohydrate. D-Arabinose. Aniline oxalate spray reagent.29

537

538

TECHNIQUES FOR ISOTOPE STUDIES

[22]

Methyl-D-arabinopyranoside. Silver carbonate. Ethyl acetate, c.p. 4, 6-Ethylidine-D-glucose. Paraldehyde. Ammonium hydroxide.

Reagents for the Determination of the C 14Activity in the Glucose Carbons 10% hydrobromic acid. Phenolphthalein indicator. Mercuric acetate. Lead tetraacetate. Glacial acetic acid. Hydrochloric acid solution, standardized at 0.10 N. Pyridine, c.p. 1 N hydrochloric acid. Sodium arsenite solution (30 g. of As203 and 15 g. of NaOH in 500 ml. of solution, giving a solution of approximately 1.2 N Na2HAsO3 lo4). Potassium permanganate. a . Methyl-D-Glucopyranoside. Methyl glucoside is prepared in a sealed ignition tube according to the method of Fischer. 81 Ten milliliters of a 0.25% solution of dry HCI (0.086 N) in pure, anhydrous methyl alcohol is added to 1 g. of the dry, crystalline glucose in the tube. The mixture is refluxed with frequent shaking for about 30 minutes on a steam bath in which time all the glucose dissolves. The contents of the tube are frozen with liquid nitrogen, and the bomb tube is carefully sealed with a torch. The tube is then placed in a lead pipe as a precautionary measure. Glucoside formation is carried out at 100° for 50 hours in an oven. At the end of that time the sealed tube is immersed in liquid nitrogen. The tube is then cautiously opened, the contents removed from the tube with a minimum amount of anhydrous methyl alcohol, and the solution concentrated in a vacuum desiccator. When crystals of the a isomer separate out, the small amount of mother liquor is poured off and the crystals are washed with absolute ethyl alcohol. This first crop of methyl glucoside crystals is dissolved in water, treated with decolorizing carbon, and filtered. The solution is then concentrated to a thick sirup in a vacuum oven at 40 °, and crystallization is induced with anhydrous ethyl alcohol. The pure methyl-a-D-glucopyranoside melts at 166° and gives [a]~° = ~-158.9 ° (water, c = 10). Weight of dry crystals, 0.60 g.

~04W. C. Pierce and E. L. Haenisch, "Qualitative Analysis," 2nd ed., p. 203. John Wiley & Sons, New York, 1946.

[29.]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

539

The mother liquors and alcohol washings are combined and concentrated. F u r t h e r t r e a t m e n t of the concentrate b y the method described above yields a second crop of crystals. T h e pure methyl-~-D-glycopyranoside melts at 105 ° and gives [~]eo = _ 3 4 . 2 ° (water, c = 10). Weight, 0.20 g. The two crops of crystals are dried and combined. Total yield, 0.80 g., or 75 % of theoretical. b. D-Glucose Phenylosazone. To 1 g. of glucose in a large test tube is added a solution of 5 g. of recrystallized phenylhydrazine hydrochloride and 8 g. of sodium acetate in 20 ml. of water. T h e mixture is heated on a steam bath for 1 hour. During this time, golden yellow crystals of the phenylosazone are formed. At the end of the hour, the tube is cooled to room t e m p e r a t u r e and then stored at 0 °. T h e crystals of the glucosazone are then separated b y filtration and dried. The crude crystals are dissolved in boiling ethanol (95%), and water is added until the osazone begins to crystallize. Crystallization is allowed to proceed at 0 °. The glucosazone is filtered and washed, first with small amounts of ice water and then with ether. T h e crystals are dried in a v a c u u m oven at 40 °. The yield of pure osazone is 1.2 g. (62% of theoretical). c. D-Glucose Phenylosotriazole. The osotriazole is prepared according to the m e t h o d of H a n n and Hudson. 1°5 An aqueous solution of 100 ml. of CuSO4 (7.0 g. of CuSO4'5H20 in 600 ml. of H~O) is added to 0.83 g. of finely divided, recrystallized osazone, and the resulting suspension, after the addition of about 2 ml. of n-butyl alcohol (wetting agent for the osazone), is refluxed for 2 hours. ]V[ost of the osazone dissolves after the first hour and, as the color of the solution changes from blue to green, a small a m o u n t of copper or copper oxide precipitates. At the end of the second hour, the solution, now dark in color, is filtered while hot. Crude osotriazole crystals appear when the solution is stored at 0 °. The solution is filtered, and the crystals are dissolved in a minimum of boiling water. Activated carbon is added, and the boiling solution is filtered in a steamheated Biichner funnel. T h e solution is allowed to cool to room tempera° ture and stored at 0 ° overnight. During t h a t time, crystallization of pure osotriazole is completed. Concentration of the filtrate from the crude osotriazole i n vacuo yields a second crop of the triazole; this is recrystallized in the aforementioned manner. T h e total yield of pure, asbestos-like crystals is 0.32 g. (50% of theoretical); m.p., 195 to 196 °. d. 2-Phenyl-4-formylosatriazole. A suspension of 0.3 g. of the osotriazole in 9.6 ml. of water is refrigerated for 1 hour. To this suspension is added 9.6 ml. of a saturated (0.37 M) aqueous solution of NaIO4, ~05 R. M. H a n n and C. S. Hudson, J. Am. Chem. Soc. 66, 735 (1944).

[@.2]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

541

in the Erlenmeyer flask is warmed on the steam bath, and 2.4 ml. of 30% hydrogen peroxide is added from the dropping funnel. With the appearance of gas bubbles, the flask is removed from the steam bath and the reaction allowed to continue. The reaction is complete with the appearance of a purple color. A second 2.4-ml. portion of peroxide is added in the same manner, whereupon a similar reaction takes place. The dark reaction mixture is then filtered and passed over the large Duolite C-3 and A-4 ion exchange columns (3 X 25 cm.). The neutral effluent is concentrated to a thick sirup under reduced pressure, and crystallization is induced by the addition of absolute methyl alcohol. Subsequent recrystallization produces white crystals of D-arabinose. Approximate yield, 1.0 g. Fifty milliliters of a BaC12 solution (10 g. BaCI~'H20 in 100 ml. of H~O) is added to the NaOH in the trap. The resulting BaCO3 is filtered off, washed, dried, and weighed. The amount obtained is 1.8 g. (theoretical yield, 2.5 g.). This barium carbonate cannot be used as a valid measure of the activity of carbon 1.1~ The arabinose prepared in this manner is examined chromatographically 29 and gives a single C14-containing spot which occupies an area identical with that of the colored spot that appears after the filter paper is sprayed with a solution of aniline oxalate. g. Methyl-D-arabinopyranoside. The methyl arabinoside is prepared by a modification of the method of Hudson. 108One-half gram of arabinose is refluxed for 3 hours with 6.7 ml. of pure, anhydrous methyl alcohol containing 1.5% of dry HC1 (0.516 N). The acid is neutralized with about 0.6 g. of solid Ag2CO3; the solution is filtered, treated with charcoal, and refiltered. On reducing the volume of the filtrate in a vacuum desiccator, a gumlike material is obtained. This material consists of a mixture of the a and ~ isomers of methyl-D-arabinopyranoside. To separate these isomers, the mixture is extracted with hot ethyl acetate and the insoluble isomer is then crystallized from absolute ethyl alcohol. Re-extraction with hot ethyl acetate and recrystallization from ethyl alcohol yields pure methyl-~-D-arabinopyranoside with m.p. 169° and gives [a]~° = -245.5 ° (water, c = 7). The alcohol mother liquors are evaporated in a vacuum desiccator to a thin sirup which crystallizes slowly on standing. This crystalline mixture of a and ~ isomers is separated and fractionally recrystallized from hot ethyl acetate as previously described. Since the ~ isomer is less soluble in ethyl acetate, it crystallizes first, and the a isomer accumulates in the mother liquors. The total weight of the pure f~ isomer is 0.23 g., 43% of theoretical. lo8 C. S. Hudson, J. Am. Chem. Soc. 47~ 265 (1925).

542

TECHNIQUES FOR ISOTOPE STUDIES

[22]

h. 4,6-Ethylidine-D-glucose. One gram of crystalline glucose is converted to 4,6-ethylidine-D-glucose according to the method of Hockett et al. 109 A solution consisting of 1 ml. of paraldehyde and 2 drops of concentrated sulfuric acid is added to 1 g. of glucose in a 125-ml. glassstoppered flask, and the mixture is placed on a mechanical shaker. The contents of the flask are shaken for 24 to 30 hours at room temperature. At the end of that time the reaction mixture usually sets to a solid mass. On the addition of about 30 ml. of ethyl acetate, the flask is stored at 0 ° overnight. The crude crystals are then filtered and washed successively with ethyl acetate and ether. They are recrystaliized from 25 ml. of ethanol to which about 3 drops of aqueous ammonium hydroxide has been added. The solution should be neutral at this time. A small amount of insoluble salt is removed by filtration through a pad of Celite and the resulting clear solution concentrated to a small volume in vacuo at room temperature. The concentrated alcohol solution, about 4 to 5 ml., is then placed in the icebox to induce crystallization of the pure 4,6-ethylidine-Dglucose. Yield is approximately 310 mg. and has a melting point of 179 to 180 °. 3. The Quantitative Recovery of Carbon Dioxide in the Lead Tetraacetate Oxidation of Glucose Derivatives Principle. The action of 3 moles of lead tetraacetate on compounds which contain three adjacent hydroxyl groups leads to the formation of the corresponding dialdehyde with the middle carbon splitting out as COs. This reaction can be used in the oxidation of the various glucose derivatives for the isolation of some of the individual carbon atoms. 11° A schematic representation of this reaction is as follows:

R

R

I

I

I I

R'

HCOH CHO I 3Pb(C,H,O2)~ HCOH CHO + CO2 HCOH

I

R' It should be noted, however, that under these conditions the oxidation of vicinal primary hydroxyl groups yields formaldehyde which is not further oxidized. 109 R. C. Hockett, D. V. Collins, and A. Scattergood, J. Am. Chem. Soc. 73, 599 (1951). tl0 S. Abraham, J. Am. Chem. Soc. 72, 4050 (1950).

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

543

Apparatus and Reagents Lead tetraacetate (prepared according to McClenanhan and Hockettm). Glacial acetic acid. Phenolphthalein indicator. Methyl orange-methylene blue mixed indicator. 1 M barium chloride solution. 0.10 N hydrochloric acid solution. 0.10 N sodium hydroxide solution (C02-free). Ascarite, C02-absorbing column. The apparatus is shown in Fig. 7. A column (at G) is filled with ascarite and serves to obtain carbon dioxide-free air. The reaction tube (A) is 2 cm. in diameter and 17 cm. long. The inlet which is placed 1.5 ram. from the bottom of tube A has a diameter of 4 mm. (drawn down to 2 ram. at the bottom) and emerges from tube A about 8 cm. from the bottom. A 24/40 standard taper glass joint connects the reaction tube (A) with the gas washing tube (B). The buret inlet at H has a 5-ml. capacity and a stopcock (a) with a 2-ram. bore. The tip of this buret should extend below the glass joint 24/40. The bulb on tube B has a diameter of 3 cm. and is 15 cm. long. Connected to the gas washing tube (B) with a 10/30 standard taper glass joint are burets D and E ring-sealed on a 50/50 standard taper glass joint (F). Buret D has a 35-ml. capacity which is graduated into tenths, and buret E has a capacity of 5 ml. Both stopcocks b and c have a 2-ram. bore. The tips of both burets should go below the 50/50 joint. Buret E has a driptip with a 4-ram. diameter; D has a regular drawn-out buret tip made from standard 2-ram. capillary. The 50/50 glass joint (F) is now connected to the receiver (C). The top 7 cm. of C has a diameter of 4.5 cm. and is drawn down to 2 cm. for the bottom 9 cm. The gas bubbling tube (d) which has the 10/30 joint is made from standard l-ram, capillary and reaches through the ring seal (F) to within 1 cm. of the bottom of the receiver (C). The outlet tube which is 4 ram. in diameter is about 1 cm. down from the bottom of the 50/50 joint. The suction is applied at this point when the apparatus is all assembled. Procedure. The oxidizing agent, lead tetraacetate, is prepared according to McClenanhan and Hockett, TM and stored in a desiccator over phosphorus pentoxide. When needed, it is weighed out and put into 2 ml. of glacial acetic acid in the reaction tube (A). Two additional milliliters of the reagent is used to wash down the sides of the tube. The slight brown color, due to decomposition of the reagent, has no effect on the reaction as a large excess of the oxidizing agent is used in the reactions. 111 W. S. McClenanhan and R. C. Hockett, J. Am. Chem. Soc. 60, 2061 (1938).

[29.]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

545

phthalein h a v e been added, is sucked into the receiver (C) through buret (c). A precipitate of b a r i u m carbonate is formed in the receiver (C). After increasing the r a t e of bubbling, the excess sodium hydroxide is titrated to the phenolphthalein end point, with care to avoid local concentration of acid. (In the case of C14-1abeled compounds, the b a r i u m carbonate m a y be assayed for a c t i v i t y at this point.) T h e yield of carbon TABLE III I~ECOVERY OF CARBON DIOXIDE a

Compound I)-Glucoseb D-Arabinoseb Methyl-I)-glucoside b Methyl-D-arabinoside b D-Glucose phenylosotriazoleb Glycerol ¢ 4,6-Ethylidine-D-glucose ~ Formaldehydeb

Amounts used, Time, g. hr. 0.012 0.025 0. 068 0. 0125 0. 044 0. 062 0. 0343 0. 060

8.0 6.0 4.0 3.0 5.0 4.0 4.0 4.0

CO2 calculated, %

Carbons of sugar as BaCO3

95.3 99 97.7 !)9.5 98.7 99.4 96.5 0.0

1,2,3,4,5 2,3,4,5 3 4 4,5 2 1,2 --

The temperature is maintained at 40 to 42° throughout these reactions. b S. Abraham, J. Am. Chem. Soc. 72, 4050 (1950). c D. Kritchevsky and S. Abraham, Arch. Biochem. and Biophys. 39~ 305 (1952). d S. Abraham, I. L. Chaikoff, and W. Z. Hassid, J. Biol. Chem. 195, 567 (1952). dioxide is then determined b y titration of the precipitated b a r i u m carbonate with 0.1 N hydrochloric acid to the m e t h y l orange end point. W h e n the sample was 0.0120 g. of glucose, the carbon dioxide yield as determined b y this m e t h o d was 95.3% of the theoretical. 110 I n the case of the D-glucose phenylosotriazole, the c o m p o u n d is dissolved in 1 ml. of pyridine and introduced into the reaction tube. W a t e r (0.5 ml.) is added to the reaction t u b e at the beginning of the oxidation, since water is needed to produce the carbon dioxide. Blanks are run before each reaction to determine t h e c o n t a m i n a t i n g carbon dioxide. An alkali absorption column (ascarite) is used to obtain carbon dioxide-free air which is sucked t h r o u g h the whole apparatus. T h e blanks thus obtained are usually negligible. 4. D e t e r m i n a t i o n of C 14 Activity of Individual Glucose Carbons Isolation of Carbon 1 (Fig. 6, Equation I). This m e t h o d is a slight modification of t h a t of Sowden. 1~ One g r a m of crystalline glucose is heated with 10 ml. of 10% h y d r o b r o m i c acid in a sealed t u b e at 130 ° for 112j. c. Sowden, J, Am. Chem. Soc. 71, 3568 (1949).

546

TECHNIQUES FOR ISOTOPE STUDIES

[22]

24 hours. The black residue obtained is filtered off and washed with water. Sufficient solid silver sulfate is added to the filtrate to precipitate the unreacted hydrobromic acid and bromide ion. The precipitate is filtered and washed well with water. The filtrate is made acid with 1 N sulfuric acid and steam-distilled; 1500 to 2000 ml. is collected in a period of 3 to 4 hours. The major portion of the formic acid distills over in the first 1000 ml. The distillate is titrated with standard sodium hydroxide to the phenolphthalein end point. The yield of formic acid is approximately 83 %. The formic acid is oxidized to CO2 with mercuric acetate,11~ and the COs collected in sodium hydroxide solution. The carbonate is precipitated by addition of barium chloride, mounted on filter paper, and assayed for radioactivity. 1~° Isolation of Carbons 1 plus 2 (Fig. 6, Equation II). An aqueous solution of 4,6-ethylidine-D-glucose containing 34.3 mg./ml, is prepared and oxidized with lead tetraacetate ~° for 4 hours. The recovery of C02 is about 96 to 97%.

Isolation of Carbon 3, Carbon ~, and Carbons ~ plus 5 (Fig. 6, Equations III, IV, and V). The lead tetraacetate oxidations of methyl glucopyranoside, methyl arabinopyranoside, and D-glucose phenylosotriazole are carried out as previously described. The resulting BaCOa precipitates are mounted on filter paper, TM and C 1~ contents determined. Isolation of Carbon 6 (Fig. 6, Equation VI). A sample of glucose, when oxidized with periodate, yields 5 moles of formic acid and 1 mole of formaldehyde. T M This formaldehyde, which is derived from C6, is isolated and oxidized to COs with KMn04. To 0.18 g. of glucose dissolved in 15 ml. of water is added 15 ml. of 0.37 M NaI04. The solution is allowed to stand overnight at room temperature; by this time the oxidation is about 98% complete. The excess periodate is decomposed by the addition of 30 ml. of 1 N HC1 and enough sodium arsenite reagent to remove all the iodine color from the solution, usually about 40 ml. The solution is made basic to phenol red with NaOH, and distilled. The distillate is collected in an iced container, and the distillation continued until crystals begin to form in the distilling flask. Two grams of KMnO4 is added to the distillate, and the mixture is refluxed while a stream of CO2-free air is passed through the system. The CO2 produced from the reaction is collected in NaOH. After 1 hour of refluxing, the system is flushed out for 20 minutes, and the carbonate precipitated with 2 ml. of 1 M BaCl~ solution. The excess base is titrated lla H. D. Weihe a n d P. B. Jacobs, Ind. Eng. Chem. Anal. Ed. 8, 44 (1936) ; J. D. Reid a n d H. D. Weihe, Ind. Eng. Chem. Anal. Ed. 10, 271 (1938). H~ R. E. Reeves, J. Am. Chem. Soc. 63, 1476 (1941).

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

547

to the phenolphthalein end point, and the BaC03 formed is mounted on filter paper and its C 14 content measured. TM Isolation of Carbons 1 through 6 and Carbons 2 through 6. Glucose is oxidized by the wet combustion method of Van Slyke and Folch H5 or by the persulfate method described on page 549, H6 converted to barium carbonate, mounted, and assayed for the activity of the complete hexose molecule. T M Arabinose representing carbons 2 through 6 of the starting glucose, obtained from the decarboxylation of calcium gluconate, is also converted to barium carbonate and assayed for C ~4 in a similar manner. The values for C J4 activity of four of the six glucose carbons, namely 1, 3, 4, and 6, are based on a procedure involving their individual isolation as BaCO3. The values for the other two carbons are determined indirectly, i.e., by difference. The isotopic content of carbon 2 can be obtained in two ways. Since carbons 1 and 3 are measured individually, the C ~4 content of carbon 2 can be obtained either by the lead tetraacetate oxidation of 4,6-ethylidine-D-glucose (a reaction yielding COs derived from carbons 1 and 2) or by the combustion of 2-formyl-4-phenylosotriazole (a reaction yielding COs derived from carbons 1, 2, and 3). This method yields activity measurements for four individual carbon atoms--l, 3, 4, and 6. In addition, values are also obtained for (1) the sum of carbons 1 and 2; (2) the sum of carbons 1, 2, and 3; (3) the sum of carbons 4 and 5; (4) the sum of carbons I through 6; and (5) the sum of carbons 2 through 6. Thus by this method it is possible to determine the activity of each carbon atom of the glucose molecule without assuming that the activity of carbon 3 is equal to that of carbon 4, that of 2 equal to 5, or that of 1 equal to 6.

C. Other Chemical Methods of Glucose Degradation Aronoff and Vernon 1~7 have employed a chemical method for the degradation of glucose based on the oxidation of glucosazone with periodate. However, certain difficulties with this procedure have been noted by Vittorio et al. TM The assumption that carbon 3 is equal to carbon 4, made in order to obtain the activity of all the glucose carbons, is a limitation of the procedure as outlined by Topper and Hastings. t~8 The scheme devised by Bishop H9 utilizes some of the reactions described in the method presented in the previous section. In this method periodate oxidation of glucose phenylosotriazole yields 1 mole of formaldehyde (C-6), 2 moles 11~ D. D. Van Slyke and J. Foleh, J. Biol. Chem. 156, 509 (1940). 1~ J. Katz, S. Abraham, and N. Baker, Anal. Chem. 26, 1503 (1954). 117 S. Aronoff and L. Vernon, Arch. Biochem. 28, 424 (1950). 118 y . Topper and A. B. Hastings, J. Biol. Chem. 179, 1255 (1949). ~19 C. T. Bishop, Science 117, 715 (1953).

548

TECHNIQUES FOR ISOTOPE STUDIES

[22]

of formic acid (C-4+5), and 1 mole of 2-phenyl-4-formylosotriazole ( C - 1 + 2 + 3 ) . On nitration of the latter compound with fuming nitric acid at room temperature, 1~0 it yields 2-(p-nitrophenyl)-4-formylosotriazole, m.p. 136 to 137 °. Alkaline permanganate oxidation T M produces the acid 2-(p-nitrophenyl)-4-carboxyosotriazole, m.p. 236 to 2370,12° which is converted to the silver salt with ammoniacal silver nitrate solution. The silver salt is decarboxylated to yield COs (C-3) and 2-(p-nitrophenyl)osotriazole, m.p. 183 to 184 °12° (C-1+2). Bishop finds that initial nitration is essential to this scheme as the corresponding 2-phenylosotriazole is an oil and is difficult to purify. A new chemical procedure for the determination of the C14-distribu tion in labeled glucose has been described by Boothroyd et al. 1~ By this method analysis may be performed on as little as 2 mM. of glucose. The method is based on the degradation of methyl-a-D-glucopyranoside with periodate. 12~ Oxidation of the glucoside with periodate yields carbon 3 as formic acid, and a dialdehyde which is oxidized with bromine and isolated as the strontium salt of D'-methoxyhydroxymethyldiglycolic acid. The purified salt is then hydrolyzed to a mixture of glyoxylic and glyceric acids. The two-carbon acid (C-142) is isolated from the mixture as the 2,4-dinitrophenylhydrazone and thermally decarboxylated to yield carbon dioxide from carbon 2. From the complete combustion of the hydrazone and the direct determination of carbon 2, the C 14 activity residing in carbon 1 may be determined by difference. Carbon 1 may also be assayed directly by the procedure outlined in the previous section (page 545). The glyceric acid ( C - 4 + 5 + 6 ) is then oxidized by periodate to yield carbon dioxide (C-4), formic acid (C-5), and formaldehyde (C-6). This method can also be applied to other hexoses with minor modifications. Thus, the complete degradation of labeled galactose can be accomplished by first converting it to the methyl-D-galactopyranoside and then oxidizing the glycoside with periodic acid. The subsequent steps wit1 be the same as those given in the procedure of Boothroyd et al., 122 since the removal of carbon 3 as formic acid from the galactose derivative will result in the production of a dialdehyde which is identical with the one resulting from the corresponding methyl glucopyranoside. Methods for Pentose Degradation. Using a similar method to that applied to glucose or galactose, Brown ~4 has assayed the individual i~o H. von Pechmann, Ann. 262, 265 (1891). 121 R. L. Shriner and R. C. Fuson, " T h e Systematic Identification of Organic Compounds," p. 164. John Wiley & Sons, New York, 1945. 122 B. Boothroyd, S. A. Brown, J. A. Thorn, and A. C. Neish, Can. J. Biochem. Physiol. 33, 62 (1955). l~S E. L. Jackson and C. S. Hudson, J. Am. Chem. Soc. 59, 994 (1937). 1~4S. A. Brown, Can. J. Biochem. Physiol. $3, 368 (1955).

550

TECHNIQUES FOR ISOTOPE STUDIES

[22]

persulfate. 128 The former method has been adopted for the oxidation of C14-1abeled compounds by Barker. 1.9 Many modifications of this procedure have appeared in the literature. 13°-13~ This method is limited to the oxidation of dry and nonvolatile compounds; it involves the handling of a highly corrosive fluid, constant attention during combustion, and the use of special apparatus. Although it has been modified for the combustion of certain volatile compounds, TM the procedure and apparatus are too complex for routine laboratory analysis. The persulfate oxidation as described by Osburn and Werkman l~s can be used only for water-soluble compounds; it has been adopted by Weinhouse 133 and Anthony and Long TM for C 14 assay. This method may be employed for the oxidation of volatile as well as nonvolatile compounds with the limitation that the compounds must be water-soluble. ~°° This reaction can be expressed by the following equation: H20 -}- K2S20s--* 2KHS04 -}- 1~O2

Apparatus and Reagents 50-ml. Erlenmeyer flask with a center well (see Fig. 5). Rubber serum cap (see Fig. 5). Siphon (see Fig. 5). Potassium persulfate, reagent-grade. 4 % silver nitrate solution. Standardized sodium hydroxide (C02-free) solution, approximately 1N. Standardized hydrochloric acid solution, approximately 0.2 N. Dilute sulfuric acid. Saturated barium chloride solution.

Procedure. About 500 to 600 mg. of solid potassium persulfate is placed into the main compartment of the flask so that none of the salt is introduced into the center well. This is conveniently performed with a porcelain spoon and a small large-mouthed funnel. The sample (which should yield from 10 to 80 mg. of BaC03) and water are added to the main comll80. L. Osburn and C. H. Werkman, Ind. Eng. Chem. Anal. Ed. 4, 421 (1932). 1,9 It. A. Barker, in "Isotopic Carbon" (Calvin et al., eds.), p. 93. John Wiley & Sons, New York, 1949. is0 A. Lindebaum, J. Schubert, and W. D. Armstrong, Anal. Chem. 20, 1120 (1948). 181j. A. Them and P. Shu, Can. J. Chem. 29, 558 (1951). 13~E. A. Evans and J. L. Huston, Anal. Chem. 24, 1482 (1952). lss S. Weinhouse, in "Isotopic Carbon" (Calvin et al., eds.), p. 94. John Wiley & Sons, New York, 1949. 18, D. S. Anthony and M. V. Long, Oak Ridge Natl. Lab. Rept. 1303 (1952).

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

551

p a r t m e n t t o g i v e a v o l u m e of 5 t o 15 ml. T h e flask is t h e n s w i r l e d in o r d e r to p a r t i a l l y d i s s o l v e t h e p e r s u l f a t e . T h i s m i x t u r e c a n b e acidified w i t h a few d r o p s of d i l u t e H2S04 in o r d e r t o l i b e r a t e a n y c a r b o n a t e p r e s e n t in t h e s a m p l e . O n e m i l l i l i t e r of 4 % s i l v e r n i t r a t e s o l u t i o n is a d d e d to t h i s TABLE IV CARBON RECOVERY ¥¢ITH PERSULFATE OXIDATIONa'b Compound Glucose Fructose Inositol Glycerol Sodium acetate Lithium lactate Succinic acid Citric acid Benzoic acid Potassium acid phthalate Ethyl acetoacetate Acetaldehyde Acetaldehyde + 20 mg. NaHSO3 Methanol Ethanol Histidine hydrochloride Thymine Sodium oxalate Zinc lactate Fumaric acid Acetone Adenine hydrochloride

Carbon taken, rag.

Recovery of C()2, %

2.40 2.40 2.40 4.40 2.40 3.60 2.40 2.40 2.82 2.40 1.72 2.80 2 80 2 34 2.52 2.40 2.10

100 97 100 98 99 98 98 100 95 99 98 100 100 94 102 97 96 101 c 97 ° 99 c 98c 80

2.05

All samples were prepared by weighing out analytical reagent-grade compounds and dissolving them in water. Aliquots of these solutions were used throughout The yields were determined by titration of the alkali (after the addition of barium chloride) to the phenolphthalein and the methyl orange-methylene blue end points. Each value is an average of at least two separate determinations. The duplicate titrations agreed within 2%. b j. Katz, S. Abraham, and N. Baker, Anal. Chem. 26, 1503 (1954). c D. S. Anthony and M. V. Long, Oak Ridge Natl. Lab. Rept. 1303 (1952). mixture, and enough standard C02-free sodium hydroxide to absorb the C 0 2 f o r m e d is d e l i v e r e d i n t o t h e c e n t e r well. T h e flask is s t o p p e r e d w i t h t h e r u b b e r cap. T h e vessels a r e e v a c u a t e d b y i n s e r t i n g a 20- or 2 2 - g a g e h y p o d e r m i c n e e d l e w h i c h is c o n n e c t e d t o a v a c u u m line. T h e v a c u u m s e r v e s t o r e m o v e a n y d i s s o l v e d CO2 a n d p r o v i d e s a t i g h t seal for t h e flask d u r i n g c o m b u s t i o n . S e v e r a l s u c h flasks m a y be p r e p a r e d in t h i s w a y .

552

TECHNIQUES FOR ISOTOPE STUDIES

[22]

T h e flasks a r e p l a c e d in a w a t e r b a t h a t 40 to 50 ° a n d b r o u g h t u p t o a b o u t 70 ° in t h e p e r i o d of 15 to 20 m i n u t e s . T h e c o n t e n t s of t h e m a i n well will d a r k e n a n d gas e v o l u t i o n c o m m e n c e . T h e r e a c t i o n p r o c e e d s s m o o t h l y a t 70 t o 75 ° a n d is u s u a l l y c o m p l e t e d w i t h i n 30 m i n u t e s . A t t h e e n d of t h i s p e r i o d t h e d a r k color d i s a p p e a r s a n d t h e s o l u t i o n b e c o m e s w a t e r - c l e a r . A l o n g e r o x i d a t i o n t i m e m a y be r e q u i r e d w i t h s o m e c o m p o u n d s , a n d in s u c h cases t h e flasks s h o u l d be h e a t e d u n t i l t h e m i x t u r e is clear. TABLE V COMPARISON OF VAN SLYKE AND 1)ERSULFATE COMBUSTION OF C 14 COMPOUNDS a,b

Persulfate oxidation

van Slyke oxidation

Barium carbonate, rag.

Specific activity, c.p.m./mg,

Barium carbonate, rag.

Specific activity, c.p.m./mg.

Glucose-C ~4 Glucose-C '4

35.1 37.3

9.1 9.4

37.5 36.8

9.2 9.3

Succinic acid-P.-C I' Succinie acid-2-C '4

18.2 16.4

21.1 20.5

18.3 18.0

21.2 20.7

Blank

0.8

0.8

The glucose was photosynthetically prepared ¢ and was shown to be evenly labeled, d The succinic acid used was labeled with C 14in the 2 position.' Identical aliquots were combusted in each case. The van Slyke combustion was carried out according to Barker,S and all samples after the addition of barium chloride and titration of the excess alkali were mounted on filter paper according to Entenman et al.g and assayed for radioactivity on a conventional end-window counter. b j . Katz, S. Abraham, and N. Baker, Anal. Chem. 26, 1503 (1954). E. W. Putman and W. Z. Hassid, J. Biol. Chem. 196, 749 (1952). S. Abraham, E. W. Putman, and W. Z. Hassid, Arch. Biochem. and Biophys. 41, 61 (1952). * E. C. Jorgenson, J. A. Bassham, M. Calvin, and B. M. Tolbert, J. Am. Chem. Soc. 74, 2418 (1952). s H. A. Barker, in "Isotopic Carbon" (Calvin et al., eds.), p. 93. John Wiley & Sons, New York, 1949. o C. Entenman, S. R. Lcrner, I. L. Chaikoff, and W. G. Dauben, Proc. Soc. Exptl. Biol. Med. 70~ 364 (1949). T h e flasks a r e left for a n a d d i t i o n a l 15 t o 20 m i n u t e s in t h e w a t e r b a t h t o e n s u r e c o m p l e t e CO2 a b s o r p t i o n . T h e flasks r e q u i r e l i t t l e a t t e n t i o n ; h o w e v e r , if t h e t e m p e r a t u r e is p e r m i t t e d t o i n c r e a s e t o o f a s t or exceed 75 ° t h e e v o l u t i o n of O3 m a y b e c o m e t o o v i g o r o u s a n d f o r c e t h e c a p out. I n l i e u of a w a t e r b a t h t h e c o m b u s t i o n m a y also b e c a r r i e d o u t in a n o v e n

[22]

C A R B O H Y D R A TSYNTHESIS E AND DEGRADATION

553

initially set at 75 °. The reaction proceeds somewhat slower than in the water bath and requires about 1 ~ hours. After the flasks have been cooled to room temperature, the vacuum is released by the insertion through the rubber cap of a hypodermic needle connected to an ascarite tower. The flasks are opened and the contents of the center well are rapidly and quantitatively transferred, by means of the siphon, into a suitable vessel for titration of the alkaline carbonate solution. Addition of about 1 ml. of saturated barium chloride solution and titration of the excess base with standardized HCI solution affords a measure of completeness of oxidation. An analyst can easily combust and titrate the BaCO3 from over 20 samples in a few hours. The above procedure has been used extensively for the combustion of C14-1actate and C~4-acetate and to some extent for radioactive assay of C~4-ethyl acetoacetate and C14-glycerol with highly satisfactory results. ~00 This method is applicable to a large variety of water-soluble compounds including carbohydrates, organic acids, amino acids, alcohols, and some heterocyclic compounds, as demonstrated in Table IV. It gives satisfactory results with volatile compounds such as acetaldehyde, methanol, and the like. As can be seen from Table IV, the recovery of carbohydrate ranges from 94 to 100% in all compounds tested with the exception of adenine which gives a value of about 80%. The results obtained with C14-glucose and C~4-succinate in a comparative study using the Van Slyke and Folch combustion fluid ~1~and persulfate can be seen in Table V. The radioactive assay of these compounds by these two methods was found to be identical.~°°

III. Isolation of Labeled Carbohydrates from Biological Sources

A. Glucose-C TM 1. As the p-Nitrophenylhydrazone

Principle. This method of glucose isolation from urine is based on the preparation of its p-nitrophenylhydrazone, the removal of the p-nitrophenylhydrazine with benzaldehyde, and the subsequent crystallization of the glucose. ~35 Reagents 10% HgSO4 in 5% sulfuric acid. Barium carbonate. 95% ethanol. IV[ethanol. 135 S. Gurin, A. M. Delluva, and D. W. Wilson, J. Biol. Chem. 171, 101 (1947).

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

555

Saturated zinc sulfate solution. Duolite cation exchange resin C-3 anion exchange resin A-4. Phenylhydrazine. Glacial acetic acid. Ether. Ethanol. Procedure. Approximately 4 to 8 ml. of blood is withdrawn from the left ventricle of a rat into a heparinized syringe. Plasma, obtained by centrifugation, is deproteinized with Ba(OH)2 and ZnS04 by the method of Somogyi ~4~ and passed successfully through a Duolite C-3 cation and A-4 anion exchange column. The columns are then washed with 5 to 6 vol. of distilled water to remove all adhering glucose. The combined neutral effluent and washings are concentrated to about 4 to 5 ml., under reduced pressure, and transferred to a 15-ml. centrifuge tube. To this glucose solution, 100 rag. of phenylhydrazine and 186 rag. of glacial acetic acid are added and the mixture heated on the steam bath for 1 hour. Phenylglucosazone crystals usually appear within 15 minutes. The mixture is then cooled, first slowly to room temperature and then to about 4 ° . After centrifugation, the supernatant liquid is removed by decantation, and the crystals washed first with water (to remove electrolytes) and finally twice with ether (to remove aniline and excess phenylhydrazine). The glucosazone is dissolved in 2 to 3 ml. of hot ethanol, filtered, and recrystallized by the addition of water. The golden yellow needles thus obtained are dried in a vacuum oven set at 45 ° for several hours.

3. As the Pentaacetate This procedure as outlined on page 501 consists of forming glucose pentaacetate from the dried urine residue by treatment with pyridine and acetic anhydride. The pentaacetate is recovered in the crystalline form and hydrolyzed by refluxing with 0.1 N H2SO4. The contaminating ionic material is removed with ion exchange resins and the resulting glucose crystallized. 12,28,~43 C~4-Glucose may also be isolated from tissue slice experiments in this way through the use of unlabeled carrier glucose. 139

4. Chromatographically The method used by Katz and Chaikoff 144 for the isolation of glucose as well as a great number of other compounds from tissue slice experiments with C~4-1abeled substrates is a combination of paper chroma14~ M. Somogyi, J. Biol. Chem. 160, 69 (1945). 1~3 M. R. S t e t t e n a n d Y. Topper, J. Biol. Chem. 203, 653 (1953). 144 j . Katz a n d I. L. Chaikoff, J. Biol. Chem. 206, 887 (1953).

556

TECHNIQUES FOR ISOTOPE STUDIES

[22]

t o g r a p h y in conjunction with r a d i o a u t o g r a p h y . T h e bulk of the inorganic salts in tissue extracts is easily r e m o v e d b y an electrolytic desalting procedure without loss of the labeled compounds. 145 This desalted solution is then subjected to two-dimensional c h r o m a t o g r a p h y first in waters a t u r a t e d phenol and then in butanol-acetic acid-water2 48 T h e chromat o g r a m s thus obtained are used to prepare r a d i o a u t o g r a p h s on X - r a y film. With the use of a S t a n d a r d X - r a y illuminator the radioactive areas seen on the negative m a y be located on the p a p e r c h r o m a t o g r a m and thus assayed for C ~4 a c t i v i t y as well as eluted and degraded. 147 B. Ribose-C 14

See Vol. I V [23]. C. Isolation of Conjugated Glucuronides f r o m Urine Principle. Glucuronic acid can be isolated f r o m the urine of a v a r i e t y of experimental animals, chiefly in the f o r m of conjugation products with phenolic substances and steroids. 148-167 T h u s the m e t h o d of isolation a n d purification of the glucuronide depends on the aglucone m o i e t y administered. I n the studies of the precursors of glucuronic acid, P a c k h a m and Butler 16~ h a v e administered Ci4-1abeled substrates to a - n a p h t h o l - t r e a t e d rats and h a v e isolated the uronic acid conjugate. Subsequent hydrolysis and t r e a t m e n t with b a r i u m hydroxide yielded b a r i u m glucuronate. Reagents

a-Naphthol. p-Toluidine hydrochloride. 6 N hydrochloric acid. 14~R. Consden, A. H. Gordon, and A. J. P. Martin, Biochem. J. 41, 590 (1947). 14sA. A. Benson, J. A. Bassham, M. Calvin, T. C. Goodale, V. A. Haas, and W. Stepka, J. Am. Chem. Soc. 72, 1710 (1950). 147j. Katz, S. Abraham, R. Hill, and I. L. Chaikoff, J. Biol. Chem. 214, 853 (1955). 148E. H. Mosbaeh and C. G. King, J. Biol. Chem. 185, 491 (1950). 149W. Deiehmann and G. W. Thomas, J. Ind. Hyg. Toxicol. 25, 286 (1943). 150R. T. Williams, "Detoxieation Mechanisms." John Wiley & Sons, New York, 1947. 151H. Meyer and C. Neuberg, Z. physiol. Chem. 29, 256 (1900). 15~M. A. Packham and G. C. Butler, J. Biol. Chem. 194, 349 (1952). 153F. Eisenberg, Jr., and S. Gurin, J. Am. Chem. Soc. 73, 4440 (1951). 1~4E. Fromm and P. Clemens, Z. physiol. Chem. 84, 384 (1901). 15~M. Berenbom, Thesis, University of Toronto, 1947. x56N. E. Artz and E. M. Osman, "Biochemistry of Glucuronic Acid." Academic Press, New York, 1950. 1~ F. Eisenberg, Jr., and S. Gurin, J. Biol. Chem. 195, 317 (1952).

[22]

CARBOHYDRATE SYNTHESIS AND DEGRADATION

557

1 N sulfuric acid. Ethyl ether. Ethyl alcohol. Phenolphthalein indicator. Saturated barium hydroxide. Procedure. A suspension of 150 mg. of a-naphthol in water is administered by stomach tube to each of 4 well-fed 200-g. rats. One hour later the rats are given the C~4-1abeled compound, either by intraperitoneal injection or by stomach tube, and immediately placed in a cage fitted with a metal funnel to allow collection of the urine..The rats are supplied with water but are given no food during the following 8 hours during which time the pooled urine sample is collected. The urine is adjusted to pH 8 to 9 and stored overnight at 5 °. It is then filtered and the washed precipitate discarded. The combined urine and water washings are concentrated to about 5 ml. at 50 to 60° in vacuo, neutralized, and mixed with 600 mg. of p-toluidine hydrochloride. After chilling, the crude p-toluidine naphthol glucuronide is collected by filtration, washed with cold water, and dissolved in a minimum of boiling water. The resulting solution is treated with decolorizing carbon, filtered while hot, and then chilled. The white crystals which form are collected by filtration, washed, and dried. The crystals are then converted to naphthol glucuronide by the addition of 6 N hydrochloric acid (1 ml. per 100 rag. of p-toluidine naphthol glucuronide). The naphthol glucuronide is collected by filtration, washed with cold water, dried, and recrystallized, if necessary, from hot water (m.p. 197 to 198°). The glucuronide is hydrolyzed at 100 ° with 1.0 N sulfuric acid (2 ml. per 100 mg.) for 6 hours. After extraction of the a-naphthol with ether, the hydrolysis mixture is made alkaline to phenolphthalein with a saturated barium hydroxide solution. Carbon dioxide gas is then bubbled through the mixture for a few minutes, heated to 100 ° for 10 minutes, filtered, and the precipitate washed with boiling water. The combined filtrate and washings are concentrated to 2 ml. in vacuo and mixed with 100 ml. of ethanol. The barium glucuronate is collected by filtration and washed with alcohol. This barium glucuronate may be converted to the sodium salt by dissolving the precipitate in water and passing the resulting solution through a cation exchange column in the sodium form. Degradation of C ~4 Glucuronic Acid. In order to determine the location and specific activity of C 14 in biosynthetic glucuronic acid derived from administered radioactive precursors, a degradation scheme has been developed by Eisenberg and Gurin 1~3,157which yields a series of fragments sufficient to permit the measurement or calculation of C 1~ in all the

558

TECHNIQUES

FOR ISOTOPE

STUDIES

[22]

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560

TECHNIQUES FOR ISOTOPE STUDIES

[22]

carbon atoms of glucuronic acid. These workers have isolated the hexuronic acid as its menthol conjugate. Oxidative hydrolysis of methyl glucuronide with Br2 and HBr yields saccharic acid, which when treated with HI04 breaks into 2 moles of formic acid originating from C-3 and C-4. Glyoxylic acid is further degraded by known procedures to C02 and formic acid representing C-1~-6 and C-2-~5 respectively. Methyl methyl glucuronidate on oxidation with HI04 yields a substituted diglycolic aldehyde which on hydrolysis with semicarbizide hydrochloride yields the semicarbazones of glyoxal, representing C-1-~2 and mesoxaldehyde methyl ester, representing C-4-[-5~6. Decarboxylation of the substituted glucuronide yields C-6. T M D. Glycogen-C 14

Methods for the isolation of glycogen-C TM have been presented elsewhere (Vol. III [7]). One of the methods of isolation consists in digestion of the tissue in hot 30% potassium hydroxide solution and precipitation of the crude glycogen with ethanol in the presence of sodium sulfate. The polysaccharide is dissolved in water and dialyzed against distilled water to remove any contaminating amino acids and any other dialyzable substances. The dialyzed starch solution is then filtered into a volumetric flask and diluted to volume with distilled water. Aliquots of this solution may be plated directly on aluminum disks, the water evaporated, and the sample assayed for C14-activity, or they may be oxidized with potassium persulfate ~°° (see page 549) to barium carbonate and counted in any conventional manner.

IV. Miscellaneous C~4-Labeled Carbohydrates Table VI (pp. 558-559) summarizes procedures for the preparation of a number of C14-1abeled carbohydrates.

[23]

ISOTOPIC CARBON PATTERNS IN CARBOHYDRATE

561

[23] Determination of Isotopic Carbon Patterns in Carbohydrate by Bacterial Fermentation B y I. A. BERNSTEIN 1 and HARLAND G. WOODla

The bacterial fermentation, when applicable, would appear to be the m e t h o d of choice for determining the pattern of tracer carbon in carbohydrates. Available chemical procedures, generally, are more involved, require greater time, and necessitate a larger sample. For example, the level of C ~4 in each carbon of glucose can be determined on as little as 0.5 millimole with Leuconostoc mesenteroides. Before a particular fermentation is used for degrading carbohydrate, the source of each carbon in the products should be ascertained. In addition, one must know the amounts and directions of "cross contamination," if any, which result from the organism's own metabolism. The following fermentations meet these criteria and make possible the determination of the level of isotope in each carbon of D-glucose and D-ribose. I t seems likely that the isotopic carbon patterns of D-galactose, D-xylose, and L-arbinose m a y also be determined in this manner. Other monosaccharides as well as other fermentations could possibly be included, but sufficient critical data do not seem to be available to ensure their validity for the intended purpose.

Fermentation of Glucose by LactobaciUus casei L. casei CH~OHCHOHCHOHCHOHCHOHCHO ----* 2CHaCHOHCOOH (C-l, 6)(C-2, 5)(C-3, 4)

This fermentation was first used to degrade hexose by Wood et al. TM when t h e y determined the distribution of C 13 in glycogen isolated from the livers of rats which had received NaHC13Oa. Lactobacillus casei, A T C C 7769, has since been extensively employed to determine the patterns of C 1~ and C TM in glucose from various sources. The carboxyl carbon of the lactic acid arises from carbons 3 and 4 of the glucose, the carbinol carbon from 2 and 5, and the methyl carbon from 1 and 6. 1Department of Dermatology, Medical School, University of Michigan, Ann Arbor, Michigan. 1, Department of Biochemistry, School of Medicine, Western Reserve University, Cleveland, Ohio. lb H. G. Wood, N. Lifson, and V. Lorber, J. Biol. Chem. 159, 475 (1945).

562

TECHNIQUES FOR ISOTOPE STUDIES

[23]

M a i n t e n a n c e of Cultures

Medium 1% glucose. 0.5% Difco yeast extract. 0.5 % Bacto-tryptone. 0.6% sodium acetate.3H~O. Distilled H20.

Procedure. Ten milliliters of medium is inoculated with several drops of the stock culture and incubated at 37 ° for 24 hours. Cultures are stored at about 5 ° and are transferred every two to three months. P r e p a r a t i o n of B a c t e r i a for F e r m e n t a t i o n of Glucose

Medium. The medium is the same as described above. Procedure. F i f t y milliliters of medium is inoculated with 1 ml. of stock culture and incubated at 37 ° for 24 hours. This entire culture is then added to 1.5 1. of medium in a 2-1. flask and incubated for 48 to 60 hours. The cells are harvested b y centrifugation and washed three times by suspending each time in 20 vol. of distilled H20. The wet weight of cells is determined, and the bacteria are dispersed 2 in 4 vol. (4 ml./g.) of H20 to make a 20% suspension. P r e p a r a t i o n of G l u c o s e for F e r m e n t a t i o n

The sample of glucose to be fermented is dissolved in distilled H20. If a glycogen hydrolyzate is to be used, the H2SO4 should be neutralized to p H 7 with N a O H and the molar concentration of S O 4 - - adjusted so as to be less t h a n 0.06 in the fermentation mixture.lb A nonalcoholic solution of b r o m o t h y m o l blue m a y be used as an internal indicator for the p H adjustment. Fermentation Procedure

Reagents 0.4 M NaHCOs. Glucose sample in H20. Distilled H20.

Procedure. The fermentation is carried out in a 125-m]. W a r b u r g In case it is necessary to prepare the cells the evening prior to use, the cells are not resuspended at this point but are stored as a paste at about 5 °. Before use, they are washed, centrifuged, weighed, and dispersed in H20 as a 20% suspension.

[9.3]

ISOTOPIC CARBON PATTERNS IN CARBOHYDRATE

563

respirometer vessel with two side arms. 8 Four milliliters of the 20% cell suspension is divided between the two side arms. Six milliliters of 0.4 M NaHCOa and 1 millimole or less of glucose in 20 ml. of distilled H20 are put in the main compartment. The flask is " g a s s e d " with 100% C02 for 10 to 15 minutes 4 with frequent shaking to facilitate saturation with C02, is closed off from the atmosphere, and is immersed in the water bath at 37 ° . After equilibration, the fermentation is started b y transferring the bacteria to the main c o m p a r t m e n t and is followed manometrically b y the evolution of COs from the buffer resulting from the formation of lactic acid. 6 When the fermentation is finished (theory: 2 moles of COs per mole of glucose fermented), 2 ml. of 2 N H2S04 is added to acidify the medium. Manometric measurements generally indicate 1.8 to 1.9 millimoles of acid formed per millimole of glucose originally present.

Isolation and Purification of Lactic Acid After separation of the cells b y centrifugation, the lactic acid is removed from the supernatant solution (which should be acid to Congo red) b y continuous extraction with ether for 24 hours. About 25 ml. of H20 is initially p u t into the receiver, as this appears to decrease the destruction of the extracted lactic acid by impurities in the ether. The ether in the receiver is removed b y evaporation, and the aqueous solution is concentrated b y distillation to 20 ml. after which 200 ml. is distilled with steam at constant volume to remove any steam-volatile contaminants resulting from the fermentation or introduced with the ether. About 5 % of the lactic acid m a y be lost in the distillate during this procedure. The yield of lactic acid (usually 85% of the a m o u n t of glucose submitted to the bacteria) is determined b y titration with N a O H and with phenolphthalein as indicator. The lactic acid m a y be further purified by a number of procedures including preparation of the guanidine salt 8 and partition chromat o g r a p h y on Celite No. 535. 7 It has been found, however, that additional These vessels may be obtained from E. Machlett and Son, 220 East 23rd Street, New York 10, New York. The side arms have joints to facilitate introduction of materials and venting plugs for better "gassing." 4 If bromothymol blue is present internally, "gassing" may be terminated when the color of the solution changes from greenish blue to yellowish green. For a detailed description of the Warburg respirometer technique see W. W. Umbreit, R. H. Burris, and J. F. Stauffer, "Manometric Techniques," Burgess Press, Minneapolis, 1949. 6 M. A. Packham and G. C. Butler, J. Biol. Chem. 194, 349 (1952). 7 H. E. Swim and L. O. Krampitz, J. Bacteriol. 67, 419 (1954); H. J. Saz and L. O. Krampitz, ibid. 67, 409 (1954).

564

[23]

TECHNIQUES FOR ISOTOPE STUDIES

purification of the lactic acid is unnecessary when purified glucose is fermented. Purification through Guanidinc Lactate. To prepare the guanidine salt of lactic acid, 2 molar equivalents of guanidine as the sulfate is added to the aqueous solution of sodium lactate and the H20 is removed by lyophilization. The guanidine lactate is extracted from the dried residue with hot absolute alcohol and crystallized. The salt is recrystallized from alcohol until the melting point (162 °) coincides with and is not depressed by mixture with an authentic sample. The free acid is then quantitatively recovered in the effluent when an aqueous solution of the salt is passed through a small column of Dowex 50 (hydrogen form). Purification by Partition Chromatography. Lactic acid may be purified by partition chromatography according to the procedure described in detail on page 563 for the purification of acetic acid. For elution of the lactic acid, however, 150 ml. each of 100% CHC13, 97.5% CHC13-2.5% n-butanol, 95% CHC13-5% n-butanol, and 92.5% CHC13-7.5% nbutanol (each equilibrated with 0.18 N H~SO4) in that order is used. Under the conditions described, the lactic acid first appears after about 75 ml. of the ]ast solvent has been put through the column.

Chemical Degradation of Lactic Acid (see also Vol. IV [22])

Principle. The isotopic level in each carbon of the lactic acid may be determined by a modification of the method of Wood et al. TM involving the following reactions: CHsCHOHCOOH MnO.~ CH3CHO ~- CO2 (C-l)

CH3CHO NaOI) CHI3 ~- HCOONa CHIa

Ag+

"iodic

HNOa

sulfate"

-~ CO - -

* CO2

HCOOH

Hg ++

--' CO2

Reagents Lactic acid sample in H20. MnSO4-HaPO4 reagent. Dissolve 100 g. of MnSO4.4H:O and 25 ml. of 85% H3PO4 in distilled H20 and make to 1 1. Aqueous NaHSO3, prepared fresh immediately prior to use. A 1% solution is used when 0.5 millimole of lactic acid is being degraded, 2% for 0.5 to 1.5 millimoles, and 2.5% for 1.5 to 2.5 millimoles. Acidified 0.25 saturated KMnO4. Add 30 ml. of distilled H20 and 1 ml. of 2 N H2SO4 to 10 ml. of saturated KMnO4. Approximately 2.5 N NaOH (carbonate-free).

[23]

ISOTOPIC CARBON PATTERNS IN CARBOHYDRATE

565

0.02 M KS/[nO4 in carbonate-free H20. K2HPO4, solid. 1 N NaOH. Solution chilled to about 2 °. 0.1 N Is. Solution chilled to about 2 °. 2 N HsSO~. 2 M NaAsOs. 1 N acetic acid. 0.3 M mercuric acetate in dilute acetic acid. Dissolve 100 g. of reagent mercuric acetate and 30 ml. of glacial acetic acid in distilled H20 and make to 1 1. Boil gently for 30 minutes to remove any dissolved CO2. 0.2 M AgN03 containing 4 ml. of concentrated HNO3 per 100 ml. Absolute alcohol. Ascarite 8 or other CO2 absorbent. Anhydrous magnesium perchlorate. ZnCls, granular. Dry at 140° for several days prior to use. "Iodic sulfate." This is a mixture of IsO~ and concentrated HsSO~ suspended on silicic acid ~ and prepared as follows: 180 g. of IsOs, 45 g. of silicic acid, and 50 ml. of concentrated HsSO4 are mixed in a mortar and heated for 2.5 hours at 220 ° in a round-bottomed flask while being aerated with dry air brought into the flask with a vacuum pump. The material is adequate for use when it is evenly yellow throughout and must be protected from moisture. For a newer method of preparation see the abstract of Smiley. ~o

Decarboxylation of Lactic Acid. Lactic acid is converted to COs (carboxyl carbon) and acetaldehyde by a modification of the procedure of Friedemann and Kendall 11 in the apparatus 12 shown in Fig. 1. The sample in 75 to 100 ml. of H20, a boiling chip, and 10 ml. (per millimole of lactic acid) of the MnSO4-HsP04 reagent are put in the 200-ml. reaction flask (A) which is attached to the "degradation h e a d " (B). 1~ Bead tower F is charged with 30 ml. of NaHSO:~ (for concentration, 8 A COs absorbent m a r k e t e d b y A r t h u r H. T h o m a s Co., Philadelphia, Pennsylvania. 9 M. Sehultz, Bet. 77B, 484 (1944); Chem. Abstr. 40, 4858 (1947). 10 W. G. Smiley, Nuclear Sci. Abslr. 8, 391 (1949). IL T. E. F r i e d e m a n n a n d A. I. Kendall, J. Biol. Chem. 82, 23 (1929). ~2 The glassware shown m a y be purchased from the Corning Glass Works (Attention: Special A p p a r a t u s Section), Corning, New York. The numbers in Fig. 1 a n d 2 refer to the catalogue n u m b e r s of the pieces. is Instead of the degradation head (B) and r o u n d - b o t t o m e d flask (A), a round-bottomed flask with three necks (M) equipped with a condenser (bulb type) (O), a dropping funnel with stopcock (P), and an inlet tube (N) for aeration m a y be substituted.

566

TECHNIQUES

••c,

FOR

ISOTOPE

[23]

STUDIES

~iiii:!i:~i!i::i~i!:i.~:i.!.!:.!i~!iil~,¢~._.~ (E-4)

.3)

J B (A.I)" F (,4.51

a

b

c

d

e

I

n

FIG. 1. Degradation train and accessories. Apparatus for decarboxylation of lactic acid: (A) reaction flask, round-bottomed, single-necked, 300 ml.; (B) "degradation head" with condenser (Allihn), dropping funnel, and inlet tube attached; (C) stopcock, double oblique bore; (C r) side view of stopcock (C); (D) drying tube with (a) anhydrous magnesium perchlorate (or CaCl2) and (c) CO2 absorbent (glass wool used at each end of tube and between a and c) ; (F) bead tower, with stopcock, containing glass beads (4 or 5 mm. in diameter); (G) same as (F) but without stopcock; (H) drying tube with anhydrous magnesium perchlorate (glass wool used at each end) and CO2 collector consisting of (I) "bubbler" containing 2.5 N NaOH (carbonate-free) and (J) drying tube with powdered anhydrous magnesium perchlorate (glass wool at each end). For a determination of yield, I and J with open ends covered by rubber "policemen" are weighed as a unit (wire hook shown on J used to mount collector in balance). CO2 collector (K) with coils partially immersed in liquid N2 in Dewar flask (L) may be substituted for I and J. Reaction flask (A) and "degradation head" (B) may be replaced by (M) flask, round-bottomed, three-necked, 300 ml.; (N) adapter for aeration; (0) condenser, Allihn; and (P) dropping funnel with stopcock. Apparatus for formic acid oxidation, same as above except that F and G are omitted. Apparatus for iodoform oxidation, as above except that F and G are replaced by tube (E) containing in order (a) CaC12, (b) ZnC12, (c) CO~ absorbent, (d) anhydrous magnesium perchlorate, (e) "iodic sulfate," and (f) anhydrous magnesium perchlorate (glass wool used at each end and between substances). All ground joints are standard taper 24/40. Designations in parentheses indicate catalog numbers of parts available commercially. 1~ The "degradation head," catalog number A-l, has been modified by replacing the condenser, West, with a condenser, Allihn.

[23]

ISOTOPIC CARBON PATTERNS IN CARBOHYDRATE

567

see a b o v e s e c t i o n on r e a g e n t s ) , a n d b e a d t o w e r G w i t h 30 ml. of acidified 0.25 s a t u r a t e d K M n O 4 . D r y i n g t u b e D is filled w i t h A s c a r i t e or o t h e r CO2 absorbent, and tube H with anhydrous magnesium perchlorate. W i t h t h e CO2 c o l l e c t o r ( I or K ) r e m o v e d f r o m t h e t r a i n a n d t h e flask c o n t e n t s r e f l u x i n g g e n t l y , t h e s y s t e m is a e r a t e d ~4 (at a r a t e of a b o u t 3 b u b b l e s p e r s e c o n d ) w i t h c a r b o n a t e - f r e e a i r or 1 0 0 % N2 for 10 t o 15 m i n u t e s t o r e m o v e a n y CO2 p r e s e n t . T h e CO2 collectoV ~ is t h e n i n t r o d u c e d i n t o t h e t r a i n a n d t h e r e a c t i o n is c a r r i e d o u t b y a d d i n g t h e 0.02 M K M n O 4 d r o p w i s e f r o m t h e f u n n e l t o t h e b o i l i n g s o l u t i o n in t h e r e a c t i o n flask u n t i l t h e p u r p l i s h b r o w n color p e r s i s t s for s e v e r a l m i n u t e s . T h i s is f o l l o w e d b y 15 a d d i t i o n a l m i n u t e s of refluxing a n d a e r a t i o n . T h e a c e t a l d e h y d e is constantly ~e b e i n g a e r a t e d i n t o t h e N a H S O u s o l u t i o n w h e r e it is t r a p p e d as t h e b i s u l f i t e a d d i t i o n c o m p o u n d while t h e C 0 2 p a s s e s on t h r o u g h t h e K M n O 4 s c r u b b e r t o t h e CO2 collector. T h e K M n O 4 s c r u b b e r is i n c l u d e d t o p r e v e n t t h e t r a n s f e r of a n y SO2 f r o m t h e N a H S O a s o l u t i o n i n t o t h e CO2 collector. T h e r e c o v e r y of CO2 in t h i s r e a c t i o n is u s u a l l y b e t t e r t h a n 9 5 % , a n d m o r e t h a n 9 0 % of t h e a c e t a l d e h y d e is g e n e r a l l y r e c o v e r e d in t h e N a H S O 3 as d e t e r m i n e d b y t i t r a t i o n w i t h I2. TM Hypoiodite Oxidation of Acetaldehyde. T h e a c e t a l d e h y d e is r e c o v e r e d f r o m t h e N a H S Q s o l u t i o n b y d i s t i l l i n g t h r e e - f o u r t h s of t h e v o l u m e a f t e r t h e a d d i t i o n of 6 g. of solid K2HPO~. A b o u t 10 ml. of H 2 0 is i n i t i a l l y p u t i n t o t h e d i s t i l l a t i o n r e c e i v e r (a 300-ml. d i s t i l l a t i o n flask i m m e r s e d in a n ~4 Carbonate-free air can be passed through the system by applying a positive pressure of air at the start of the train (at tube D) or suction at the distal end of the CO~ collector. The authors prefer the former, since in this case a leak would result in some loss of yield but would not give a lowered specific activity determination as would be the case if a vacuum system were in use and nonisotopic COo. leaked in from the atmosphere. If a vacuum system is being used a 10 to 15-minute aeration should be carried out with the CO.~ collector in the system to check for a blank CO~ yield. If a pressure system is to be used, a simple mercury pressure stabilizer should be connected into the system, before the air enters the train, to "buffer" line pressure changes. If the COs collector is a "coil" immersed in liquid N2 and the CO~. yield is to be determined manometrically in a vacuum system, t00 % N,. rather than air should be used for aeration. Use of air results in a "blank" yield of unknown condensable gas. is The COo. collector may be a "bubbler" containing 2.5 M NaOH (carbonate-free) such as is shown in Fig. 1, or some other type of glass trap containing NaOH, or it may be a "coil" trap (see Fig. 1) partially (one-third to one-half) immersed in liquid N2 or air. Recovery of CO~, as such, with a "coil" is advantageous when the isotopic content is to be determined in the gaseous state, e.g., in the mass spectrometer or in a gas phase radioactivity counter. The COs yield can be determined as an increase in weight of the trap (in the case where NaOH is used) or manometrically. is If the degradation head (B) shown in Fig. 1 is used, the 0.02 M KMnO, should be introduced into flask A by quickly rotating stopcock C 360 °, thus adding a small amount of the reagent and interrupting the aeration for a minimum length of time.

568

TECHNIQUES FOR ISOTOPE STUDIES

[23]

ice bath), and the apparatus is so arranged that the distillate is delivered directly into this liquid to minimize loss of acetaldehyde. The iodoform reaction is carried out on the acetaldehyde (at a concentration no greater than 1 millimole per 100 ml.) in the receiver (still in the ice bath) by adding (per millimole of lactic acid degraded) 25 ml. of cold 1 N NaOH at once and, while the solution is being mechanically stirred, 70 ml. of cold 0.1 N I2 dropwise. The mixture is kept at about 2 ° for 2 to 14 hours after which 2 N H2S04 is added dropwise with stirring until the color of I2 appears. The I~ is destroyed with 2 M NaAsO~. The solution is then further acidified, if necessary, to Congo red, to make certain that all the I~ has been reacted. At this point, if the CHI3 is allowed to settle, the solution should be colorless or slightly yellow. The CHI3 may be separated from the supernatant solution (containing the HCOOH) by filtration through an asbestos pad on a Gooch crucible (and washed with H~O) or by removing the supernatant solution through a filter stick having an asbestos plug (and washing the remaining precipitate three times with 10 ml. of H20). The latter method generally gives a better yield when a small amount of CHI3 is involved, since the asbestos plug can be returned to the reaction flask and the subsequent oxidation of the CHI3 to CO~ can be carried out in the same flask without transfer. Any CHI~ on the filter stick is washed back into the flask with 10 ml. of absolute alcohol. Oxidation of Formic Acid (cf. volume III, p. 285). The supernatant solution of the CHI~ is neutralized with NaOH to phenolphthalein and evaporated to about 15 ml. The concentrated solution (with any salt which may have precipitated) is transferred to a steam distillation flask, acidified with H2SO4 to Congo red, and 10 to 15 vol. steam distilled. If the distillate shows traces of I~, several drops of 2 M NaAsO2 are added. The HCOOH (a-carbon) in the distillate is then oxidized to CO~ in a modification of the apparatus shown in Fig. 1 by the specific method 1~of Weike and Jacobs. TM The distillate, a boiling chip, and 5 ml. of 1 N acetic acid are placed in a 300-ml. reaction flask (A) which is attached to the "degradation head" (B). 13 The apparatus is the same as shown in Fig. 1 except that bead towers F and G are omitted and the top of the condenser on B is attached directly to drying tube H. With the collector absent from the train, the solution is refluxed and aerated with CO2-free air or N~ for 10 minutes to remove any CO~ present. The COs collector is then added to the system, and 40 ml. of the mercuric acetate reagent is added. Boiling and aeration are continued for 20 minutes, and the CO~ yield is 17 O. L. Osburn, H. G. Wood, a n d C. H. W e r k m a n , Ind. Eng. Chem. Anal. Ed. 5, 247 (1933). is H. D. Weihe a n d P. B. Jacobs, Ind. Eng. Chem. Anal. Ed. 8, 44 (1936).

[23]

ISOTOPIC CARBON PATTERNS IN CARBOHYDRATE

569

determined. To determine whether the recovery has been complete, a further 10-minute collection period is made. T h e over-all yield of COs f r o m the a - c a r b o n of the lactic acid is a b o u t 50 %. Oxidation of Iodoform. T h e C H I 3 (E-carbon) is selectively oxidized to CO~ in the a p p a r a t u s shown in Fig. 1 as described b y Shreeve et al. 19 T h e reaction flask containing the sample in 50 ml. of 2 0 % alcohol is incorporated into the train shown in the figure. 13 T h e center t u b e of the " d e g r a d a t i o n h e a d " should extend u n d e r the solution in the flask. Bead towers F and G and t u b e H are replaced with t u b e E 2° containing, in order, CaC12 (a), ZnC12 (b), Asearite (c), a n h y d r o u s m a g n e s i u m perchlorate (d), "iodic s u l f a t e " (e), and m a g n e s i u m perchlorate (f). I n the reaction flask the CHI3 is converted to CO ~1 which is converted to COs on passage through the "iodic sulfate. ''22 With the CO~. collector out of the system, the train is aerated for 5 minutes to clear the COs from the a p p a r a t u s beyond the Ascarite (c). The collector is then put in and the mixture is refluxed gently. If the solution is boiled or aerated too vigorously some of the C H I 3 will pass b e y o n d the condenser and be lost. With the boiling and aerating continuing, 45 ml. of the acidified 0.2 M AgN03 (per millimole of lactic acid initially present) is added and the COs is collected over a 1-hour period or until there is no further recovery. The yield of CO2 for the E-carbon of lactic acid is a b o u t 50%. The specificity of this reaction procedure appears to eliminate the need for purification of the CHI3. ~9,2a Reliability of This Procedure. T h e validity of this procedure for the degradation of lactic acid is shown b y the d a t a in Table I. I n all reactions in which COs is collected, care m u s t be t a k e n to remove all extraneous COs present in the reaction mixture or in the train prior to starting the intended reaction. I t seems advisable to determine b l a n k COs values b o t h before and after the reaction to eliminate the possibility of u n k n o w n dilution of the COs sample. T h e a p p a r a t u s and reagents should be tested periodically b y determining the COs from a b l a n k run. 19W. W. Shreeve, F. W. Leaver, and I. Siegel, J. Am. Chem. Soc. 74, 2404 (1952). 2oGlass wool (not cotton) is used to separate the substances. A tube of 12-ram. inner diameter may be used to contain all the substances in the train--each occupying ~bout 10 cm. of the length. The CaC12 and ZnC12 are used to prevent any alcohol from passing on in the tube. If the alcohol is not adequately trapped, blank CO2 values will be obtained. 21 Any CO2 which may arise in the reaction flask is trapped by the Ascarite (c) prior to the conversion of the CO to CO2. ~ The part of the "iodic sulfate" which is used up changes from yellow to brown so that a tube containing the material may be used for many oxidations and need be replaced only when most of the reagent is brown. ~a I. A. Bernstein, K. Lentz, M. Maim, P. Schambye, and H. G. Wood, J. Biol. Chem. 215, 137 (1955).

570

[23]

TECHI~IQUES FOR ISOTOPE STUDIES

Other Methods for Degrading Lactic Acid (see Vol. IV [22]). Lactic acid m a y also be degraded by the procedure of Roseman. 2~ B y this method the acid is condensed with o-phenylenediamine to form the 2-(a-hydroxyethyl)benzimidazole which is purified by recrystallization. The derivative is treated with N a O I to give CHI~ representing the B-carbon of lactic acid. Another aliquot of the derivative is oxidized with K1V[nO4 to give 2-benzimidazolecarboxylic acid. This acid is decarboxylated at 190 ° to give CO~ (a-carbon of lactic acid) and benzimidazole (carboxyl carbon). T h e CHIs and benzimidazole, after suitable purification, are burned to C02 for isotopic analysis.

TABLE I ISOTOPIC DATA ON THE DEGRADATION OF SYNTHETIC LACTIC ACID-C 14 BY THE I~IETHOD OF WOOD e~ a l . a

Atoms, % excess or c.p.m./mfllimole of carbon Type of labeling

Isotope

CH~

CHOH

COOH

C14H3CISHOHCOOHb

C I~ C 14

0.00 28,600

1.34 25

0.00 0

CHsCHOHC14OOH c

Cl4

525

50

105,000

" H. G. Wood, N. Lifson, and V. Lorber, J. Biol. Chem. 159, 475 (1945). b V. Lorber, N. Lifson, H. G. Wood, W. Sakami, and W. W. Shreeve. J. Biol. Chem. 183, 517 (1950). c I. A. Bernstein, unpublished data. Using synthetic lactic acid-l-C ~4 and lactic acid-2-C 14 to check the validity of this method, Roseman found essentially po "cross contaminat i o n " of C 14 during the degradations. The yields reported for this m e t h o d are lower t h a n those generally obtainable b y the m e t h o d of Wood et al. Aronoff et al. 25 reported the oxidation of lactic acid b y CrOs to CO2 (carboxyl carbon) and acetic acid. The barium salt of this acid was then pyrolyzed to BaCOs (a-carbon) and acetone. T r e a t m e n t of the acetone with N a O I yielded CHI3 (B-carbon). Reliability of the Lactobacillus casei M e t h o d The data shown in Table I I allow an evaluation of the validity of this technique for determining the p a t t e r n of isotopic carbon in glucose. I t is 24S. Roseman, J. Am. Chem. Soc. 75, 3854 (1953). 35S. Aronoff, H. A. Barker, and M. Calvin, J. Biol. Chem. 169, 459 (1947) ; M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwick, "Isotopic Carbon," p. 250. John Wiley & Sons, New York, 1949.

[23]

571

ISOTOPIC CARBON PATTERNS IN CARBOHYDRATE

seen t h a t , a l t h o u g h t h e m e t h o d m a y r e s u l t i n q u a n t i t a t i v e i n a c c u r a c i e s , it gives a r e a s o n a b l y t r u e p i c t u r e of t h e over-all p a t t e r n . TABLE II COMPARISON OF C 14 DISTRIBUTION IN GLUCOSE DETERMINED BY CHEMICAL AND L. casei DEGRADATION

Chemical degradation

L. casei fermentation

Type of labeling Glucose_l_C,, ~.d.~ Glucose-34-C ~4d

C-3,4 C-2,5 C-1,6 9.7 361

0 7.7

294 12.2

Glucose_3,4_C,4 I.g 2560 Glucose-C ~4 (differential labeling)1.g4140

57

49

Glucose_C,4 i h

28

6920 28

C-1

C-2~

70

1290

1670

1850 1970b 7280

5950

5550

428

71b

C-2,3,4,5 b C-3,4,5 ~ C-6

815i

20

20

33 ~ 21()0~* 34

By degradation of glucobenzimidazole. b By NaIO4 oxidation of gluconate. c Synthetic sample. d Isotopic data given in millimicrocuries per milligram of carbon. M. Gibbs, R. Dumrose, F. A. Bennett, and M. R. Bubek, J. Biol. Chem. 184, 545 (1950). i Isotopic data given in counts per minute per millimole of C02. g I. A. Bernstein, K. Lentz, M. Malta, P. Schambye, and H. G. Wood, J. Biol. Chem. 215, 137 (1955). h M. Cook and V. Lorber, J. Biol. Chem. 199, 1 (1952). i By HBr oxidation of glucose.

Applicability to Other Hexoses S c h a m b y e et al. 26 h a v e r e p o r t e d t h e d e g r a d a t i o n of g a l a c t o s e - C ~4 b y this p r o c e d u r e u s i n g cells a d a p t e d to f e r m e n t this hexose b y g r o w t h on all e q u a l m i x t u r e of D-galactose a n d D-glucose. F e r m e n t a t i o n of Glucose by Leuconostoc mesenteroides L. mesenteroides

CH2OHCHOHCHOHCHOHCHOHCHO CHaCHOHCOOH + HOCH2CH8 + CQ (C-6) (C-5)

(C-4)

(C-3)

(C-2)

(C-1)

T h i s d e g r a d a t i o n of glucose yields each c a r b o n s e p a r a t e l y - - a n a d v a n t a g e over t h e f e r m e n t a t i o n w i t h L. casei. D e M o s s et al. 27 r e p o r t e d 2s p. Schambye, It. G. Wood, and M. Kleiber, J. Biol. Chem. 226, in press (1957). 27 R. D. DeMoss, R. C. Bard, and I. C. Gunsalus, J. Bacteriol. 62, 499 (1951).

[9,3]

ISOTOPIC CARBON PATTERNS IN CARBOHYDRATE

573

and are transferred at least every month. The cultures may also be maintained in 0.5% agar using the medium given below for growth of the culture. Preparation of Bacteria for Fermentation of Glucose Medium

1% glucose. The glucose is autoclaved separately as a 10 % solution. 1% tryptone. 1% Difco yeast extract. 0.5% K2HPO~. 10% (by volume) tomato juice. 2% (by volume) salt mixture (autoclaved separately), consisting of 0.96% MgSO4.7H20, 0.04% NaC1, 0.072% FeSO4.7H~O, 0.18% MnSO4-H20, and 0.002% ascorbic acid. Distilled H20. The broth (except the salt mixture and glucose) is autoclaved for 20 minutes, the denatured protein is removed by centrifugation and/or filtration, and the clear supernatant solution is sterilized by autoclaving for 20 minutes. The salts and glucose are added aseptically just prior to inoculation. Procedure. Ten milliliters of the complete medium is inoculated from a stab culture and incubated at 30 ° until "gassing" perfusely (about 24 hours). Successive subcultures are made into 10 ml. of broth using a drop of inoculum until "gassing" occurs at 10 to 12 hours. Cultures are then successively transferred into 100 ml. and 1 1. of medium, using 1 ml. and 10 ml. of inoculum, respectively. Each culture is incubated at 30 ° until the evolution of gas is obvious (8 to 12 hours). When the large culture has begun to " g a s " strongly, the cells are removed by centrifugation, washed twice with distilled H~O, resuspended in distilled H20 (4 ml./g, wet weight of cells), and used immediately. Occasionally inactive ceils have been obtained when harvesting has been delayed too long (cf. Altermatt et al. 31 for a discussion of this problem). Preparation of Glucose for Fermentation The sample of glucose to be degraded is prepared as described on page 562. Fermentation Procedure Reagents

1 M phosphate buffer, pH 6.0. 2.5 M NaOH (carbonate-free).

574

TECHNIQUES FOR ISOTOPE STUDIES

[23]

Glucose sample in HsO. Distilled HsO.

Procedure. The fermentation is carried out in a 125-ml. Warburg respirometer vessel 3 with two side arms into which is put a total of 4 ml. of the cell suspension. Fifteen milliliters of 1 M phosphate buffer and 1 millimole of glucose in 11 ml. of distilled HsO are put in the main compartment. Two milliliters of 2.5 M NaOH (carbonate-free) is placed in the center well to absorb the evolved COs. A filter paper " w i c k " is immersed in the NaOH solution to facilitate rapid absorption of COs. A small vessel containing one-tenth the quantities of each component except the NaOH is run simultaneously to observe the progress of the fermentation manometrically~ (by C02 evolution). The vessels are "gassed" with 100% Ns (COs-free) for 15 minutes, closed off from the atmosphere, and equilibrated in the bath at 30 ° . The bacteria are then transferred to the main compartment. When the evolution of CO2 has ceased in the control flask, 3s the NaOH-NaHC03 solution and the filter paper from the center well in the large flask are removed and put in a stoppered container and the cells are separated from the supernatant solution by centrifugation. The fermentation may also be carried out in a 100 ml. round-bottomed, two-necked flask equipped with two gas inlet tubes one of which is connected to a "bubbler" (as shown in Fig. 1) containing 2.5 M NaOH (carbonate-free). The equipment is set up on a shaker (e.g. the Warburg apparatus) with the flask immersed in a water bath at 30 °. The sugar and phosphate solutions are put into the flask and the flask is "gassed" as above with Ns ( C O j r e e ) . One of the inlet tubes is then removed and the bacterial suspension is quickly introduced. "Gassing" is continued while the fermentation is in progress in order to carry the C02 over into the "bubbler." The progress of the fermentation is followed by weighing the bubbler periodically. Isolation and Purification of the Fermentation Products

Carbon Dioxide. The CO2 (carbon 1 of the glucose) is recovered from the NaOH solution and filter paper " w i c k " by acidification with HC103 or lactic acid in a closed system or by the addition of BaCI~, depending on the method to be used for determination of radioactivity. The recovery is about 90 % based on the amount of glucose present at the start of the fermentation. Ethanol. The fermentation supernatant is neutralized to phenol red s~ T h e C02 yield as m e a s u r e d manometrieally in the control vessel is a p p a r e n t l y not a measure of the actual CO~ yield in the large flask.

[23]

ISOTOPIC CARBON PATTERNS IN CARBOHYDRATE

575

(added internally as an aqueous solution) with N a 0 H and three-fourths the volume distilled to recover the ethanol (carbons 2 and 3 of the hexose). The ethanol in the distillate is oxidized to acetic acid in a glass-stoppered (held in place by rubber bands), round-bottomed flask by adding 15 ml. of a potassium dichromate solution 3~ and heating in a boiling H20 bath for 30 minutes. The acetic acid is recovered by concentration of the mixture to 15 to 20 ml. by distillation and steam distillation at constant volume of 10 to 15 vol. After neutralization of the acid in the distillate with NaOH, the solution is evaporated to dryness and the acetic acid is purified by partition chromatography on a column of Celite No. 53534 with mixtures of CHC13 and n-butanol.m5 A glass tube 20 mm. in diameter containing 15 g. of ether-washed, air-dried Celite No. 535 is used to purify not more than 1.0 millimole of the acid. The Celite is thoroughly mixed (but not ground) with 7.5 ml. of 0.18 N H2SO~. Enough CHC13 (equilibrated with 0.18 N H2SO,) 38 is added to make a slurry, and the material is put in the tube in small portions--each being packed well with a glass tamper. After all the Celite has been put into the tube, the column is further packed under positive air pressure, with care being taken not to allow the column to become dry. The dried sample of sodium acetate is dissolved in 1 ml. of H20, and, with 1 drop of thymol blue as an internal indicator, the solution is acidified with 2 N H2SO4 to a definite pink color. Two grams of Celite is added with mixing, and the damp mixture is put on top of the column and tamped. Glass wool is used to transfer any residual solid to the column and is put in the tube and pushed down on top of the column. Then 150 ml. of 100% CHC13 (equilibrated with 0.18 N H2SO,) 36 is passed through the column, being collected in 15-ml. portions. To each aliquot of eluate, 1 drop of 0.1% soluble phenol red in H20 and about 5 ml. of H20 (carbonate-free) are added. The tubes are vigorously shaken to facilitate extraction of the organic acid into the aqueous layer. The extracted acid is titrated with 0.01 N NaOH (carbonate-free). Acetic acid first appears in the tenth tube under these conditions. The eluting solvent is then changed to 97.5% CHC13-2.5% n-butanol (equilibrated with 0.18 N H2SO,), 36 and collection of 15-ml. aliquots is continued. The titrations are then carried out with 0.05 M NaOH (carbonate-free). Recoveries are 3~ 134 g. of K~Cr~O7 4- 675 ml. of 10 N H2SO4 diluted to 1 1. 34 A product of the Johns Manville Co. ~ C. S. Marvel and R. D. Rands, Jr., J. Am. Chem. Soc. 72, 2642 (1950). Some details of the procedure are modifications of E. Swim and K. Lentz. 3~ The organic phase is passed through W h a t m a n No. 1 filter paper on a gravity funnel to remove suspended droplets of H~O.

576

TECHNIQUES FOR ISOTOPE STUDIES

[23]

80 % or better, based on the a m o u n t of glucose present at the start of the fermentation. After separation of the aqueous phase and removal of residual CHC18 and butanol b y aeration, the solution is evaporated to 10 to 15 ml., acidified to Congo red with H2S04, and the acetic acid recovered by steam distillation (at constant volume) of 10 to 15 vol. Barium acetate is formed b y titration with 0.1 N Ba(OH)2 (carbonate-free) to bromothymol blue. 87 The solution is then evaporated to dryness in a porcelain boat or directly into tube C of Fig. 2.

C(C.20)

J

•IceI~ Fro. 2. Pyrolysis apparatus. (A) receiver, round-bottomed, 300 ml., containing 20 ml. of H~O and immersed in an ice bath; (B) adapter with vacuum take-off and stopcock; (C) pyrolysis tube; (D) porcelain boat containing dried barium acetate; (E) electrical combustion furnace with thermostatic control, and (F) thermocouple. Designations in parentheses indicate catalog numbers of parts available commercially.l~ All ground joints are standard taper 24/40.

Lactic Acid. T h e residue from the above 3~ vol. distillation is acidified with H2S04 to Congo red and the lactic acid (carbons 4, 5, and 6 of the glucose) contained therein is recovered by continuous ether extraction as described on page 563. Chemical Degradation of Acetic Acid (see also Vol. IV [22])

Principle. The isotopic content of each carbon of the acetic acid can be determined b y the following reactions: 3~This end point is difficult to see, but titration to about pH 7 is preferable to a more alkaline pH, since there is less likelihood that carbonate will be titrated. Any BaCOs formed at this stage from unlabeled carbonate will dilute the BaC1403 formed in the pyrolysis of the barium acetate.

578

TECHNIQUES FOR ISOTOPE STUDIES

[23]

thetically prepared acetic acid-l-C 14 containing 3.1 × 106 c.p.m./ millimole of compound was degraded by the above procedure, 0.17% of the activity appeared in the CHI3 (a-carbon). Similarly, 0.88% of the activity of the molecule appeared in the BaCO3 (carboxyl carbon) when acetic acid-2-C 14 containing 5.7 X 106 c.p.m./millimole of compound was analyzed. Nevertheless it should be noted that randomization of isotope has been observed in the pyrolysis of barium acetate by others 39possibly using somewhat different procedures from those described above. Other Procedures. Other procedures by which acetic acid may be degraded include (1) decarboxylation by the Schmidt reaction and oxidation of the resulting amine to CO24o and (2) formation of 2-methylbenzimidazole, its conversion to the 2-benzimidazolecarboxylic acid, and the decarboxylation of this acid. 2~ Both procedures are reported not to result in significant "cross contamination" of isotope. In the former procedure, the dried sample of sodium acetate, NAN3, and 100% H2SO4 are warmed together to give COs (from the carboxyl carbon) which is removed by aeration with air or N2 (COs-free) and methylamine (from the methyl carbon). The reaction mixture is then brought to pH 11 to 12 with NaOH and the amine recovered by aeration into dilute H2SO4. The methylamine sulfate is neutralized and oxidized to COs with alkaline KMn04. In the second method, acetic acid is condensed with o-phenylenediamine to form 2-methylbenzimidazole which is isolated. This compound is condensed with benzaldehyde, and the resulting derivative is oxidized with KMn04 to the 2-benzimidazolecarboxylic acid. This acid is heated at 190° to give COs (methyl carbon of the acetic acid) and benzimidazole (earboxyl carbon) which is burned to C02. Chemical Degradation of Lactic Acid Lactic acid is degraded as described above and also in Vol. IV [22]. Reliability of the Leuconostoc mesenteroides Procedure Tables III and IV present data which demonstrate the reliability of the method. The entire procedure appears to give results with an error no greater than 2 to 3%. Applicability to Other Hexoses Schambye et al. ~6 used this technique to degrade galactose-C ~4. The cells were adapted to ferment galactose by being grown on 10 ml. of 39 M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwich, "Isotopic Carbon," p. 248. John Wiley & Sons, New York, 1949. 40 E. F. Phares, Arch. Biochem. and Biophys. 85, 173 (1951).

[23]

ISOTOPIC

CARBON P A T T E R N S

IN CARBOHYDRATE

wz wz ;2

o0

[-.,.

¢q

(b

g~

~d O

L)

©

z

%

O

579

580

TECHNIQUES FOR ISOTOPE STUDIES

[23]

glucose m e d i u m until " g a s s i n g " occurred in 12 hours followed b y subculturing on 10-ml." aliquots of m e d i u m containing galactose instead of glucose until " g a s s i n g " was again observed in 12 hours. At this point the cells were p r e p a r e d as usual for f e r m e n t a t i o n in 100 and 1000 ml. of m e d i u m containing galactose as the carbohydrate. TABLE IV ANALYSIS OF GLUCOSE-1-C14 RY FERMENTATION WITH L. meser~teroidesa S. A.b of products formed S. A.b of glucose added

CO~ (C-l)

9.6 9.6

57 56

Ethanol (C-2,3) Lactate (C-4,5,6) 0.01 0

0 0.02

° I. C. Gunsalus and M. Gibbs, J. Biol. Chem. 194, 871 (1952). b S. A. -- millimierocuries per milligram of carbon (specific activity). Although known isotopic glucosamine has not as y e t been degraded, f e r m e n t a t i o n of unlabeled glucosamine b y cells a d a p t e d to this comp o u n d 41 yielded C02, ethanol, and lactic acid in the usual quantities. 42

Fermentation of Ribose by Lactobacillu$ S e n t o s u s CH2OHCHOHCHOHCHOHCHO

L. pentosus

* CH~CHOHCOOH ~ HOOCCH3 (C-5) (C-4) (C-3) (C-2) (C-1)

F r e d et al. 48 showed t h a t Lactobacillus pentosus 124-2 produced 1 mole each of acetic acid and lactic acid per mole of pentose utilized. L a m p e n et al. 44 found essentially all the isotope in the m e t h y l group of the acetic acid when xylose-l-C I4 was fermented. 45 Gest and L a m p e n 4e p o s t u l a t e d t h a t this organism cleaves pentose between carbons 2 and 3 so t h a t the source of the acids would be as shown above. Bernstein 4' c o m p a r e d the p a t t e r n s of C 14 found in samples of ribose b y chemical m e t h o d s with those obtained b y f e r m e n t a t i o n with this organism and concluded t h a t the 4x Glucosamine hydrochloride was substituted for glucose in the growth procedure. 4~I. A. Bernstein, unpublished data. 43 E. B. Fred, W. H. Peterson, and J. A. Andrews, J. Biol. Chem. 48, 385 (1921). 44j. O. Lampen, H. Gest, and J. C. Sowden, J. Bacteriol. 61, 97 (1951). ,5 D. A. Rappoport, H. A. Barker, and W. Z. Hassid [Arch. Biochem. and Biophys. $1, 326 (1951)] obtained similar results in the fermentation of L-arabinose-l-C TM with LactobaciUus pentoaceticus as did Altermatt et al. 31 with Leuconostoc mesenteroides fermenting D-xylose-l-C 14. 46H. Gest and J. O. Lampen, J. Biol. Chem. 194, 555 (1952). ~ I. A. Bernstein, J. Biol. Chem. 205, 309 (1953).

[23]

ISOTOPIC CARBON PATTERNS IN CARBOHYDRATE

581

sources of the carbons in the products were as Gest and Lampen had postulated and that this fermentation could be used to determine the pattern of tracer carbon in ribose.

Maintenance of Cultures

Medium 1% xylose. The xylose is autoclaved separately as a 10% solution and is added aseptically before the medium hardens. 0.4% Difco yeast extract. 1% Difco nutrient broth. 1.6 % sodium acetate.3H20. 2% agar. 1% (by volume) salt mixture, consisting of 2% MgSO~-7H20, 0.1% NaC1, 0.1% FeSO4.7H~O, and 0.1% MnSO4.4H20. Distilled H~O.

Procedure. The agar medium is inoculated by a stab from a previous culture and incubated for 24 hours at 37 ° . Cultures are stored at about 2° and transferred at least once a month. Preparation of Bacteria for Fermentation of Ribose

Medium. The bacteria are grown for fermentation on the same medium (prepared in the same way) as is used for maintenance of the cultures except that no agar is used and the first transfer is made into a medium containing glucose instead of xylose. Procedure. Five milliliters of medium containing 1% glucose as carbon source is inoculated from a stock culture and incubated at 37 ° for 24 hours. Two drops of this culture is used to inoculate 10 ml. of medium containing 1% xylose. After 24 hours at 37 °, this entire culture is added to 100 ml. of "xylose" medium. The new culture is allowed to grow for 12 hours at which time it is added to 3 1. of medium containing xylose. The cells are harvested by centrifugation after 36 to 40 hours and are washed twice with 40 ml. of 1% KC1 and twice with the same amount of distilled H20. The wet cell weight is determined, and the cells are suspended in 0.1 M NaHCO3 (4 ml./g.) to make a 20% suspension. Preparation of Ribose for Fermentation The sample of ribose to be fermented is dissolved in distilled H20.

Fermentation Procedure

Reagents 0.1 M NaHC03.

582

TECHNIQUES FOR ISOTOPE STUDIES

[23]

Ribose sample in H20. Distilled HsO. Procedure. The fermentation is carried out in a 125-ml. Warburg respirometer vessel with two side arms.S Five milliliters of cell suspension in 0.1 M NaHCO~ is divided between the two side arms. Then 7.5 ml. of 0.1 M NaHC03 and 0.5 millimole of ribose in 17.5 ml. of HsO are put in the main compartment. A separate vessel is prepared in a similar manner without the ribose to measure the endogenous metabolism of the cells. 48 The vessels are "gassed" for 15 minutes with 5% CO2--90% Ns, closed, and equilibrated for temperature in the bath at 37 ° . The bacteria are then transferred to the main compartment, and the fermentation is followed manometrically by the evolution of COs from the buffer resulting from acid formation 5 (theory: 2 moles of COs evolved per mole of ribose utilized). When no more COs is being evolved, 2 ml. of 2 N H~SO, is added to acidify the medium. I s o l a t i o n and Purification of F e r m e n t a t i o n A c i d s

Acetic Acid. The bacteria are removed by centrifugation and the acetic acid is recovered by concentrating (by distillation) the supernatant solution (acid to Congo red) to about 15 ml. and then distilling with steam at constant volume until a total of 15 vol. of distillate is obtained. The distillate is titrated with NaOH to phenolphthalein and evaporated to about 15 ml. The acid is then purified as described on page 575. Lactic Acid. The lactic acid is recovered by continuous ether extraction from the combined steam distillation residues as described on page 563. C h e m i c a l D e g r a d a t i o n s of A c e t i c Acid and Lactic Acid

Acetic acid may be degraded as described on page 576, and lactic acid as on page 564. R e l i a b i l i t y of the

Lactobacillus pentosus P r o c e d u r e

The validity of the procedure is shown by the comparative data given in Table V. The C ~ patterns of labeled ribose samples were determined by this procedure and compared with data obtained by chemical methods only. When the values for carbons 1 and 2 by the fermentation were 48 The specific activity of the acetate formed from labeled ribose in the fermentation is corrected for the endogenous production of acetate as measured manometrically in this vessel. Gest and Lampen 4s report the endogenous metabolism of this organism to result almost entirely in the production of acetate.

584

TECHI~IQUES FOR ISOTOPE STUDIES

[24]

The cells were grown on a medium containing 0.5 % glucose and 0.5% xylose. Although the fermentation was carried out and the products isolated and degraded by techniques different from those discussed in this article, it seems likely that the detailed procedures given here could be conveniently adapted to this fermentation if desired.

[24] Isotopic Experimentation with Intermediates of the Tricarboxylic Acid Cycle By H. E. SWIM and M. F. UTTER I. General Considerations The objective of this report is to discuss certain procedures applicable to isotopic tracer experiments with intermediates of the tricarboxylic acid cycle and the related compounds, lactic and acetic acids. Primary attention is directed toward procedures which appear to be particularly well suited for tracer experiments, and no attempt is made to review the voluminous literature on biochemical methodology of this group of compounds. We have attempted to select methods which seem generally useful in tracer experiments, but the choice is necessarily somewhat arbitrary and many methods of equal merit have undoubtedly been omitted. The rationale of isotopic tracer experiments is quite straightforward: the presentation of appropriately labeled compounds to a biological system followed by the extraction, separation, and degradation of suspected intermediates and end products. For reasons of convenience the discussion of methodology relating to tracer experiments with compounds of the tricarboxylic acid cycle has been divided into four sections: extraction and separation, determination, degradation, and synthesis. The coverage of these four topics is by no means uniform. Only the first of these, the extraction and separation of tricarboxylic acid cycle intermediates, is dealt with in the detail comparable to that usually achieved in these volumes. The presentation of other aspects of isotopic methodology departs somewhat from the usual format in that it has not been possible in most cases to provide complete details. The discussion is directed toward correlating diverse procedures with respect to their applicability and limitations with only an outline presentation of actual methods in most instances. Additional details can be obtained from the appropriate references which are cited.

584

TECHI~IQUES FOR ISOTOPE STUDIES

[24]

The cells were grown on a medium containing 0.5 % glucose and 0.5% xylose. Although the fermentation was carried out and the products isolated and degraded by techniques different from those discussed in this article, it seems likely that the detailed procedures given here could be conveniently adapted to this fermentation if desired.

[24] Isotopic Experimentation with Intermediates of the Tricarboxylic Acid Cycle By H. E. SWIM and M. F. UTTER I. General Considerations The objective of this report is to discuss certain procedures applicable to isotopic tracer experiments with intermediates of the tricarboxylic acid cycle and the related compounds, lactic and acetic acids. Primary attention is directed toward procedures which appear to be particularly well suited for tracer experiments, and no attempt is made to review the voluminous literature on biochemical methodology of this group of compounds. We have attempted to select methods which seem generally useful in tracer experiments, but the choice is necessarily somewhat arbitrary and many methods of equal merit have undoubtedly been omitted. The rationale of isotopic tracer experiments is quite straightforward: the presentation of appropriately labeled compounds to a biological system followed by the extraction, separation, and degradation of suspected intermediates and end products. For reasons of convenience the discussion of methodology relating to tracer experiments with compounds of the tricarboxylic acid cycle has been divided into four sections: extraction and separation, determination, degradation, and synthesis. The coverage of these four topics is by no means uniform. Only the first of these, the extraction and separation of tricarboxylic acid cycle intermediates, is dealt with in the detail comparable to that usually achieved in these volumes. The presentation of other aspects of isotopic methodology departs somewhat from the usual format in that it has not been possible in most cases to provide complete details. The discussion is directed toward correlating diverse procedures with respect to their applicability and limitations with only an outline presentation of actual methods in most instances. Additional details can be obtained from the appropriate references which are cited.

[24]

INTERMEDIATES OF THE TRICARBOXYLIC ACID CYCLE

585

The planning and carrying out of isotopic tracer experiments depends oll a number of considerations some of which are mentioned briefly in the following paragraphs. In isotopic experiments the purity of the compounds involved is of even greater importance than in many other types of chemical studies, since the presence of even traces of a highly labeled contaminant can lead to erroneous conclusions. This requirement for high purity influences all stages of the methodology of isotopic experimentation. The problem of permissible dilution of the isotopic label faces every investigator in this field. Ultimately, the magnitude of dilution which can be sustained depends on the sensitivity of the counting methods. Dilution occurring during the biological stage and in the separation and degradation stages must be considered. In many cases it is expedient or necessary to add carrier compounds at various points during the experiment. Obviously, no set rules are possible and individual problems must be considered carefully prior to experimentation. Many isotopic experiments have been performed in a semiquantitative fashion and the data used to formulate qualitative conclusions. In such circumstances, the problems of dilution may be of lesser importance and also the specificity of the degradative procedures used need not be so rigid. II. Extraction and Separation of Acids Introduction

The procedures described in this section are applicable to the isolation and separation of intermediates of the tricarboxylic acid cycle and related compounds from a wide variety of sources. Acids are isolated from reaction mixtures by extraction with ether, acetone, or other suitable organic solvents. After removal of the solvent, a number of chromatographic procedures may be employed to resolve the mixture into its individual components. Separations are usually made on a two-phase chromatogram consisting of an immobile phase prepared by saturating a solid with water, the solid merely acting as a mechanical support, and a mobile phase which is some liquid immiscible with water. Separations on chromatograms of this type depend on differences in the partition of the compounds between two liquid phases. 1 Ion exchange techniques may also be used in the separation of organic acids. 2,8 This method, however, has not gained general acceptance primarily because of the difficulty in devising methods for determining small 1 A. J. P. Martin and R. L. M. Synge, Biochem. J. 3§~ 1358 (1951). 2 H. Busch, R. B. tturlbert, and V. R. Potter, J. Biol. Chem. 196, 717 (1952). 3 H. H. Schenker and W. Reiman, Anal. Chem. 25, 1637 (1953).

586

TECHNIQUES FOR ISOTOPE STUDIES

[24]

quantities of organic acids by titration in acidic or buffered column effluents. Water adsorbed on silicic acid, Celite, or paper has been used most frequently as the immobile phase in partition chromatography of organic acids. Excellent separations of acids of the tricarboxylic acid cycle are obtained by partition chromatography on paper, 4 but the method is of limited usefulness in isotopic experimentation because the quantity of acid that can be employed is too small to be readily determined by conventional methods. Procedures employing columns prepared with Celite or silicic acid are preferable because relatively large quantities of a mixture of acids can be resolved into its individual components. The selection of Celite over silicic acid is primarily one. of personal choice, since neither offers any outstanding advantages over the other and comparable separations are obtained when the same developing solutions are employed. The procedures described below in some detail have yielded satisfactory results in the extraction and separation of intermediates of the tricarboxylic acid from a variety of microorganisms, enzyme mixtures, and other sources. These techniques combine many features of a number of similar methods. 5-1° It is emphasized that additional procedures may offer certain advantages under specially circumscribed conditions.

Equipment and Reagents Chromatographic tube constructed from Pyrex tubing, 1.8-cm. inside diameter and 75 cm. in length, constricted at the bottom and sealed to a stopcock. A plug of glass wool at the constriction acts as a support for the column. Pressure device. A tank of nitrogen or air line fitted with a suitable pressure-regulating valve. Lyophile apparatus. Ethyl ether, free from ethyl peroxide and organic acids. The presence of peroxide can be detected by the liberation of iodine (brown coloration or blue coloration on the addition of a few drops of 1% starch solution) when a small sample is shaken with * R. J. Block, E. L. Durrum, and G. Zweig, " A Manual of Paper Chromatography and Paper Electrophoresis." Academic Press, New York, 1955. 5 F. A. Isherwood, Biochem. J. 40, 688 (1946). s C. S. Marvel and R. D. Rands, Jr., J. Am. Chem. Soc. 72, 2462 (1950). 7 C. E. Frohman, J. M. Orten, and A. H. Smith, J. Biol. Chem. 193, 277 (1951). 8 E. F. Phares, E. H. Mosbach, F. W. Denison, Jr., and S. F. Carson, Anal. Chem. 24, 660 (1952). 9 W. A. Bulen, J. E. Varner, and R. C. Burrell, Anal. Chem. 24, 187 (1952). 10F. E. Resnik, L. A. Lee, and W. A. Powell, Anal. Chem. 27, 929 (1955).

[24]

INTERMEDIATES OF THE TRICARBOXYLIC ACID CYCLE

587

an equal volume of potassium iodide solution (2 g. of KI dissolved in 100 ml. of 0.05 N HC1). If the ether contains peroxide it can be removed by shaking 1 1. with 100 ml. of an acidic solution of ferrous sulfate (50 g. of FeSO4, 5 ml. of concentrated HC1, and 100 ml. of water) and distilled unless acids are to be removed also. The presence of organic acids can be determined by titrating a sample with 0.01 N NaOH (phenol red indicator). When free from peroxide, the ether is distilled over sodium hydroxide to remove organic acids. Celite 535, Johns Manville. Dry Celite is firmly packed into a Pyrex tube of suitable diameter and washed with ether (free from peroxide and organic acids) by allowil~g approximately 1 1. per 100 g. of Celite to flow through the column. The Celite is then spread in a thin layer on aluminum foil (hood) and allowed to dry at room temperature overnight. It is then heated in an oven at 100 ° for 4 to 6 hours and finally stored in a closed container. n-Butanol, reagent grade. Most lots are satisfactory, but some are contaminated with organic acids which can be removed by washing thoroughly with water or distilling over sodium hydroxide. Chloroform, reagent grade, extracted several times with water to remove ethanol. Chloroform-n-butanol mixtures equilibrated against 0.2 N H2SO4. Each mixture is thoroughly shaken in a separatory funnel with 0.5 to 1 vol. of 0.2 N H2SO4 and the two phases are allowed to separate. It is desirable to repeat this procedure several times. To assure the complete removal of water droplets from the chloroform-butanol solutions, each is finally filtered through a dry filter paper (12 cm. in diameter, any grade, in 15-cm. filter funnel, per liter of ether). For smaller volumes of solution the size of the filter paper should be reduced accordingly to avoid the possibility of removing acid from the solvent so it is no longer equilibrated. Phenol red indicator, 0.04%, prepared by mixing 100 rag. of solid (acid form) with 2.8 ml. of 0.1 N NaOH and diluting to 250 ml. with water. Extraction of Acids Procedure A. This method involves the adsorption of an acidified mixture containing intermediates of the tricarboxylic acid cycle on Celite followed by elution with ether. The procedure is applicable to tissue homogenates, to biochemical reaction mixtures, and to solutions in general.

588

TECHNIQUES FOR ISOTOPE STURIES

[24]

The following example is based on a 10-ml. sample. The pH of the mixture is adjusted to less than 2 (congo red paper) with 10 N H~SO~. Sulfuric acid is also used in experiments where it is necessary to terminate biochemical reactions prior to extracting the acids. The solution is then mixed thoroughly with 20 g. of dry Celite and transferred quantitatively to a chromatographic tube (18-ram. inside diameter) in 2-g. portions, each portion being uniformly packed with a stainless steel pestle. The organic acids are eluted from the Celite by allowing ether to run through the column, or it may be forced through under pressure at a rate not exceeding 24 ml./min. The volume of ether required to extract the acids quantitatively varies with the composition of the sample, although 300 to 500 ml. is usually sufficient. It is therefore advisable to determine the volume of ether required by collecting aliquots of the eluate at suitable intervals and titrating each with 0.01 N NaOH (phenol red indicator). The ether is distilled i n v a c u o (without application of heat) or evaporated in a well-ventilated hood with the aid of a gentle stream of air. The residue is chromatographed on Celite as described under Separation of Acids. This method is superior to the conventional liquid-liquid extraction in that all the acids of the tricarboxylic acid cycle are recovered quantitatively in a relatively short time. Furthermore, this method can be used at temperatures as low as 0 ° if necessary. In addition the method serves as a desalting procedure. This is particularly important when the original mixture contains high concentrations of phosphates and other inorganic salts which may interfere in the chromatographic separation of acids with peak effluent volumes (Table I) greater than 500 ml. The volume of material which can be extracted in the manner described is limited only by the amount of Celite which can be conveniently extracted with ether. The salient points to be considered in adapting the extraction method to larger or smaller volumes are as follows. (1) The ratio of sample (water) to Celite must not exceed 1:2. If too much water is present in the column, some will be eluted along with inorganic acids and other water-soluble materials which interfere with chromatographic separations. (2) The volume of ether required to elute the organic acids quantitatively should be determined by titrating aliquots of eluate, collected at intervals during the extraction with 0.01 N NaOH. (3) The diameter of the tubing should be such that a column of Celite 20 to 25 cm. is obtained. The volume of the sample may also be reduced i n v a c u o at temperatures usually not exceeding 30 ° prior to ether extraction on Celite. No other alteration in the procedure is required. The degree of concentration should not exceed 100-fold, since excess acid may contribute to the

[~4]

INTERMEDIATES OF THE TRICARBOXYLIC ACID CYCLE

5~,0

decomposition of the more labile m e m b e r s of the tricarboxylic acid cycle series. A portion of the acetic acid is r e m o v e d b y distillation b u t can be readily recovered b y neutralizing the distillate and e v a p o r a t i n g to dryness on a s t e a m bath. T h e acetic acid is purified b y c h r o m a t o g r a p h y on Celite. I t will be noted t h a t at no point in the sequence is the mixture of acids neutralized. This is of considerable i m p o r t a n c e when q u a n t i t a t i v e TABLE I SUMMARY OF RESULTS IN SEPARA.TION OF ACIDS BY PARTITION CHROMATOGRAPHY ON CELITE ~

Peak number b

Acid

1 2 3 4 5 6

Propionic Acetic Pyruvic Fumaric Succinic a-Ketoglutaric

7

cis-Aconitic

8 9 10

Malic Citric d-Isocitric

Per cent (v/v) chloroform-butanol

Peak effluent volume/ ml.

Acid added, aM.

Acid recovered, aM.

100-0 95-5 90-10 90-10 85-15 80-20 70-30 70-30 60-40 50-50

70 150 210 260 360 420 630 690 860 950

40.0 20.0 30.0 35.0 35.0 25.0 10.0 20.0 20.0 10.0

39.8 20. l 29.1 34.8 35.1 23.7 9.6 19.5 19.6 9.2

" Compiled from H. E. Swim, Thesis, Western Reserve University, 1952. b Refers to chromatograph, Fig. 1. Average value obtained from several experiments. recovery of the keto acids is required. Oxalacetic acid is unstable at neutral or alkaline p H ' s . ll F u r t h e r m o r e , when mixtures are e v a p o r a t e d to dryness or lyophilized at neutral or alkaline p H ' s , only 60 to 80 ,~,~of the pyruvic and a-ketoglutaric acids is recovered on subsequent chromat o g r a p h y on Celite. T h e low recoveries are p r e s u m a b l y due to the formation of polymers of these keto acids under alkaline conditions which do not migrate on the column with the developing solutions employed. Procedure B. This m e t h o d is similar in principle to Procedure A and is well suited to the isolation of acids from d r y powders, tissues, and microbial cells. I t has been employed successfully ill the isolation of intermediates of the tricarboxylie acid cycle from microorganisms 12,~ in experiments employing 100 g. of cells (wet weight) (see ref. 14 for 1~p. Ostern, Z. physiol. Chem. 218, 160 (1933). 12j. A. DelVfoss and H. E. Swim, unpublished experiments. 13E. Fisher, Jr. and H. E. Swim, unpublished experiments. ~4H. E. Swim and L. O. Krampitz, J. Bacteriol. 67, 419 (1954); H. J. Saz and L. O. Krampitz, ibid. 67, 409 (1954).

590

TECHNIQUES FOR ISOTOPE STUDIES

[24]

examples of this type of experiment). Enzymatic reactions are terminated by adjusting the pH of the reaction mixture to 3.5 to 4 with HC1 and cooling the mixture to 0% The cells are recovered by centrifugation and washed twice with cold water. After washing, the packed cells are mixed with 150 ml. of distilled water to form a thick slurry, 4 ml. of 5 N HC1 is added, and the mixture is then lyophilized. The small quantity of HC1 vapor which is not condensed in the lyophile apparatus may be prevented from reaching the vacuum pump by employing a trap filled with sodium hydroxide pellets or one cooled in liquid nitrogen. The lyophilized material (approximately 20 g.) is mixed thoroughly with 40 g. of dry Celite. If the material contains C 14, this operation should be performed in a well-ventilated hood. The cell-Celite mixture is acidified (pH less than 2) by mixing uniformly with 15 ml. of 0.5 N H2S04. If the sample contains an unusually large quantity of various salts it may be necessary to use more concentrated H2S04 (volume not to exceed 20 ml.). The pH can be readily checked by mixing small pieces of congo red paper with the Celite. The acidified mixture is added quantitatively in 10-g. portions to a Pyrex tube (5 to 6 cm. in diameter), each portion being packed uniformly. The organic acids are eluted by allowing 3 1. of ether to flow through the column, or it may be forced through under pressure at a rate not exceeding 100 ml./min. The volume of ether required to elute the acids quantitatively may vary with the composition of the sample, and this can be determined as described in Procedure A. It is essential that the Celite remain acid (pH less than 2) throughout the extraction. Congo red paper can be used as an internal indicator by adding several small pieces to the cell-Celite mixture and by packing the column in a manner so that a number of these are visible. After evaporation of the ether (eluate), the residue is chromatographed on Celite as described under Separation of Acids. Since acetic acid is volatile, it is removed by lyophilization. This compound is readily recovered by neutralizing the distillate and evaporating to dryness on a steam bath. The acetate is purified by chromatography on Celite. A small portion of the pyruvic acid may also be removed by lyophilization, but the extent to which this occurs has not been established. The quantity of material to be extracted in this manner can be varied over a wide range. The important points to be considered in varying the quantities of sample are the same as described under Procedure A.

Separation of Acids Preparation of the Column. The preparation and characteristics of a 10-g. column are described. Ten grams of Celite 535 is thoroughly mixed

[24]

INTERMEDIATES

OF T H E TRICARBOXYLIC ACID CYCLE

591

with 5 ml. of 0.2 N H2SO4 in a 250-ml. beaker. The fine slurry prepared by flooding the mixture with chloroform (previously equilibrated with 0.2 N H2SO4) is added to the tube in 2-g. portions, each portion being packed uniformly with a stainless steel pestle. A more uniform column is obtained if 50 to 100 ml. of chloroform is forced through the column (after packing) under 10 to 15 pounds of pressure of nitrogen or air. Care must be exercised not to allow the level of the chloroform to fall below the top of the Celite, and the pressure must be reduced gradually to avoid cracking the column. Addition of Sample to Column. The mixture of acids obtained according to Procedure A or B, under Extraction of Acids is dissolved in 1 to 1.5 ml. of 0.2 N H2SO4 and thoroughly mixed with 2 to 3 g. of Celite. In the case of neutral or alkaline mixtures from other sources (when keto acids are not present) the pH is adjusted to less than 2 with H2SO4 prior to mixing with Celite. The Celite-acid mixture is transferred quantitatively to the top of the column and uniformly packed. This is conveniently accomplished by wiping the last traces of Celite from the container with a plug of glass wool which is then pushed down firmly on top Of the column. The glass wool plug serves to hold the Celite in place and should be sufficiently large to sweep down particles of Celite adhering to the upper portion of the tube. After packing, sufficient chloroform (equilibrated with 0.2 N H2SO4) is added to saturate the Celite. Separation Procedure. The developing solutions used in increasing polarity are as follows: chloroform, and chloroform containing 5, 10, 15, 20, 25, 30, 35, 40, and 50% (v/v) n-butanol. For a 10-g. column, 100 ml. of each solution is allowed to run through the column or may be forced through under pressure at a rate not exceeding 4 ml./min. The effluent is usually collected in 10-ml. fractions in 20 N 150-mm. tubes or by the use of an intermittent syphon constructed so as to deliver the correct volume. One drop of phenol red indicator and about 5 ml. of CQ-free water are added to each fraction which is then titrated with 0.01 N NaOH. During the titration, intimate contact between the two phases is obtained by bubbling CO:-free air through the mixture. It is necessary to apply a small blank correction to the titrations. These values increase gradually with increased concentrations of n-butanol (Fig. i). Heavy emulsions are encountered when the concentration of n-butanol in the eluant exceeds 40% which makes accurate titrations practically impossible. This difficulty is eliminated by addition of 5 ml. of water-washed chloroform to each tube prior to titrating. Recovery of Individual Acids from Column E~uent. After titrating with NaOH, the various acids, with the exception Of the keto acids, are recovered as their sodium salts by combining serial fractions of the effluent

592

TECHNIQUES

FOR

ISOTOPE

[24]

STUDIES

(aqueous phase plus nonaqueous phase) containing a single acid and evaporating the solvent on a steam bath (hood) with the aid of a gentle stream of air. Fractions containing keto acids are treated in a somewhat different manner to avoid possible decomposition and polymerization. The latter is of critical importance if enzymatic procedures are to be employed to degrade the keto acids. Immediately after titrating, the contents of a series of tubes containing one of the keto acids are combined. The aqueous phase containing the acid is separated from the chloroformbutanol mixture. The latter is extracted several times with small volumes of water. The combined extracts are added to the aqueous phase (keto •acid solution), the pH is adjusted to 1 with hydrochloric acid, and the solution is lyophilized.

z z

3.0" 4 ~j~ 2.0.~ ~ 1.0-

(5 .4

o-~

160

Ebo : ~

4bo e~o ~

MILLILITERS OFEFFLUENT ~

s~

9bo Ioo

FIG. l. Typical chromatograph of a mixture of known acids. The sodium salts or the free acids (in case of keto acids) are dissolved in water and transferred quantitatively to volumetric flasks. These solutions are subsequently examined for the presence of contaminants (see Discussion of Separation Procedures) and finally used for isotopic analyses. Discussion of Separation Procedures. A typical chromatograph of a mixture of known acids is illustrated in Fig. 1. The quantitative aspects of this experiment are summarized in Table I. Propionic acid is also included, since it is an intermediate in the degradation of succinic and a-ketoglutaric acids. The separation procedure is similar to that described by Marvel and Rands, e who employed silicic acid instead of Celite in the immobile phase, and the results obtained with the two types of columns are in excellent agreement. Peak effluent volume (Table I) is defined as the quantity of effluent collected while a given compound migrates to the bottom of the column and is measured at the point at which the greatest concentration of the compound is eluted. When variables such as dimensions of the column, solvents employed, strength of H2S04 used to prepare the Celite and developing solutions, and rate of change in polarity of eluant are

[9.4]

INTERMEDIATES OF THE TRICARBOXYLIC ACID CYCLE

593

maintained constant, each compound has a characteristic peak effluent volume. This constant can be used in the tentative identification of an acid component from an unknown mixture. The quantitative addition of a sample to the top of a prepared column is facilitated by adsorbing in Celite prior to transfer. On the other hand, acids are usually dissolved in tert-amyl alcohol or other organic solvents before transfer to silicic acid columns. There are, however, certain technical difficulties associated with this procedure. The results obtained by Bulen et al2 indicate that a technique similar to that described above for Celite is applicable to silicic acid columns although this method has not been in general use. Zbinovsky and Burris 15 have described an additional method for adding aqueous solutions of organic acids to silicic acid columns. Simple devices for gradually and automatically increasing the nbutanol concentration and thereby the polarity of the solvent entering the column have been described. 16,1~These methods facilitate the use of a mechanical apparatus for the collection of effluent fractions and eliminate false chromatographic peaks which occasionally are produced when the polarity of the solvent is increased in discrete steps. Furthermore, the results obtained by Marshall et al. ~8 indicate that chromatographic resolution can be improved by rigid control of the rate at which the polarity of the eluant is increased. The quantity of a mixture of acids which can be separated on a 10-g. column varies with the acids involved. For example, the amount of two acids which can be separated depends on the difference between their peak effluent volumes and the sharpness with which they are eluted. For the mixture of acids shown in Table I, quantities up to 300 micromoles (total acid) can be separated. The same procedures can be used on a smaller or larger scale by simply applying proportionality factors to determine the materials required. The amount of Cetite used to adsorb the sample may also be varied according to the size of the column. It is essential that not more than 1 ml. of sample be added for each 2 g. of Celite. When unknown mixtures are chromatographed there is usually a possibility that fractions may be contaminated with organic acids with peak effluent volumes similar to those of the tricarboxylic acid cycle. The presence of a relatively large quantity of such a contaminant is indicated by irregularities in the contour or symmetry of the curves on a 15 V. is K. 17 R. is L.

Zbinovsky and R. H. Burris, Anal. Chem. 26~ 208 (1954). O. Donaldson, V. J. Tulane, and L. M. Marshall, Anal. Chem. 24, 185 (1952). R. Allen and D. N. Eggenberger, Anal. Chem. 27~ 476 (1955). M. Marshall, K. O. Donaldson, and F. Friedberg, Anal. Chem. 24, 773 (1952).

594

TECHNIQUES FOR ISOTOPE STUDIES

[9.4]

chromatograph (Fig. 1) and by an increase in the volume of eluant required to eluate a given quantity of acid (broader peak). In certain instances there may be no indication that a contaminant is present. The following procedures are useful in checking the purity of acids isolated by partition chromatography. 1. In instances where methods are available, the quantity of acid as determined by titration is compared with that obtained by a more specific method (see Section III, Determination of Acids). 2. Chromatographic procedures employing paper and a variety of developing solutions 19-~7 may be used to examine a portion of the sample. Paper chromatography is well suited for this purpose, since minute quantities of sample are employed and a variety of developing solutions can be tested with a minimum expenditure of time and materials. The phenol red indicator that is added during the titration of fractions of effluent from the column does not interfere in isotopic analyses because the quantity of carbon introduced in this manner is ordinarily insignificant in comparison with that of the organic acid per se. The ratio of phenol red to organic acid is reduced even further when samples are diluted with carrier (see Section IV). Phenol red may interfere, however, with the spectrophotometric determination of a number of compounds, particularly the keto acids. This difficulty can be circumvented by using a pH meter or a titrimeter instead of phenol red to determine the end point of the titration. Spectrophotometric methods can also be employed in place of titration, or phenolphthalein may be substituted for the phenol red indicator. If the aqueous phase is acidified after titration and the phases are thoroughly mixed prior to the separation of the two, as already described for the isolation of keto acids, the phenolphthalein is for the most part extracted into the chloroform-butanol, since in the acid form it is sparingly soluble in water. A small quantity of the organic acid is lost in the chloroformbutanol, and it is therefore necessary to determine the acid recovered in the aqueous phase by other means (see Section III). A d d i t i o n a l Separation Procedures. 1. Lactate and succinate are not lg j. W. H. Lugg and B. T. Overell, Australian J. Sci. Research A1, 98 (i948).

~oA. A. Benson, J. A. Bassham, M. Calvin, T. C. Goodale,V. A. Haas, and W. Stepka, J. Am. Chem. Soc. 72, 1710 (1950). ~1j. B. Stark, A. E. Goodban, and H. S. Owens, Anal. Chem. 23, 413 (1951). 2~F. W. Denison, Jr., and E. F. Phares, Anal. Chem. 24, 1629 (1952). ~3M. L. Buch, R. Montgomery, and W. L. Porter, Anal. Chem. 24, 489 (1952). ~4F. A. Isherwood and C. S. Hanes, Biochem. J. 55, 824 (1953). ~5F. Bryant and B. T. 'Overell, Biochim. et Biophys. Acta 10, 471 (1953). ~6A. R. Jones, E. J. Dowling, and W. J. Skraba, Anal. Chem. 25, 394 (1953). 2~R. W. Scott, Anal. Chem. 27, 367 (1955).

[24]

INTERMEDIATES OF THE TRICARBOXYLIC ACID CYCLE

595

well separated by the procedure described, since the peak effluent volumes are 340 and 360 ml., respectively. Lactate can be removed from a mixture of succinate and lactate by treatment with permanganate, 2s and the residual succinate can be rechromatographed on Celite as already described. In instances where both lactate and succinate are required, the procedure described by Phares et al. 8 can be employed to resolve the mixture into its components. The procedure is similar to the one described except that Celite-0.5 N H2SO4 constitutes the immobile phase and the column is developed with ethyl ether which has been equilibrated with 0.5 N H2SO~. With the proper selection of eluants, silicic acid may also be employed for the separation of lactic and succinic acids. 7,0,~8,29 2. Mixtures of formic and pyruvic acids are not resolved on Celite with the chloroform-butanol solutions employed. A mixture of the two acids can be resolved by partition chromatography as described by Bulen et al. 9 The pyruvic acid can also be isolated and chromatographed as the 2,4dinitrophenylhydrazine derivative (see part 4, this section). 3. Oxalacetic acid is not sufficiently stable to be chromatographed in columns prepared with Celite-0.2 N H2SO4. Only 20% is recovered (peak effluent volume approximately 450 ml.), and the remainder appears as pyruvic acid and as an increased blank titration value between the pyruvic and oxalacetic acid peaks. This difficulty can be circumvented by using Celite containing 3 N H2SOt as described by Utter. a° Frohman et al. 7 used silica gel-4.7 N H~SO4 as the immobile phase, and the chromatogram was developed with mixtures of c h l o r o f o r m - t e r t - a m y l alcohol. The general application of procedures of this type is limited because large blank titration values are obtained with the more polar solutions which makes it difficult to determine small quantities of organic acid in the eluate by titration. This is of particular significance in the separation of c i s - a c o n i t i c , malic, citric, and isocitric acids. 4. Pyruvic, oxalacetic, and a-ketoglutaric acids may be conveniently separated by chromatography in the form of their 2,4-dinitrophenylhydrazones. 31-33 III. Determination of Acids Partition chromatography is useful not only in the isolation of pure intermediates of the tricarboxylic acid cycle from biological sources but 28H. G. Wood, C. H. Werkman, A. Hemingway, and A. O. Nier, J. Biol. Chem. 169, 377 (1941). 29A. C. Neish, Can. J. Research B27, 6 (1949). 30M. F. Utter, J. Biol. Chem. 188, 847 (1951). 3i G. A. LePage, Cancer Research 10, 393 (1950). 32D. Cavallini and N. Frontali, Biochim. et Biophys. Acta 13, 439 (1954). 33A. I. Virtanen, J. K. Meittinen, and H. Kunttu, Acta Chem. Stand. 7, 38 (1953).

[24]

INTERMEDIATES OF THE TRICARBOXYLIC ACID CYCLE

597

also in the determination of these compounds. The values obtained in the determination of individual acids by titrating fractions of effluent from partition chromatograms are satisfactory for most purposes particularly when conditions are arranged in such a manner that the blank titration values are reduced to a low level. The method when properly applied is relatively specific and sufficiently sensitive that 5 to 10 microequivalents of acids can be determined quantitatively without sacrificing any of the sample. The latter is of particular importance in situations where all the experimental material is required for further isotopic analysis. The usefulness of partition chromatography in quantitating acids is evidenced by its extensive application in diverse fields and by the fact that few recent advances have been made in the development of additional methods. On the other hand, supplementary methods are frequently useful in detecting gross contamination of acids isolated by partition chromatography, in compensating for losses which may occur in the recovery of acids from column effluents prior to isotopic analysis, and in the routine analysis of large numbers of samples, e.g., during the course of preliminary experiments. It is apparent that such procedures should not only be simple and specific, but also applicable on a micro- or semimicro scale. 5~[ethods which have been employed frequently are listed in Table II. Few of these techniques satisfy all the requirements of ideal analytical procedures but are nevertheless suitable for a variety of investigations. The choice of a given method is largely dependent on factors peculiar to a given analytical problem. It is beyond the scope of this report to consider the applications and limitations of each method. For information of this type in addition to technical details, the references appended to Table II should be consulted.

IV. Degradation of IsotopicaUy Labeled Compounds Introduction The degradation of isotopically labeled compounds ordinarily represents the application of conventional techniques of organic chemistry, bt~t the small amounts of compounds involved and the special aims of isotopic experiments often necessitate modifications of the usual techniques. As noted earlier, the requirements for purity of the compounds under study are very stringent in many circumstances. In most tracer experiments it is necessary to localize the labeling within a compound by the use of specific procedures whereby the labeled carbon atoms representing a single position or group of positions within the molecule can be isolated and analyzed. The possibility of cross contamination with other

598

TECHNIQUES FOR ISOTOPE STUDIES

[24]

positions can be detected in many cases only by the degradation of compounds with known labeling. As will become apparent in the following discussion, information of this type is available for only a limited number of degradative procedures, although the limits of cross contamination in other methods can be inferred to some extent by examining data obtained from compounds whose labeling is not known with certainty. The practice of balancing the sum of the isotopic content of the individual carbons against the content of the entire compound provides a useful safeguard against many types of experimental error. Examples of this type of calculation are presented in the reports of Leaver et al. "~4 and Bernstein et al.3S The preparation of samples for degradation, the conversion of compounds to a form in which they can be counted, and degradative methods for individual acids are considered in this section.

Preparation of Samples for Degradation Before proceding with the degradation and isotopic analysis of the various compounds isolated by partition chromatography, it is advisable to establish the purity of each compound by independent means (see Separation of Acids under Section II). Once the criteria for purity are satisfied, it is usually necessary to dilute the labeled compound with a known amount of unlabeled material (carrier), since most of the following methods require from 0.25 to 5 millimoles of material with the apparatus usually available. It is apparent that the magnitude of such dilutions must be anticipated in planning experiments in order to avoid shortages of material containing sufficient isotope to be measured accurately. Such factors as the relative yields of products formed from a given substrate, dilution from endogenous sources, and the quantity of material required for a particular degradation procedure must be considered in the calculations. Much of this information can be obtained from preliminary experiments of a semiquantitative nature. On the other hand, situations arise where it becomes difficult to predict the yield or the specific activity of metabolic products obtained from a given substrate. It is therefore expedient to establish the specific activity of each compound prior to the addition of carrier to avoid excessive dilution of isotope. In the case of C 14, an aliquot of the sample may be evaporated in metal planchets and counted. This method is superior to converting the compound to CO~, s~F. W. Leaver, H. G. Wood, and R. Stjernholm, J. Bacteriol. 70, 521-530 (1955). 35I. A. Bernstein, K. Lentz, M. Malm, P. Schambye, and H. G. Wood, J. Biol. Chem. 215, 137-152 (1955).

[~4]

INTERMEDIATES

OF T H E TRICARBOXYLIC ACID CYCLE

599

since none of the material is sacrificed. The procedure is sufficiently accurate for the purpose, and the results m a y be expressed (approximately) in terms of BaCO3 by applying a correction factor for the difference (due to alterations in the geometric arrangement of the counting chamber) in counting efficiency between the two methods.

Combustion Methods Although compounds m a y be counted on occasion without conversion to C02 (see above), in most cases the compound or the fractions derived from it are converted to CO2 b y appropriate combustion techniques and

counted as CO2 or BaCO3. The two most commonly used combustion procedures employ either chromic acid ~e or persulfate. 37 The first of these is the method of choice, since it has been tested extensively and found to be effective for a wide variety of organic compounds. It has been found expedient by many investigators in these laboratories to employ double the concentration of chromic acid rather than that originally proposed. ~6 This method requires anhydrous samples, whereas aqueous solutions of pure compounds are conveniently combusted by the persulfate method. Ma~ny compounds, including all mentioned in this report, appear to be oxidized completely by persulfate on the basis of yield of CO2. On the other hand, Sakami ~7~ has found that a few compounds such as choline are not oxidized completely. In addition, Wood et al. 38 have observed that the values obtained with propionate-2-C 14 and propionate-3-C 14 by the persulfate method are lower than when chromic acid is employed, which suggests that carbons 2 and 3 are not converted quantitatively to CO2. This type of discrepancy may have been overlooked previously because it is within the error of CO2 measurements by the usual procedures. Special combustion procedures have been employed for several compounds which are intermediates in various degradative schemes; these include formic acid by mercuric ions; 39 CO by iodic sulfate; 4°,41 iodoform by hot silver nitrate; 42 and methylamine by alkaline KR'[nO4. 43 36D. D. Van Slyke and J. Folch, J. Biol. Chem. 136, 509 (1940). 37O. L. Osburn and C. H. Werkman, Ind. Eng. Chem. Anal. Ed. 4, 421 (1932). 37~W. Sakami, personal communication. 38 H. G. Wood, R. Stjernholm, and F. W. Leaver, J. Bacteriol. 70, 510 (1955). 39 N. Pirie, Biochem. J. 40, 100 (1946~. 40 M. Schutze, Ber. 77A, 484 (1944). 41 W. G. Smiley, Nuclear Sci. Abstr. 3, 391 (1949). 4~W. W. Shreeve, F. W. Leaver, and I. Siegel, J. Am. Chem. Soc. 74, 2404 (1952). 43 E. F. Phares, Arch. Biochem. and Biophys. 33, 173 (1951).

600

[24]

TECHNIQUES FOR ISOTOPE STUDIES

Degradation of Individual Acids

Succinic Acid. Succinic acid has been degraded by modifications of the Schmidt reaction by several investigators 44-4~ to yield C02 from the carboxyl carbons and ethylenediamine from the methylene carbons as indicated in equation 1. 4 3 2 1 NaN3~ 1,4 3 2 HOOC'CH2"CH2"COOH ........ 2C0~ -{- NH~CH2"CH2NHs oxidation 2,3 NHsCH2"CH2NH2 ............ * 2C02

(1) (2)

The method described by Phares and Long 44 appears to be the most satisfactory modification of this procedure in .terms of the methylene carbons. The yield of COs is approximately 70% of theoretical, and the yield of ethylenediamine obtained by vacuum distillation after treatment with azide is 40 to 45 %. The results obtained by the use of succinate-l-C ~4 and succinate-2-C 14 44 indicate that cross contamination between the carboxyl and methylene carbons is less than 1%. When succinic acid was converted to its anhydride prior to the Schmidt reaction, the yield of ethylenediamine was increased to 70% and the cross contamination virtually eliminated. The ethylenediamine was oxidized to CO~ by treatment with persulfate. 37 Succinic acid has also been degraded by a combination of biological and chemical techniques which are based on the observation of Johns 47 that Micrococcus lactilyticus (VeiUoneUa gazogenes) decarboxylates succinate quantitatively to propionate and COs. Swim 4s has utilized this observation for the degradation of succinate as follows. 43

2

1

M. lactilyticus

3

2

1,4

1,4

HOOCCH~CHsCOOH ................... * CH3CHsCOOH ~ COs

(3)

CH3CH2COOH ......... N a N ~ CH3CHsNH2 3 2 1,4 W CO2 alkaline KMn04 CH3CH~NHs ...................... ~ CH3COOH oxidation 2,3 CH3COOH ............ * 2COs

(4) (5) (6)

In order to avoid the possibility of endogenous dilution of isotope in reaction 3, it is desirable to use a minimum quantity of a highly active suspension of M. lactilyticus. To ensure highly active preparations, the culture should be grown from a young inoculum (12 hours) that has been 44 E. F. Phares and M. V. Long, J. Am. Chem. Soc. 77, 2556 (1955). 4~ M. Strassman and S. Weinhouse, J. Am. Chem. Soc. 75, 1680 (1953). 4~ H. G. Wood and F. W. Leaver, Biochim. et Biophys. Aeta 12, 207 (1953). 47 A. T. Johns, J. Gen. Microbiol. 5, 326 (1951). 4s H. E. Swim, Thesis, Western Reserve University, 1952.

[24]

INTERMEDIATES OF THE TRICARBOXYLIC ACID CYCLE

601

TABLE III DEGRADATION OF LABELED SUCCINATEa

Quantity, ~M. Reaction Succinate ~M. lactilyticus

b

Propionate -{- C02 Propionate l HNz Acetate -t- COs Succinate I M. lactilytlcus

b

Propionate ~- CO2 Propionate l I~N~ Acetate T CO~

Specific activity c.p.m./~M.

Compound

Initial

Final

Initial

Final

Succinate_l,4_C~4 c Propionate CO~ Acetate

560 ----

90.6 455 470 22.6

258 ----

--254 0.03

Acetate CO2

800 ~ 800 d

726 750

---

0.5" 248"

Succinate-2,3-C '4 ! Propionate CO2 Acetate

560 ----

81.4 470 484 19.9

Acetate CO2

600 d 600 d

462 480

1453 ----

---

--11.8 0.12

1400' 11.6'

° H . E. Swim, Thesis, Western Reserve University, 1952. b Reaction volume, 25 ml. : 4.0 ml. of 10% suspension of Micrococcus lactilyticus, 0.1 M potassium phosphate buffer, p H 6.0, 0.004% sodium sulfide, 560 micromoles of isotopic sodium succinate. Two milliliters of 3 N N a O H in center well and 10 millimoles of H~SO~ in side arm. Gas phase, helium. Temperature, 37 °. Incubation period, 3 hours. c Synthesized from acetate-l-C 1~ by Escherichia coli under anaerobic conditions in the presence of fumarate [see H. E. Swim and L. O. Krampitz, J. Bacteriol. 67, 426 (1954), for discussion]. d Quantity of propionate degraded after dilution with unlabeled compound. ° Corrected for dilution of C14-propionate with unlabeled propionate. I Synthesized from acetate-2-C ~4 as described in (c); under the conditions employed, about 1% of the C 1~ appears in the 1,4 carbons. t r a n s f e r r e d d a i l y a t l e a s t t w i c e p r i o r t o use. F r e s h l y h a r v e s t e d cell s u s p e n s i o n s s h o u l d b e u s e d . P r e p a r a t i o n s ( 1 0 % s u s p e n s i o n in 0 . 0 1 % s o d i u m sulfide) can be stored at room temperature under helium for 4 to 6 hours w i t h o u t s e r i o u s loss of a c t i v i t y . T h e a c t u a l p r o t o c o l f o r a b a c t e r i a l d e g r a d a t i o n is s h o w n in t h e l e g e n d of T a b l e I I I w h i c h a l s o i l l u s t r a t e s t h e v a l i d i t y of t h i s s e r i e s of r e a c t i o n s a s t e s t e d o n s u c c i n a t e - 2 , 3 - C 14 a n d s u c c i n a t e - l , 4 - C ~4. T h e p r o p i o n a t e

602

TECHNIQUES FOR ISOTOPE STUDIES

[24]

produced from succinate by M . lactilyticus was chromatographed on Celite and degraded further (equations 4 to 6) by a modification of the procedure described by Phares. 43 The ethylamine produced by treating with azide was oxidized by permanganate and the resulting acetic acid was isolated by chromatography and converted to C02 by treatment with chromic acid. 38 It is apparent from the results shown in Table III that the yields of propionate and C02 from the bacterial decarboxylation are equivalent and that there is essentially no dilution of any fraction, and that cross contamination is insignificant. Suspensions of M . lactilyticus also yield a small amount of acetate of endogenous origin (unlabeled). The presence of acetate makes it necessary to separate the propionate by chromatography prior to degradation in order to avoid dilution of both the carboxyl and methylene carbons in later steps of the procedure. The Hunsdiecker procedure 49 for degrading carboxylic acids has been used successfully by Dische and Rittenberg, ~° who report that a sample of succinate-2,3-C 14 yielded 76% of the theoretical C02 from the carboxyls with a contamination from the methylene carbons of less than 1%. M a l i c Acid. Malic acid can be degraded by MnO2 oxidation, followed by degradation of the resulting acetaldehyde. 43

2

1

MnO2

1,4

3

2

H O O C C H s C H O H C O O H ......... 2COs + CHaCHO K2Cr,O7 CH3CHO ............. * CH3COOH

CHsCOOH

_.Na_N,_~ CH,,NH2 3 2 ~- CO2 CH,NHs _K__MnO~COs

(7) (8)

(9) (10)

This series of reactions (equations 7 to 10) is an adaptation of the oxidation of lactate by MnOs described by Friedemann et al., 51 coupled with the degradation of acetic acid as carried out by Phares. 43 The entire procedure has been used by Wood et al. 5s in a study of malate formation from COs in the propionic acid fermentation. The results obtained by these investigators when biologically formed malate was degraded by the above procedure and by an independent determination of the 1 position with HsSO4 as described below indicates that the contamination of CO2 (from positions 1 and 4) by the 2, 3 carbons is of the order of 3%. Cross contamination might be expected if some malonic acid is produced 49 H. Hunsdiecker and C. Hunsdiecker, Ber. 75A, 291 (1942). 60 R. Dische and D. Rittenberg, J. Biol. Chem. 211, 199 (1954). 61 T. E. Friedemann, M. Gotonio, and P. A. Shaffer, J. Biol. Chem. 73, 335 (1927). 52 H. G. Wood, R. Stjernholm, and F. W. Leaver, J. Bacteriol. 72, 142 (1956).

[2~]

INTERMEDIATES OF THE TRICARBOXYLIC ACID CYCLE

603

during the initial oxidation which can then be decarboxylated to acetate in a random fashion. In this way, the 2 position may contribute to the C02. However, this difficulty is minimized by avoiding a large excess of KMnO4 and by using relatively mild conditions. 52 The 2 and 3 positions seem to be relatively free from contamination by the carboxyl carbons as might be expected, since the acetMdehyde is removed from the reaction mixture. The yields of COs and acetaldehyde are nearly theoretical and 70 to 90%, respectively. 52 The foregoing procedure has the disadvantage that it does not distinguish between the two carboxyl carbons of malic acid. These positions can be determined separately, however, by other methods. The 4 position (fl-COOH) can be obtained as CO2 by the use of a nmlate-adapted strain of L a c t o b a c i l l u s a r a b i n o s u s as reported by Korkes and Ochoa. 53 4 3

2

1

HOOCCH2CHOHCOOH

L. arabinosus 4

3

2

1

............. * CO2 + CH~CHOHCOOH

(1l)

The initial observations were made with whole cells, but Ochoa et al. 54 and Korkes et al. 55 describe the preparation of cell-free extracts and partially purified enzyme systems which can carry out the above reaction. Nossal ~6 has carried out extensive studies of the conditions of the decarboxylation reaction as carried out by whole cells. Freshly harvested cells contain fumarase in addition to malic enzyme, and, therefore, CO~ will be produced from the 1 position as well as from the 4. The fumarase can be reduced or eliminated by fractionation of the cell-free preparations or by storage of the whole cells in the cold. None of the above reports deals with this reaction as a tool in isotopic analysis. However, Utter a6 utilized intact cells (aged in the cold) of L. a r a b i n o s u s for this purpose. The 4 position of malate was found to randomize with the 1 position to the extent of about 10%, presumably because of the presence of residual fumarase activity. In addition, as with most preparations of whole cells, some dilution by endogenous materials undoubtedly occurs. The difficulties of endogenous dilution and randomization via fumarase could be obviated by the use of fractionated cell-free systems. As is frequently the case in degradations based on enzymatic procedures, the amounts of enzyme required to produce sufficient COs for counting purposes makes the method somewhat laborious. The lactate produced in the above reaction (equation l l) ~3 S. K o r k e s a n d S. Ochoa, J. Biol. Chem. 176, 463 (1948); see Vol. I I I [71]. ~4 S. Ochoa, J. B. V. Salles, a n d P. J. Ortiz, J. Biol. Chem. 187, 863 (1950) ; see Vol. I

[124]. 5~S. Korkes, A. del Carapillo, and S. Ochoa, J. Biol. Chem. 187, 892 (1950). 5s p. M. NossM, Biochem. J. 49, 407 (1951); 50, 349, 591 (1952).

604

TECHNIQUES FOR ISOTOPE STUDIES

[24]

could be isolated and degraded. T o the authors' knowledge this procedure has not been used, probably because of the serious obstacles introduced b y the endogenous production of lactate. The 1 position (a-COOH) of malate can be obtained as CO b y treatm e n t with concentrated H2S04'° and the CO converted to COs (equation 13) as described b y Schutze. 4° 43

2

1

H~SO4 1

4(?)

H O O C C H s C H O H C O O H .......... , CO + C02 iodi¢ 1 CO ........ * COs sulfate

(12) (13)

I t would appear from equation 12 t h a t the COs is derived from position 4, but in practice this fraction is contaminated b y other carbons. B y a combination of this method with the K M n 0 4 procedure described earlier (equations 7 to 10) it is possible to obtain an analysis of the individual carbons of malic acid, s~ although the 4 position must be calculated b y difference. Fumaric Acid. Fumaric acid can be converted to succinic acid b y reduction with Zn 5~ or b y hydrogenation. 5° The succinic acid can then be degraded b y one of the methods described in the section dealing with t h a t acid. Zn HOOCCH-----CHCOOH ............. ~ H O O C C H ~ C H s C 0 0 H (14) or Pd(H~) I t is significant t h a t Dische and Rittenberg s° report t h a t the Hunsdiecker m e t h o d 49 for the degradation of carboxylic acids is not effective with fumaric acid, since the yield of COs from fumaric acid-2,3-C 14 was only 25% and the COs was contaminated b y carbons 2 and 3 to the extent of about 4 %. Oxalacetic Acid. Krebs ~8 reported t h a t oxalacetic acid was decarboxylated rapidly in acid solution in the presence of a n u m b e r of metal ions of which A1+++ is one of the most effective. 43 2 1 Al+++ 4 3 2 1 H O O C C H s C O C O O H ......... * COs ~- CH~COCOOH

(15)

This method has been adapted 8° for the determination of isotopic content of the 4 position (f~-COOH) of oxalacetate. The p y r u v a t e can be decarboxylated b y ceric sulfate 59 to yield the 1 position of oxalacetate as COs. 3 2 1 Ce(S04)2 3 2 1 C H s C O C O O H ............. * CH3COOH ~- CO~ 5, H. A. Krebs, D. H. Smyth, and E. A. Evans, Biochem. J. 34, 1041 (1940). 5s H. A. Krebs, Biochem. J. $6, 303 (1942). 6~C. Fromageot and P. Desnuelle, Biochem. Z. 279, 174 (1935).

(16)

606

TECHNIQUES FOR ISOTOPE STUDIES

[24]

can be obtained as CO or C02 b y t r e a t m e n t with concentrated H2S04 according to Weinhouse et al. 65 54 3 2 1 cone. H2SO4 6 1,5 4 3 2 H O O C C H 2 C O H C H s C O O H ................ * CO -~ 2CO2 -{- CH3COCH3

(22)

I

COOH 6 The t e r t i a r y carboxyl is converted to CO which can be oxidized to CO~. 4° T h e p r i m a r y carboxyls are obtained as C02 and the remainder of the molecule as acetone, although the latter was not isolated from this particular procedure. T h e d a t a obtained b y Weinhouse et al., 6~ employing the quinidine salt of citric acid, indicate t h a t there is little contamination of the CO b y the p r i m a r y carboxyls. The C02 formed f r o m the p r i m a r y carboxyls m a y be diluted perhaps as m u c h as 6 % b y other portions of the molecule or b y impurities. Weinhouse et al. 65 obtained the noncarboxyl carbons of citrate as acetone b y t r e a t m e n t with dilute dichromate and isolation of the acetone as the Deniges complex in a b o u t 70% yield. 54 3 2 1 K2Cr207 4 3 2 H O O C C H 2 C O H C H ~ C O O H ............. * 3C02 ~ CH3COCH3

L

(23)

COOH 6

The acetone is free of c o n t a m i n a t i o n f r o m the p r i m a r y carboxyl carbons. Acetone can be further degraded b y the iodoform reaction. 66 4 3 2 NaOI 4,2 4,2 3 CH3COCH3 ......... * CH3I -~ C H 3 C O O H

(24)

T h e iodoform and acetate can be degraded as described in preceding sections. T h e a b o v e methods h a v e the d i s a d v a n t a g e t h a t t h e y do not distinguish between the 2,4 and the 1,5 positions of citrate which h a v e been shown to be stereospecific with respect to synthesis and breakdown in biological systems. Several investigators h a v e carried out partial 67,~8 or complete degradations 69 of citrate in which the stereospecificity of the a b o v e positions is maintained. All these methods necessarily require a biological component, usually pigeon breast muscle or pigeon liver s5 S. Weinhouse, G. Medes, and N. F. Floyd, J. Biol. Chem. 166, 691 (1946). 60L. F. Goodwin, J. Am. Chem. Soc. 42, 39 (1920). s7 V. R. Potter and C. Heidelberger, Nature 164, 180 (1949). 68V. Lorber, M. F. Utter, H. Rudney, and M. Cook, J. Biol. Chem. 18[i, 689 (1950). s9 E. H. Mosbach, E. F. Phares, and S. F. Carson, Arch. Biochem. $3, 179 (1951).

[9.4]

INTERMEDIATES OF THE TRICARBOXYLIC ACID CYCLE

607

extracts, for the initial conversion of citric acid to a-ketoglutaric acid. 54 3 2 1 pigeon tissue H O O C C H ~ C O H C H 2 C O O H ...............

I

COOH 6

54 3 2 1 6 HOOCCH2CH2COCOOH + CO2

(25)

The resulting a-ketoglutaric acid can be degraded stepwise as shown in equations 26 to 30 below, thereby yielding a complete analysis of the citric acid molecule. Isocitric and cis-Aconitic Acids. Isocitric and cis-aconitic acids can be degraded by the stereospecific procedure described above for citric acid to yield CO2 and a-ketoglutaric acid and the latter compound degraded as shown below. (For preparation of isocitric dehydrogenases, see Vol. I [116].) a-Ketoglutaric Acid. Mosbach et al. ~9 have described a complete and apparently specific method for the degradation of a-ketoglutaric acid. 54 3 2 1 NH~ • 54 3 2 l HOOCCH2CH2COCOOH .......... H O O C C H 2 C H 2 C H N H 2 C O O H Pd(H~) chloramine T H O O C C H 2 C H 2 C H N H 2 C O O H ............... 1 H O O C C H 2 C H : C H O + CO2 NH2NH~ HOOCCH2CH2CHO ................... ~ HOOCCH2CH2CH~ (Wolf-Kishner) NaN3 5 HOOCCH2CH2CH3 ............ • CO2 + NH2CH~CH2CH3 (Schmidt) KMnO4 43 2 NH~CH2CH2CH3 ......... ~ HOOCCH~CH2

(26)

(27) (28) (29) (30)

The a-ketoglutarate is converted to glutamate (60 to 80% yield) by reductive amination. The glutamate is then converted to succinic semialdehyde by chloramine T, thereby yielding the 1 position of a-ketoglutaric acid as CO2. The succinic semialdehyde is not isolated but is reduced to butyric acid by a modified Wolf-Kishner reaction. The overall yield of these two steps (glutamate to butyrate) is 45 to 55%. The b u t y r a t e is treated by the Schmidt procedure yielding CO2 from the original 5 position of the a-ketoglutarate with propylamine as the other product. The latter is oxidized by KMnO4 to propionic acid which can be further degraded as described earlier (equations 4, 5, 9, 10). Mosbach et al. 69 tested this procedure on a-ketoglutaric acid-l,2-C :4 and found negligible dilution in any of the fractions and no cross contamination greater than 1 to 2 %.

608

TECHNIQUES FOR ISOTOPE STUDIES

[24]

a-Ketoglutaric acid can be oxidized with acid p e r m a n g a n a t e to C02 and succinic acid as shown below. 7° 54 3 2 1 acid KMn04 H O O C C H ~ C H ~ C O C O O H ................ 54 3 2 1 H O O C C H ~ C H ~ C O O H + CO~

(31)

The succinic acid can be degraded by one of the methods described in TABLE IV SYNTHESIS OF TRICARBOXYLIC ACID INTERMEDIATES

Compound

Reference

CH..CO-COOH

Anker •

CHa.CHOH-C00H

Cromer and Kistiakowsky ~

H00C.CHrC0.C00H

Heidelberger and Hurlbert ~

*

H00C.CHrCHOH'C00H

Jorgensen et al. ~

HOOC.CH=CH-COOH

San Pietro •

H00C'CH~'CHrC00H

Jorgensen et al. d

HOOC-CH2.CH2.COOH

Jorgensen et al. d

H00C-CH2-CHrC0.C00H

Kogl et a l /

H00C-CHrC(0H).CHrC00H

Rothchild and Fieldsg

~00H H00C.CHrC(0H)'CHrC00H

Wilcox et al. ~

~00H H. S. Anker, J. Biol. Chem. 176, 1333 (1948). b R. D. Cramer and G. B. Kistiakowsky, J. Biol. Chem. 137, 599 (1941). C. Heidelberger and R. B. Hurlbert, J. Am. Chem. Soc. 72, 4704 (1950). a j. A. Jorgenson, J. A. Bassham, M. Calvin, and B. M. Tolbert, J. Am. Chem. Sac. 74, 2418 (1952). • A. San Pietro, J. Biol. Chem. 198, 639 (1952). / F. Kogl, J. Halberstadt, and T. J. Barendregt, Rac. tray. ehim. 68, 387 (1949). g S. Rothchild and M. Fields, J. Am. Chem. Sac. 74, 2401 (1952). i p. E. Wilcox, C. Heidelberger, and V. R. Potter, J. Am. Chwn. ,goc. 72, 5019 (1950). the section dealing with t h a t compound. This m e t h o d has the obvious disadvantage t h a t it does not distinguish between the 5,2 and 4,3 carbons. The same method has also been used with the semicarbazide derivaT0H. G. Wood, C. H. Werkman, A. Hemingway, and A. O. Nier, J. Biol. Chem. 142, 31 (1942).

[9.5]

GLYCOLIC, GLYOXYLIC, A N D OXALIC ACIDS

609

rive of a-ketoglutaric acid by Buchanan et al. ~1 In the reaction, two moles of C02 are formed: one from position 1 of the a-ketoglutaric acid and one from the semicarbazide portion of the molecule. The 2,4-dinitrophenylhydrazone derivative of a-ketoglutarate has also been degraded by a modification of the above method. ~2

V. Synthesis of Labeled Substrates Chemical procedures for the preparation of most of the acids connected with the tricarboxylic acid cycle have been adapted for use with isotopes, and some of these compounds are available commercially in labeled form. For convenience, some of the procedures have been summarized in Table IV. Each compound can be labeled in several different positions or with combinations of C 14 and C is, but the compilation given merely lists procedures for one or two types of labeling with each compound although many of these procedures can be adapted to yield other types of labeling if so desired. Needless to say, the strictures concerning purity of compounds apply to substrates for isotopic experiments and appropriate procedures for establishing their purity must be used in the same way as in other phases of isotopic experimentation. ~1j. M. Buchanan, W. Sakami, S. Gurin, and D. W. Wilson, J. Biol. Chem. 159, 695

(1945). 7~H. J. Saz, Thesis, Western Reserve University, 1952.

[25] Synthesis and Degradation of IsotopicaUy Labeled Glycolic, Glyoxylic, and Oxalic Acids B y I~ATHARINE F. LEWIS and SIDNEY WEINHOUSE

Degradation Procedures Glycolic Acid Glycolic acid can be degraded to carbon dioxide and formic acid by several oxidizing agents, such as hydrogen peroxide, 1 cerie sulfate, ~ and the enzyme, glycolic acid oxidase 3 (equation 1). HOCH2COOH + 20--~ HCOOH + C02 + H20 1 H. A. Spoehr, Am. Chem. J. 43, 227 (1910). H. H. Willard and P. Young, J. Am. Chem. Soc. 52, 132 (1930). I. Zelitch and S. Ochoa, J. Biol. Chem. 201, 707 (1953).

(1)

[9.5]

GLYCOLIC, GLYOXYLIC, A N D OXALIC ACIDS

609

rive of a-ketoglutaric acid by Buchanan et al. ~1 In the reaction, two moles of C02 are formed: one from position 1 of the a-ketoglutaric acid and one from the semicarbazide portion of the molecule. The 2,4-dinitrophenylhydrazone derivative of a-ketoglutarate has also been degraded by a modification of the above method. ~2

V. Synthesis of Labeled Substrates Chemical procedures for the preparation of most of the acids connected with the tricarboxylic acid cycle have been adapted for use with isotopes, and some of these compounds are available commercially in labeled form. For convenience, some of the procedures have been summarized in Table IV. Each compound can be labeled in several different positions or with combinations of C 14 and C is, but the compilation given merely lists procedures for one or two types of labeling with each compound although many of these procedures can be adapted to yield other types of labeling if so desired. Needless to say, the strictures concerning purity of compounds apply to substrates for isotopic experiments and appropriate procedures for establishing their purity must be used in the same way as in other phases of isotopic experimentation. ~1j. M. Buchanan, W. Sakami, S. Gurin, and D. W. Wilson, J. Biol. Chem. 159, 695

(1945). 7~H. J. Saz, Thesis, Western Reserve University, 1952.

[25] Synthesis and Degradation of IsotopicaUy Labeled Glycolic, Glyoxylic, and Oxalic Acids B y I~ATHARINE F. LEWIS and SIDNEY WEINHOUSE

Degradation Procedures Glycolic Acid Glycolic acid can be degraded to carbon dioxide and formic acid by several oxidizing agents, such as hydrogen peroxide, 1 cerie sulfate, ~ and the enzyme, glycolic acid oxidase 3 (equation 1). HOCH2COOH + 20--~ HCOOH + C02 + H20 1 H. A. Spoehr, Am. Chem. J. 43, 227 (1910). H. H. Willard and P. Young, J. Am. Chem. Soc. 52, 132 (1930). I. Zelitch and S. Ochoa, J. Biol. Chem. 201, 707 (1953).

(1)

610

TECHNIQUES FOR ISOTOPE STUDIES

[25]

In the method of Spoehr, 1 which utilizes hydrogen peroxide in the presence of ferric ions, it is difficult to establish conditions in which stoichiometric amounts of products are produced. Consequently, the carbon dioxide, which should represent only the carboxyl carbon, is contaminated somewhat with methylene carbon. However, the formic acid arises solely from the methylene carbon. The oxidation of glycolic acid with ceric sulfate gives quantitative, isotopically pure yields of carbon dioxide and formic acid if the conditions of acidity, heating, temperature, time, and concentration of reactants established by Willard and Young ~ are employed. The following procedure was found to give stoichiometric amounts of carbon dioxide and formic acid. The apparatus consists of a 300-ml. three-necked roundbottomed flask through one neck of which there is inserted an inlet tube reaching to the bottom of the flask. This tube is attached to a drying tube filled with ascarite and a bubble counter containing 1 M sodium hydroxide. The center neck of the flask is attached to a reflux condenser to the top of which is connected an absorbing system. This is a 125-ml. Erlenmeyer flask closed with a two-holed rubber stopper; one hole carries a tube leading to the top of the condenser. The other carries a tower 25 cm. long and approximately 2 cm. in diameter filled through about 20 cm. of its length with solid glass beads of 3-ram. diameter. A 10-mm.-diameter extension of the tower leads to the bottom of the flask. The beads are held in place by a plug of glass wool. The third neck of the reaction flask is fitted with a dropping funnel equipped with a stopcock. Ten milliliters of COs-free 0.5 M sodium hydroxide solution is placed in the Erlenmeyer flask, and sufficient vacuum is applied at the top of the bead tower to draw one bubble of air per second through the apparatus. The sodium hydroxide is thereby drawn up into the bead tower where the effluent gases make contact at the surface of the beads. One millimole of glycolic acid in 65 mh of C02-free water, 32 ml. of 60% sulfuric acid (specific gravity 1.5), and 6.3 ml. of 0.1 N ceric sulfate are added successively to the reaction vessel through the dropping funnel. The reaction vessel is heated with a free flame until the contents boil, then are kept just below the boiling point (90 to 95 °) for 1 hour, and finally cooled to 50 °. The vacuum is broken,:allowing the absorbed COs to drain into the flask, the tower is washed down from the top with three successive 20-ml. portions of freshly boiled water, and the COs is precipitated as barium carbonate by the addition of 5 ml. of 1 M barium chloride solution. The contents of the reaction vessel are submitted to distillation with steam until no more volatile acid distills. The distillate is neutralized with standardized 0.1 N sodium hydroxide and evaporated to a volume of 20 ml. The solution is then quantitatively transferred with water to

[25]

GLYCOLIC, GLYOXYLIC, AND OXALIC ACIDS

(~l 1

another three-necked 125-ml. flask which is part of an apparatus identical with that described above. While CO2-free air is drawn through the flask by application of suction at the top of the bead tower, 2 ml. of 50% sulfuric acid is added to the flask and aeration is continued for 10 minutes to ensure removal of CO2. Ten milliliters of 0.5 M sodium hydroxide is then added to an Erlenmeyer flask, which is attached to the bead tower, and 10 ml. of a 10% mercuric sulfate solution is added to the formate solution through the dropping funnel. The mixture is heated to boiling, and air is drawn through the flask and bead tower for 30 minutes while the solution is refluxing. The bead tower is then washed down with CO2-free water, and the carbonate precipitated by addition of 5 ml. of 20% barium chloride solution. 4

Glyoxylic Acid Glyoxylic acid, in dilute aqueous solutions, can be oxidized quantitatively to formic acid and carbon dioxide by hydrogen peroxide. 5 However, since glyoxylic acid is most conveniently isolated from biological material by conversion to one of its carbonyl derivatives, a method utilizing one of these is preferable. The following procedure has been successfully employed 6 to determine the distribution of radioactivity in the carbons of glyoxylic acid isolated as the 2,4-dinitrophenylhydrazone from metabolism experiments. The glyoxylic acid portion of the hydrazone molecule is reduced to acetic acid, which is degraded to methylamine and carbon dioxide. The method of reduction is the Huang-Minlon micromodification of the Wolff-Kishner reaction. 7 The 2,4-dinitrophenylhydrazone (approximately 0.5 millimole) is transferred to a 200-ml. flask containing 1 g. of KOH pellets, 5 ml. of 85% hydrazine solution, and 25 ml. of diethylene glycol (redistilled). The solution is refluxed for 1 hour, then water is distilled off until the temperature of the vapors reach 180 to 190°. Refluxing is continued at this temperature for 1 hour. After cooling, the reaction mixture is acidified with dilute sulfuric acid and steam-distilled. Twenty-five-milliliter fractions are collected and titrated with base until no more volatile acids are recovered. About 90% of the hydrazone is recovered as acetic acid. The neutralized solution is evaporated to dryness on a steam bath. The recovered sodium acetate is degraded to methylamine and CO2 by the Phares s modification of the Schmidt reaction. The apparatus con4S. Weinhouse and B. Friedmann, J. Biol. Chem. 19"/, 733 (1952). 5 W. H. Hatcher and G. W. Holden, Trans. Roy. Soc. Can. Sec. I I I [3] 19, 11 (1925), H. I. Nakada, Thesis, Temple University, 1953. 7 Huang-Minlon (M. Huang), J. Ant. Chem. Soc. 68, 2487 (1946). s E. F. Phares, Arch. Biochem. and Biophys. 33, 173 (1951).

612

TECHNIQUES FOR ISOTOPE STUDIES

[25]

sists of a conical 50-ml. reaction flask equipped with an inlet tube leading to the bottom, and a condenser, the top of which is connected to two bead towers in series, the first containing 0.27 M KMn04 in 1 N H2S04 (to remove SO2 which is formed during the reaction by the reduction of H~S04 by hydrazoic acid) and the second to trap the evolved COs arising by decarboxylation of the acetic acid. The dry sodium acetate is placed in the flask, which is cooled in an ice bath, and 0.3 ml. of 100% H~S04 is carefully added. The salt is dissolved by warming and shaking, and 50 mg. of sodium azide is added to the cooled flask, which is then connected to the apparatus and slowly heated in a water bath to 60 to 70 °. After 1 hour at this temperature, the system is swept with a slow stream of air for 20 minutes. The contents of the bead tower containing the alkali is collected and the trapped COs precipitated as BaCO3. The methylamine is recovered from the residual solution by transferring the reaction flask together with the inlet tube to an aeration apparatus. This consists of an air condenser, bead tower, and 125-ml. Erlenmeyer flask of the same design as the oxidation apparatus described on page 610. Three milliliters of water is added to the reaction mixture, 5 ml. of 0.2 N H2S04 is added to the Erlenmeyer flask, and vacuum is applied to the top of the bead tower. The methylamine is liberated by the slow addition of 5 ml. of 4 N sodium hydroxide through the inlet tube. The flask is then heated in a boiling water bath for 30 minutes while air is drawn through the flask and into the sulfuric acid solution in the bead tower. The contents of the bead tower are washed into the Erlenmeyer flask, the pH is adjusted to 5.0 by addition of approximately 1.2 ml. of 1.0 N NaOH, and the methylamine is oxidized to COs by the persulfate wet combustion procedure) The yield of methylamine varies from 50 to 75%.

Detection of Glycolic~ Glyoxylic, and Oxalic Acids by Paper Chromatography The solvent formula developed by Jeanes et al. l° for the separation of mono- and disaccharides can be used for the detection of the three twocarbon acids by paper chromatography. The material is spotted on filter paper and irrigated overnight in a solvent mixture consisting of 3.0 vol. of butanol, 2.0 vol. of pyridine, and 1.5 vol. of water. After drying, the paper is sprayed with a 0.05% solution of bromophenol blue. Organic acids appear as yellow spots on a blue background. The Rj values are: 9 M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwich, "Isotopic Carbon," p. 94. John Wiley & Sons, New York, 1949. ~0A. Jeanes, C. S. Wise, and R. J. Dimler, Anal. Chem. 23, 415 (1951).

[25]

GLYCOLIC, GLYOXYLIC, AND OXALIC ACIDS

613

oxalic acid, 0.13; glycolic acid, 0.37; glyoxylic acid, 0.39. Though the RI values of glycolic and glyoxylic acids are quite close, this difficulty can be overcome by developing a second spot of the mixture on the paper and spraying this with a 0.2 % solution of o-phenylenediamine in ethyl alcohol containing 1% nitric acid. 11 On heating to 70 ° for 30 minutes, glyoxylic acid appears as a characteristic yellow spot on a steel-gray background. This color reaction is specific for carbonyl compounds.

Synthesis of Radioactive Compounds Oxalic Acid C14-Labeled sodium oxalate is conveniently prepared in essentially quantitative yield by heating dry sodium formate at 440 ° for 15 minutes. This is accomplished in the authors' laboratory by placing a test tube containing the dry salt in a vertically set-up tube furnace thermostated at the desired temperature.

Glycolic Acid Glycolic acid labeled in either carbon atom may be prepared from the correspondingly labeled glycine according to the method of N a k a d a ) Fifty microcuries of labeled glycine is washed into a beaker with sufficient carrier glycine to give a total of 6.64 mM. The calculated amount of barium nitrite (821 mg. of Ba(NO2)~.H~O) is added, and the volume is brought to 50 ml. With mechanical stirring, 10 ml. of 0.66 N H2SO4 is added over a period of 1 hour. The mixture is allowed to stand overnight, the barium sulfate is removed by centrifugation, and the clear supernatant solution is acidified with HC1 and extracted continuously with ether for 24 hours. Then 75 mg. of carrier glycolic acid is added to the aqueous layer, and extraction is continued for an additional 24 hours. The ether solution is evaporated by means of a jet of dry air until fumes of HC1 are no longer noticeable; the straw-colored sirup is transferred to a beaker with the aid of acetone and a wash solution of 125 mg. of glycolic acid in ether. The combined sirup and washings are evaporated to a small volume and allowed to crystallize. Three additional crystallizations from ether gives colorless crystals that melt sharply at 77 ° (recorded for glycolic acid, 78 to 79°). Approximately 30% of the initial radioactivity is recovered in pure glycolic acid. Singly labeled glycolates may also be prepared from the corresponding acetates according to the method of Hughes et al. TM The details of the chemical procedure are available in Document 3567 from the American 11 H. J. Koepsell, F. I-I. Stodola, and E. S. Sharpe, J. Am. Chem. Soc. 74, 5142 (1952). 12 D. M. Hughes, R. Ostwald, and B. M. Tolbert, J. Am. Chem. Soc. 74, 2434 (1952].

614

TECHNIQUES FOR ISOTOPE STUDIES

[26]

Document Institute (1719 N. Street, N.W., Washington, D.C.). The yield of calcium glycolate is 60 to 65%, starting with 20 millimoles of sodium acetate.

Glyoxylic Acid Doubly labeled glyoxylic acid can be prepared from labeled oxalic acid by the method previously described (Vol. III [49]). Sodium glyoxylate-2-C 14 may also be prepared from tartaric acid-2,3-C 14 according to the method of Weissbach and Sprinson. 13 In a typical run described by these authors 0.5 g. (3.3 millimoles) of tartaric acid in 10 ml. of water is treated with 7.8 ml. of 0.42 N sodium periodate for 10 minutes. The solution is extracted continuously with alcohol-free ether for 24 hours, and the ether solution (about 30 ml.) is extracted successively with a solution of 340 rag. (4 millimoles) of NaHCOa in 20 ml. of water and with 10 ml. of water. The combined aqueous extracts are neutralized carefully with NaHC03 to pH 7.0, concentrated i n vacuo to 1 to 2 ml., and treated with absolute ethanol until precipitation occurs. After storage overnight in the refrigerator, 442 rag. of sodium glyoxylate is obtained by centrifugation and washing with alcohol. An additional 158 mg. of sodium glyoxylate is obtained by concentration of the supernatant and washings; the total yield is 80 %.

Isolation by Column Chromatography Oxalic acid can be isolated in pure form by precipitation with calcium ions, after ether extraction to remove interfering inorganic anions. The first calcium oxalate precipitate may contain impurities, but these can usually be removed by subsequent reprecipitations. Oxalic and glycolic acids in amounts from 0.1 to 1.0 millimole can be separated from other earboxylie acids by the chromatographic procedure of Phares et al. ~4 These authors obtained satisfactory isolations and separations of a number of acids of biological importance by using Celite 545 as support for the immobile phase, and two solvent systems: butanol in chloroform-0.5 N sulfuric acid; and ethyl ether-0.5 N sulfuric acid. The choice of solvent system is dependent on the composition of the material to be separated, but, generally, pairs of acids which are not resolved by one system can be separated by rechromatographing with the other. Oxalic and glycolic acids are not separated by the chloroformbutanol system but are completely resolved in the ether system. la A. Weissbach and D. B. Sprinson, J. Biol. Chem. 205~ 1023 (1953). 14 E. F. Phares, E. tI. Mosbach, F. W. Denison, Jr., and S. F. Carson, Anal. Chem. 24, 660 (1952).

[2~]

PURINES AND PYRIMIDINES

[26] Purines and

615

Pyrimidines

B y EDWARD D. KORN

Synthesis For m a n y biochemical studies the use of uniformly labeled purine or pyrimidine compounds is satisfactory. These are most readily prepared, especially in the case of the nucleosides and nucleotides, by growing yeast on a medium containing labeled bicarbonate or ammonium salts. The R N A and D N A from this yeast m a y then be isolated and h y d r o l y z e d to the nucleotides or nucleosides, whichever m a y be desired. M a n y studies, however, require the use of purines and pyrimidines selectively labeled in specific positions. For these, it is usually best to resort to chemical syntheses. ~

Synthesis

of Adenine, Guanine, and 2,6-Diaminopurine (Fig. 1)

Adenine, guanine, and 2,6-diaminopurine m a y all be synthesized by the same general procedure. 2-5 This method is most applicable to the synthesis of 1,3-N 1~- and 8-C14-purines, although it may also be used to introduce labeled carbon into the 2 or into the 4 and 6 positions. An alternate procedure for the synthesis of 2-CI4-adenine is described later under Synthesis of Hypoxanthine. Formamidine Hydrochloride (I). Generate H C N from 10 g. of N a C N with acid, and bubble it into a flask containing 30 ml. of anhydrous ether and 10 ml. of absolute ethanol maintained at a temperature of - 1 0 to - 2 0 °. Then bubble a stream of anhydrou s HC1 through the solution until 10 minutes after the appearance of turbidity (about 30 minutes altogether). Maintain continual stirring an¢l anhydrous conditions throughout this reaction. Allow the mixture to warm gradually to 10 ° overnight, then cool it to - 2 0 °, remove the solvent, wash the residue with ether, and air-dry the crystalline product. The yield of ethyl formimino ether hydrochloride is 90 to 100%. Place 17 g. of this material in a bomb tube with 35 ml. of absolute ethanol, and cool the tube in a dry ice-ethanol bath. Then generate NH3 from 8.8 g. of NH4NO.~ by the addition of KOH, dry it over NaOH, and 1For the synthesis of nueleosides and nucleotides, see Vol. III [113]. 2L. F. Cavalieri, J. F. Tinker, and A. Bendich, J. Am. Chem. Soc. 71, 533 (1949). 3 V. M. Clark and H. M. Kalckar, J. Chem. Soc. 1950, 1029. 4M. F. Mallette, E. C. Taylor, Jr., and C. K. Cain, J. Am. Chem. Soc. 69, 1814 (1947). 5 A. Bendich, S. S. Furst, and G. B. Brown, J. Biol. Chem. 185, 423 (1950).

616

[26]

TECHNIQUES FOR ISOTOPE STUDIES

I

II 1

z--c~

II

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+

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[26]

PURINES AND PYRIMIDINES

617

bubble it into the solution. Seal the tube, heat it to 100° with occasional shaking, cool, and aerate the contents at 50 to 60° for 30 minutes to remove any excess NH3 which might be present. If a precipitate of NH4C1 has formed, filter the hot mixture and then evaporate the filtrate to dryness in vacuo over P205 and H~SO4. To synthesize 2-C14-adenine, use NaC~4N in the above procedure; for 1,3-N ~5-adenine, use N ~SH4NO3. Guanidine Nitrate ( V I I ) . Slowly add a solution of 10.5 g. of KCN in 48 ml. of water to 6.4 ml. of bromine in 2 ml. of water, maintaining the temperature below 30 °. Distill the reaction mixture at 58 to 68 °, collect the distillate (cyanogen bromide), and dry it over P20~. The yield is about 50%. Generate NHs from 12.4 g. of NH4NO3 in 30 ml. of water, dry it over NaOH, and pass it into 65 ml. of ethanol cooled in a dry ice-ethanol bath. Add 9 g. of the cyanogen bromide dissolved in 50 ml. of ether, stopper the flask, and leave the mixture at room temperature overnight. Remove any excess NH3 by aeration and NH4Br b y filtration, and evaporate the filtrate to dryness in vacuo. The yield of cyanamide is approximately 85 %. Dissolve 1.65 g. of the cyanamide in 10 ml. of a solution containing 4.72 g. of NH4NO3, and heat the resulting mixture in a bomb tube at 155 ° for 3 hours. Chill the tube, and collect the guanidine nitrate. The yield is about 50%. As in the synthesis of formamidine, both C 14 and N ~5 may be introduced into the guanidine for the synthesis of 2-C 14- and 2-amino-l,3-N 1.~_ guanine, or 2,6-diaminopurine. N~5-Guanidine may also be readily synthesized by reacting N15H4NO3 with dicyandiamide. 6 Ethylcyanoacetate ( V I I I ) and Malononitrile (II). Add 740 mg. of NaCN to 5 ml. of 3.2 M sodium chloracetate, and boil the mixture gently for 5 minutes. After the mixture cools, add 2 ml. of concentrated HC1, and evaporate the resulting solution to dryness at 60 to 70 ° i~ vacuo. Repeatedly extract the sodium cyanoacetate from this residue with about twenty 5-ml. portions of ether. For the synthesis of ethylcyanoacetate/ add a slight excess of HC1 to the ethereal solution of sodium cyanoacetate, and remove the precipitated NaC1. Distill off the ether, wash the residue with ethanol, distill off the excess ethanol at 50 to 60°, and boil the residue under reflux for 3 hours in a mixture of 5 ml. of absolute ethanol and 1 ml. of concentrated H2SO4. Remove the ethanol and water by distillation in vacuo, 6 T. L. Davis, in "Organic Syntheses" (Gilman and Blatt, eds.), Coll. Vol. I, p. 302. John Wiley & Sons, New York, 1941. 7 j. K. H. Inglis, in "Organic Syntheses" (Gilman and Blatt, eds.), Coll. Vol. I, p. 254. John Wiley & Sons, New York, 1951.

618

TECHNIQUES FOR ISOTOPE STUDIES

[26]

and neutralize the H2SO4 with Na2CO~. Extract the ester with ether, and distill off the ether in vacuo. The ethyl cyanoacetate is in the distillate obtained at 94 to 99 ° (16 mm.). The yield is about 75%. Ethyl cyanoacetate is used for the synthesis of guanine. The ethereal solution of sodium cyanoacetate, the preparation of which was described above, may also be used for the synthesis of malononitrile. ~ Add the sodium cyanoacetate to an ethereal solution containing 2 g. of diazomethane. Slowly evaporate the solution at room temperature, treat the residual oil (methyl cyanoacetate) with 2 ml. of concentrated NH4OH, and warm the mixture gently. Add an additional 2 ml. of concentrated NH4OH, and leave the solution at room temperature overnight. Then evaporate it to dryness over P205. The product is cyanoacetamide. Place 400 rag. of the cyanoacctamide in a sublimation apparatus with 400 mg. of PC15, evacuate the apparatus, and place it in a boiling water bath. After the reaction has subsided, cool the cold finger to - 5 °, and heat the reaction mixture to 160 ° for 10 minutes. Collect the malononitrile in the cold finger. The yield is about 30 %. Malononitrile is used for the synthesis of adenine and 2,6-diaminopurine. To prepare 4,6-C14-adenine, 4,6-C~4-2,6-diaminopurine, and 4-C TMguanine, use NaC~4N in the preparation of sodium cyanoacetate. This is not a particularly efiicient synthesis, however, since the isotope is introduced at an early stage of the reaction sequence. ~,6-Diaminopyrimidine (III). Add 8 g. of malononitrile dissolved in 30 ml. of dry n-butanol to about 8 g. of formamidine hydrochloride. Then add 125 ml. of sodium butoxide (1 M sodium butoxide in anhydrous n-butanol) in several portions with continual stirring, and boil the mixture gently for 4 hours. Cool the mixture in ice, collect the precipitate, wash it with water and then with ethanol, and dry it at 110 °. The yield is 70 to 80 %. $,5,6-Triaminopyrimidine Bisulfile (IV). Prepare a homogeneous suspension of 5 g. of III in 15 ml. of water. Add glacial acetic acid until the mixture is neutral to litmus, and then an additional 3 ml. of glacial acetic acid to dissolve the material completely. Then add a solution of 3.4 g. of NAN02 in 8 ml. of water. Stir the solution vigorously during this addition. Quickly collect the bright red precipitate which forms, wash it with water, and suspend the solid in 30 ml. of water. Heat this mixture to 60 to 70 °, stirring continually, and add solid sodium hydrosulfite until the red color completely disappears. Then heat the mixture to boiling, filter it, cool the filtrate, and collect the precipitate. The yield is 50 to 60%. ~,6-Diamino-5-formamidopyrimidine Hydrochloride (V). Dissolve 400 mg. of sodium formate in 4.5 ml. of water, add 1.2 ml. of 5 N HC1, and

[26]

PURINES AND PYRIMIDINES

619

then add 1 g. of IV. Let the solution stand at room temperature for 2 hours, then cool it in ice and collect the crystals which form. The yield is about 70%. To synthesize 8-C14-adenine, use C14-formate in this step. Adenine Hydrochloride (VI). Add 117 mg. of V to a tube containing 0.75 ml. of freshly distilled 4-formylmorpholine, and heat the mixture at 200 ° for 80 minutes. Cool the tube, and leave it at 0 ° overnight. Collect the precipitate, wash it with ethanol, and recrystallize it from 2 N HC1. Dry the product at 70 °. The yield is about 60%. The formyl group of V exchanges with the formylmorpholine to a significant extent. This results in about a 20% reduction in the specific activity of the adenine when isotope is introduced into position 8. This can be avoided by using diethanolamine as the solvent instead of formylmorpholineJ~ Except for the differences in the initial reactants, as indicated in Fig. 1, guanine and 2,6-diaminopurine may be synthesized by this same procedure.

Synthesis of 4-Amino-5-imidazolecarboxamide (Fig. 2) The following procedure has been described by Shaw and Woolley. 8 It is most applicable to the synthesis of the 2-C~4-carboxamide. The original procedure has been modified slightly at the point of introduction of the formyl group in order to adapt the method to the synthesis of labeled material2 Imino Ether of Ethyl Cyanoacetate (I). ~° Place 11.3 g. of ethyl cyanoacetate, 7 ml. of absolute ethanol, and 7 ml. of anhydrous ether into a flask cooled in an ice-salt bath. Bubble dry HC1 through the solution until a weight increase of 4.1 g. is reached. Then stopper the flask, and leave it for 24 hours in the cooling mixture. Collect the product, and wash it with anhydrous ether. The yield is about 80%. Labeled ethyl eyanoacetate (see above) may be used at this point to prepare 4-C14-carboxamide. Malonamamidine Hydrochloride (II). Suspend 3 g. of I in 30 ml. of absolute ethanol, which has been previously saturated with NH3, and bubble NH~ through the mixture for 4 hours at room temperature with occasional shaking. Leave the flask at room temperature for about 5 days, and then collect the precipitate. The yield is 75 %. Phenyldiazomalonamamidine Hydrochloride (III). Dissolve 1 ml. of aniline in 6.5 ml. of 6 N HC1, and add 0.85 g. of NaNO2 in 5 ml. of water, maintaining the temperature below 5°. Pour this solution of phenyldiazochloride into a solution of 1.5 g. of II dissolved in 7.5 ml. of water, and 7~ R. A b r a h m s a n d L. Clark, J. Am. Chem. Soc. 73, 4609 (1951). 8 E. Shaw a n d D. W. Woolley, J. Biol. Chem. 181, 89 (1949). 9 I. Lin, personal communication. ~0 S. A. Glickman a n d A. C. Cope, J. Am. Chem. Soc. 67, 1017 (1945).

620

TECHNIQUES FOR ISOTOPE STUDIES

q~

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[26]

[26]

PURINES AND PYRIMIDINES

621

adiust the mixture to pH 4 with concentrated sodium acetate. Leave this mixture at room temperature for several hours and then at 0 ° overnight. Collect the yellow needles, and dry them in vacuo. The yield is 90%. Formamidomalonamamidine Hydrochloride (IV). Add 300 mg. of III, 180 mg. of zinc dust, and 0.7 ml. of anhydrous formic acid to a small centrifuge tube, and heat the mixture at 60 to 65 ° for 15 minutes with continual stirring. Add an additional 30 mg. of zinc dust every 3 or 4 minutes and 0.1 to 0.2 ml. of formic acid if the mixture becomes dry. Chill the tube, centrifuge down the precipitate, and wash it well with water. Evaporate the combined supernatant fluids to dryness at 40 to 50°, and take the residue up in 20 ml. of water. Bubble H~S through the solution to remove any zinc ions which might be present, and reconcentrate the combined supernatant fluid and water washes of the zinc sulfide precipitate. Add 2 mh of absolute ethanol to the oily residue, and evaporate it to dryness. Repeat this procedure twice. Take the resulting yellow oil up into 10 ml. of absolute methanol, chill the solution, acidify it with methanolic HC1, and add 60 ml. of cold anhydrous ether to precipitate the product. Collect the precipitate, and dry it at room temperature. It may be recrystallized from about 10 ml. of 85% ethanol by the careful addition of ether. The yield is 65 %. 4-Amino-5-imidazolecarboxamide Hydrochloride (V). Slowly heat IV to 125 to 130°, and maintain it at that temperature for 1.5 hours, removing the water formed during the cyclization with a vacuum pump at 15minute intervals. Heat the residue with 4 ml. of saturated methanolic HC1 for 15 minutes at 80 to 90°, and then repeatedly evaporate the mixture to dryness at 40 to 50° to remove the HC1. Then dissolve the final residue in 1.5 ml. of water, and add 4 ml. of saturated sodium picrate. Chill the solution overnight at - 15°, and collect the carboxamide picrate which may be decolorized with charcoal and recrystallized from 85% ethanol. Dry this material in vacuo. The carboxamide hydrochloride, which is more useful in biological experiments, may readily be prepared from the picrate. Suspend the carboxamide picrate in 0.6 ml. of acetouic HC1, and collect the crystalline hydrochloride which precipitates. Repeat this procedure until the supernatant fluid is completely free of yellow color. Dry the hydroehloride in vacuo over P20~,. The yield is 60%. To synthesize 2-C14-4-amino-5-imidazolecarboxamide, use HC~OOII in the preparation of formamidomalonamamidine.

Synthesis of Hypoxanthine (Fig. 3) The following procedure is a modified version ~ of Shaw's. H If 2-C~*-4amino-5-iinidazolecarboxamide is used as the starting material, 8-C ~1~ E. Shaw, J. Biol. Chem. 185, 439 (1950).

622

TECHNIQUES FOR ISOTOPE STUDIES

[26]

hypoxanthine will be the product; if HC~4OONa is used in the first step, 2-C~4-hypoxanthine will be obtained. Note that the system of numbering is different for the carboxamide and hypoxanthine. 4-Formamido-5-imidazolecarboxamide (I). Add 100 mg. of 4-amino-5imidazolecarboxamide hydrochloride and 0.3 ml. of acetic anhydride to 50 mg. of sodium formate dissolved in 0.12 ml. of 98% formic acid. Heat this mixture at 70 ° for 1 hour with occasional stirring and then at 80 ° for 15 minutes. Evaporate the mixture to dryness at 50 °, wash the residue with three 3-ml. portions of water at 45 °, once with ethanol, and finally with acetone. Dry the product in vacuo. The yield is 80 %.

H2N--C--NH H2NC--

N

0 II HC--NH--C--NH H~N

C--C

N

I

HN--C=O HCL ~ - N H --

N II

Fia. 3. Synthesis of hypoxanthine.

Hypoxanthine (II). Dissolve 75 mg. of I in 4.5 ml. of sodium ethoxide (0.1 M sodium ethoxide in absolute ethanol), and heat for 3 hours at 75 to 80 ° with occasional stirring. Chill the mixture in ice, and collect the precipitate. Take the residue up in a few milliliters of HC1, and heat the mixture at 80 ° for 15 minutes. Chill, collect the precipitate, and dissolve it in the minimal amount of warm 1 N KOH. Then adjust the solution to pH 4 with glacial acetic acid, chill overnight, collect the hypoxanthine, wash it with water, absolute ethanol, and ether, and then dry it in vacuo. The yield is 75 %. This procedure may also be used for the synthesis of adenine ~ from 4-amino-5-imidazolecarboxamidine dihydrochloride. This latter compound is synthesized by the method described for the preparation of 4-amino-5-imidazolecarboxamide with the substitution of malononitrile for ethyl cyanoacetate. This synthesis is especially useful for the preparation of 2-Ci4-adenine. S y n t h e s i s of Xanthine

Xanthine may be synthesized from urea and ethyl cyanoacetate by the procedure described for adenine. With appropriately labeled urea (the synthesis of which is described below) 2-C '4- and 1,3-N15-xanthine may be synthesized. Xanthine may also be synthesized from 4-amino-5imidazolecarboxamide hydrochloride. 1' Fuse equal amounts of the carboxamide and urea at 175° for 2 hours. Cool the melt, grind it with water, and collect the precipitate. Dissolve the xanthine in 1 N KOH, 12 A. R. P. Paterson and S. H. Zbarsky, J. Am. Chem. Soc. 75, 5753 (1953).

[26]

PURINES AND PYRIMIDINES

623

decolorize it with charcoal, and precipitate it with glacial acetic acid. Dry the product i n vacuo. The yield is about 75%. If 2-C~4-carboxamide is used, 8-C~4-xanthine is obtained; C~4-urea results in 2-C~4-xanthine. Note again the difference in numbering of the carboxamide and purine.

Synthesis of Isoguanine This compound may be synthesized from 4-amino-5-imidazolecarboxamidine dihydrochloride and urea by the procedure described above for the synthesis of xanthine from 4-amino-5-imidazoleearboxamide.

Synthesis of Uric Acid (Fig. 4) By the following procedure label may be introduced into the 2, the 8, or the 4 and 6 carbon and into the 1 and 3 or the 9 nitrogen positions of uric acid. Urea. Dissolve 300 mg. of cyanamide in 3 ml. of water and 0.5 ml. of concentrated HC1.13 Boil the solution under reflux for 10 minutes, cool, and neutralize it with Na2COa. Evaporate the neutralized solution to dryness i n vacuo, and extract the residue with small portions of boiling ethanol. Evaporate the combined ethanol extracts to dryness, and dry the residue over P~05 in vacuo. Extract this residue with acetone, evaporate the solution to dryness, and complete the drying over P~O~. The yield is 90%. Both C 14- and N15-1abeled urea may be synthesized from suitably labeled cyanamide, the preparation of which was described above. Diethylmalonate. Heat the residue of sodium cyanoacetate (prepared as described for the synthesis of ethyl cyanoacetate) in 5 ml. of a 1 : 1 mixture of ethanol and concentrated H2SO~ on a boiling water bath for 1 hour.I4 Cool the flask rapidly, add about 2 ml. of water, and remove the insoluble residue. Wash this undissolved salt with ether, and extract the supernatant fluid with ether. Distill off the ether i n vacuo from the combined extracts, and collect the product which distills at 92 to 94 ° (16 mm.). The yield is 50%. By using labeled cyanoacetate, diethyl malonate may be synthesized with C 14 in both carboxyl groups. Barbiluric A c i d ( I ) . The condensation of urea and diethylmalonate is carried out in the same manner as the condensation of formamidine and malononitrile described for the synthesis of adenine. If Cl*-urea is employed in this step, 2-C~4-uric acid will result; 4,6-C~4-uric acid is obtained from carboxyl-labeled diethyl malonate. 13S. H. Zbarsky and I. Fischer, Can. J. Research B27, 81 (1949). 14A. I. Vogel, "Practical Organic Chemistry," p. 469. Longmans, Green and Co., New York, 1951.

624

[26]

TECHNIQUES FOR ISOTOPE STUDIES

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[26]

PURINES AND PYRIMIDINES

625

Uramil (II). The amination of barbituric acid may be accomplished by the procedure described above (see p. 618) for the synthesis of 4,5,6-triaminopyrimidine. Uric Acid ( I V ) . Fuse 55 mg. of urea with 500 mg. of uramil at 150 to 170° for 45 minutes. 15 Extract the cooled melt with 20 ml. of boiling water, remove the insoluble material, decolorize the filtrate with charcoal, concentrate it to 6 ml., and collect the precipitated ammonium pseudourate (III). Dissolve this in aqueous NaOH, and precipitate the uric acid with HC1. The yield is about 35%. If C~4-urea is used in this step, 8-C14-uric acid is obtained; if N~B-urea is used, only position 9 of uric acid should be labeled. In practice, however, some N ~5 enters nitrogen 7.

Synthesis of Uracil~ Thymine, Isocytosine, and 5-Methyl Isocytosine (Fig. 5) These four compounds may be synthesized by the same general procedure. 16-1s It is most applicable to the synthesis of 2-C ~4- and 1,3-N ~6pyrimidines, although, through the use of appropriately labeled malic or methyl malic acid, isotope may be introduced into the 4, 5, or 6 positions. fl-Melhyl Malic Acid (ii).17.~9 Under anhydrous conditions, add 2.5 to 3.0 g. of ether-washed, powdered sodium to 7 ml. of ether. Add dropwise 175 ml. of absolute ethanol to this solution. When the reaction stops, immerse the flask in an ice bath, and slowly add a mixture of 12 g. of ethyl propionate and 17.5 g. of ethyl oxalate. Distill off the ether and ethanol, and solidify the residue by cooling. Dissolve the residue in 25 ml. of cold, 33 % acetic acid, and let the solution stand at room temperature for several hours. Extract this solution with four 20-ml. portions of ether, and then wash the combined ethereal extracts with water, 10% NaHC03, and water again. Remove the ether by distillation, and distill over the product between 114 and 116° (10 mm.). The yield of ethyl ethyloxalylpropionate is 70 %. Shake 15 g. of this material and 75 mg. of platinum oxide catalyst for 2 to 3 hours under a hydrogen pressure of 3 to 4 arm. Diethyl-~-methylmalate is obtained in 90% yield. To remove the ethyl groups, boil 10 g. of the ester in 40 ml. of 0.15 N HC1 under reflux for about 5 hours. Then 15L. F. Cavalieri, V. E. Blairi and G. B. Brown, J. Am. Chem. Soc. 70, 1240 (1948). 16D. Davidson and O. Baudisch, J. Am. Chem. ~oc. 48, 2379 (1926). 17H. W. Scherp, J. Am. Chem. Soc. 68, 912 (1946). 18W. T. Caldwell and H. B. Kime, J. Am. Chem. Soc. 62, 2365 (1940). 19R. F. B. Fox and S. M. McElvain, in "Organic Syntheses" (Gilmanand Blatt, eds.), Coll. Vol. II, p. 272. John Wiley & Sons, New York, 1951.

626

TECHNIQUES FOR ISOTOPE STUDIES

[26]

concentrate the solution to a sirup i n vacuo, and dry the product over NaOH again i n vacuo. The yield is about 95 %. f~-Methyl malic acid is used in the synthesis of thymine (III) and 5-methyl isocytosine (V). Uracil ( I ) . Chill 2 ml. of fuming sulfuric acid to 0 °, and gradually add 500 rag. of urea with stirring. Then add 500 mg. of malic acid, s° and heat the mixture on a steam bath for 1 hour. Cool, and pour the reaction mixture into 6 ml. of water. The uracil precipitates on further cooling. It may be decolorized with charcoal and recrystallized from water. The yield is about 50%. If C14-urea is used in this synthesis, 2-C14-uracil is the product; N 15urea results in 1,3-NlS-uracil. C ~4- and N~5-Guanidine may be used to synthesize similarly labeled isocytosine (IV) and 5-methyl isocytosine. This procedure has also been used for the synthesis of methyl-labeled thymine starting from sodium propionate-C14. ~°! Uracil and thymine may also be synthesized by the condensation of thiourea with ethyl formylacetate and ethyl formylpropionate, respectively, and the subsequent conversion of the 2-thio analogs to the desired pyrimidines. 2~

Synthesis of Cytosine and 2~4-Diaminopyrimidine (Fig. 6) Cytosine and 2,4-diaminopyrimidine may be synthesized by a procedure which differs only slightly from that described above for the other pyrimidines. 2~,~a By this method, the 2-C ~4 and 1,3-N ~5 compounds may be synthesized. Cyanoacetaldehyde Diethylacetal (1). 54 Cool a mixture of 11.5 g. of sodium, 30 ml. of absolute ethanol, and 200 ml. of absolute ether to - 17°. Then add 44 g. of ethyl formate (previously cooled to - 1 5 °) over a 30-minute period with continual stirring. Leave the solution for 3 hours at - 15° and 12 more hours at room temperature. Collect the solid, wash it twice with anhydrous ether, and dry it over H~SO4. This material contains the desired sodium ethyl formylacetate and the by-products sodium ethyl acetoacetate and sodium formate. The yield is about 50 %. Add 50 g. of the above product, in small portions, to 460 ml. of absolute ethanol containing 60 g. of dry HC1. Stir the mixture for 3 hours, neutralize it with NaHCO3, and remove the insoluble residue. Wash this residue with ethanol, evaporate the combined supernatant fluids to s0 For synthesis of labeled malic acid, see Vol. IV [24]. ~oi R. B. Henderson, R. M. Fink, and K. Fink, J. Am. Chem. Soe. 77, 6381 (1955). 21A. A. Plentl and R. Schoenheimer,J. Biol. Chem. 153, 203 (1944). ~ A. Bendieh, H. Getler, and G. B. Brown, J. Biol. Chem. 177, 565 (1949). 23A. Bendieh, W. D. Geren, and G. B. Brown, J. Biol. Chem. 185, 435 (1950). ~4S. M. MeElvain and R. L. Clarke, J. Am. Chem. Soe. 69, 2657 (1947).

[26]

PURINES AND PYRIMIDINES

627

dryness at 60° in vacuo, and distill over the residual oil. Ethyl/~fl-diethoxypropionate is obtained in 35 % yield. Stir 25 g. of this compound with 150 ml. of concentrated NH4OH at room temperature until the mixture becomes homogeneous. Then remove the water and excess NH3 at 50° in vacuo. Heat the residue at 60 ° (10 mm.) for 30 minutes, then dissolve it in 20 ml. of benzene and add 60 ml. of warm (50 °) ether. Cool this mixture to 0 ° , and collect the precipitated /3fl-diethoxypropionamide. The yield is about 80 %. NH2 CN NH~ CN N=-C--NH2 I I I L I I C---~O + CH~. --~ O = C CH2--~ O=-C CH I I L I I ]L NH2 CH(OC2Hs)2 N=CH HN--CH I

II

NH.2 CN N=C--NH2 I I I I C = N H ~ + CH2 --~ H2NC CH I I II tl NH2 CH(OC2H~)2 N--CH III

FIG. 6. Synthesis of cytosine and 2,4-diaminopyrimidine.

Under anhydrous conditions, slowly add 13.5 g. of P205 to a mixture of 11.5 g. of the above product, 25 ml. of benzene, and 14.5 g. of triethylamine. Stopper the flask, and stir the contents with a sealed stirrer. Heat the mixture at 80 to 90° until the exothermic reaction begins, and then remove the heat. When the reaction stops, boil the reaction mixture under reflux for 1 hour. Then distill off the benzene and triethylamine at 120° . When this is completed, gradually reduce the pressure and raise the temperature, and collect the resultant distillate in a dry ice-acetone bath. Redistill this material, and collect the portion which boils between 90 and 95 ° (12 mm.). Add this distillate to 34 ml. of absolute ethanol containing 5 mg. of sodium, and leave the mixture for 1 hour at room temperature. Neutralize the solution to phenolphthalein with glacial acetic acid, remove the ethanol, and redistill the product as above. The yield of cyanoacetaldehyde diethylacetal is approximately 80 %. Cytosine (II). Add 700 mg. of urea and 1.7 ml. of I to 9 ml. of n-butanol containing 270 mg. of sodium. Boil this mixture under reflux for 2 hours. Then chill the vessel, collect the sodium salt of the intermediate, and wash it with n-butanol and ether. (In the synthesis of 2,4-diaminopyrimidine, only sodium nitrate precipitates at this point. Acidify the supernatant with 6 N H2SO4, and proceed as below.) Dissolve the intermediate product in 15 ml. of hot 2 N H~SO,, decolorize the solution with charcoal, and add 2 vol. of hot ethanol. Cool

628

TECHNIQUES FOR ISOTOPE STUDIES

[26]

the mixture, and collect the precipitated cytosine sulfate. Dissolve it in the minimal amount of water, make the solution alkaline with NH3, decolorize with charcoal, and adjust the solution to pH 7 to 7.5 with glacial acetic acid. On cooling, cytosine crystallizes in approximately 50% yield. To synthesize 2,4-diaminopyrimidine (III), substitute guanidine for the urea. The use of C 14- or N15-1abcled urea and guanidine results in 2-C 14- or 1,3-N15-pyrimidines. In the case of 2,4-diaminopyrimidine, the 2-amino group will also be labeled. Synthesis of Orofic Acid (Fig. 7) The following procedure ~5 is applicable to the synthesis of 1-N ~5- and 4,5- or 6-C14-orotic acid from appropriately labeled aspartic acid ~6 and 3-N 1~- and 2-C14-orotic acid from labeled KCNO. Potassium Cyanate. Mix 0.875 g. of finely powdered dihydrous K2C08 and 1 g. of urea in a large porcelain crucible. ~7 Gently fuse this mixture over an open flame until the evolution of NH3 ceases and a clear melt is obtained. On cooling, the molten KCNO crystallizes. The yield is 98%. N 15- or C~4-Labeled KCNO may be synthesized from appropriately labeled urea. Ureidosuccinic Acid (I). Dissolve 1.64 g. of aspartic acid in about 10 ml. of 20% KOH, and adjust the solution to pH 5.5 with concentrated HC1. Chill the solution, and dissolve in it 1 g. of finely powdered KCNO. Allow the solution to stand for 45 hours at room temperature. Acidify the solution to pH 2 with concentrated HC1, cool it, and collect the crystalline product after 2 hours. The ureidosuccinic acid may be recrystallized from water. The yield is about 70 %. For the synthesis of orotic acid, DL-aspartic acid is used, since the final product is not optically active. If the ureidosuccinic acid, itself, is to be used in biological experiments, it is best to have the L-isomer, in which case L-aspartic acid must be used in the synthesis. L-Ureidosuccinic acid will not crystallize on acidification of the reaction mixture, but it may readily be isolated. 28 Adjust the solution to pH 4 to 5, and chromatograph it on a Dowex 2 resin column (chloride form, 200 to 500 mesh, 10 X 4 cm.). Wash the column with approximately 1500 ml. of 0.005 N HC1, and then elute the L-ureidosuccinic acid with 0.05 N HC1. The compound may be detected 25 j . F. Nyc and H. K. Mitchell, J. Am. Chem. Soc. 69, 2s For the synthesis of labeled aspartic acid see Vol. IV 37 A. Scattergood, in "Inorganic Syntheses" (Fernelius, Hill Book Company, New York, 1946. 28 p. Reiehard and U. Lagerkvist, Acta Chem. Scand. 7,

1382 (1947). [28]. ed.), Vol. II, p. 86. McGraw1207 (1953).

[26]

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Isolation Excretory Products of Purine and Pyrimidine Metabolism Uric Acid. Extract the excreta with 100 ml. of 97% ethanol on a boiling water bath for 10 minutes. 31,~2 Filter and grind the moist residue to a uniform powder. Repeat this extraction three times, and then boil off the remaining ethanol. Boil the dry powder in 0.15 N HC1 for 3 to 4 minutes, cool the solution, and add enough 0.25 N piperidine to make the contents alkaline to phenolphthalein. Heat the mixture on a steam bath for 45 minutes, cool, and filter it. Add 9 vol. of water to the filtrate and 16.5 g. of NH~C1 per 50 ml. of this solution. Heat the solution at 60 to 70 ° for 20 minutes, and allow it to cool slowly for 24 hours. Collect the crystals, wash them with 10% NH4SO4, then water, and finally dissolve them in sufficient HC1 to make the resulting solution acid to litmus. Concentrate this solution, and collect the crystalline uric acid. Allantoin. Add 35 ml. of 35% phosphotungstic acid (recrystallized from ether) to 50 ml. of urine, and remove the precipitate thus formed. 33 Add a little more phosphotungstic acid to ensure complete precipitation. Then add 35 ml. of 5 % basic lead acetate to the supernatant fluid, remove the precipitate, and again test for completeness of precipitation. Mix this supernatant fluid with 35 ml. of 5 % H2S04, and remove the PbSO4. Neutralize the filtrate to litmus with 5% NaOH, and precipitate the allantoin with 100 ml. of a solution containing 1 g. of mercuric acetate and 10 g. of sodium acetate. Leave the mixture overnight, and collect the mercury allantoin precipitate. Wash this with water, and dry it over P205 i n vacuo. Grind the dry powder up in water, and bubble H~S through the mixture. Mercuric sulfide precipitates, and the free allantoin dissolves. Remove the precipitate, aerate the solution to remove the H2S, and concentrate it to 2 to 5 ml. in vacuo. Leave the solution in the cold overnight, and collect the crystalline allantoin. This may be decolorized with charcoal and recrystallized from water. Urea. Add 2 ml. of urine to 24 ml. of water, and then add 50 ml. of glacial acetic acid followed by 5 ml. of a freshly prepared 5 % methanolic solution of xanthydrol. 34 A precipitate begins to form in a few minutes. After 30 minutes, add 10 ml. of water and leave the mixture overnight. Collect the dixanthydrol urea, and recrystallize it from glacial acetic acid.

n j. L. St. Johns and O. Johnson, J. Biol. Chem. 92, 41 (1931). 3~R. B. Fisher, Biochem. J. 29, 2192 (1935). 3s G. B. Brown, P. M. Roll, A. A. Plentl, and L. F. Cavalieri, J. Biol. Chem. 172, 469 (1948). 34O. Fosse and R. D. Bell, Ann. 6, 13 (1916).

632

TECHNIQUES FOR ISOTOPE STUDIES

[26]

Ammonia. Add Permutit to 5 ml. of filtered urine. '~5 Wash the Permutit well with water, and then release the NHa with NaOH and collect it in HC1.

Isolation of Nucleic Acid Purines and Pyrimidines Procedures for the isolation and hydrolysis of nucleic acids and for the separation of the resulting purine and pyrimidine derivatives have been discussed in some detail in Vol. III [100-103, 106]. Only a brief outline of such methods will be presented here, in which will be indicated some special precautions applicable to studies with labeled materials. The procedure of Schmidt and Thannhauser, 86 as modified by LePage and Heidelberger, 37 is very satisfactory for the separation of R N A and DNA from most animal tissues. Homogenize a weighed amount of tissue in cold 4% HC104, and centrifuge the mixture in the cold. Wash the residue with cold 4 % HC104 (to remove acid-soluble purine and pyrimidine derivatives), twice with ethanol (to remove the acid), and then remove the lipids with three extractions with ethanol :ether (3:1) at 40 °. The combined acid solutions may be retained for the further separation of the acid-soluble compounds. Suspend the dry, extracted tissue in 1 N NaOH (10 ml. per 2 g. of the fresh tissue), and incubate the mixture at 34 ° for 16 hours. Under these conditions, everything is dissolved and the R N A is hydrolyzed to its constituent nucleotides. Cool the solution in ice, and add 3.5 ml. of 65 % TCA for every 10 ml. of NaOH used in the hydrolysis. The protein-DNA complex precipitates, and the RNA-nucleotides remain in solution. Wash the precipitate with cold 5% TCA, neutralize the combined supernatant fluids to pH 8 with Ba(OH)2, and add 4 vol. of cold ethanol to precipitate the barium ribonucleotides. Wash the protein-DNA residue with 10 vol. of ethanol, and then suspend it in 10 ml. of a neutral 0.3 M barium perchlorate solution. Heat this suspension in a boiling water bath for 20 minutes, cool it to room temperature, add 70 % HC104 to a final concentration of 2 %, and remove the precipitated protein at room temperature. Neutralize the supernatant fluid to pH 8 with Ba(OH)~, and add 4 vol. of cold ethanol to precipitate the barium DNA. This procedure completely separates R N A and DNA, as can be shown by the fact that no thymine is detectable in the R N A fraction and no uracil in the D N A fraction. It does not work well for bacterial nucleic acids for which special methods are necessary. 35 O. Folin and R. D. Bell, J. Biol. Chem. 29, 329 (1917). 3e G. Schmidt and S. J. Thannhauser, J. Biol. Chem. 161, 83 (1945). 37 G. A. LePage and C. Heidelberger, J. Biol. Chem. 188, 593 (1951),

[26]

PURINES AND PYRIMIDINES

633

Hydrolyze 50 to 100 mg. of the barium ribonucleotides or barium DNA in 1.5 ml. of 1 N HC1 at 100° for 1 hour. The products of this hydrolysis are the free purine bases and the pyrimidine nucleotides. Add a slight excess of H2SO4 to remove the barium ions, and chromatograph the solution on a Dowex 50 resin column (hydrogen ion form, 200 to 400 mesh, 90 × 9 mm.). Elute the column with 2 N HC1. The pyrimidine nucleotides come off together in the first few milliliters, since they are not retained by the resin. The guanine and adenine are eluted separately, in that order. Guanine and adenine may be further purified by rechromatographing them on Dowex 50, eluting with less concentrated HC1 in order to bring them off more slowly. Paper chromatographic procedures may be used for further purification if necessary2 s The pyrimidine nucleotides, which are not retained on Dowex 50, are readily separable by chromatography on both Dowex 1 anion exchange resin and paper. 3s If desired, the pyrimidine nucleotides may be hydrolyzed to the free bases which may then be separated by chromatographic techniques. To perform this hydrolysis, add about 50 rag. of the mixed pyrimidine nucleotides to 1 ml. of 12 N HC104, and heat the solution in a glassstoppered tube at 100° for 40 to 160 minutes with occasional shaking. After the hydrolysis, most of the excess perchlorate can be removed as the relatively insoluble potassium salt by the addition of KOH. If this solution is passed through a Dowex 50 resin column, cytosine is retained and uracil and thymine are not. The cytosine may then be eluted with 0.5 N HC1. Uracil and thymine are separable on Dowex 1 resin.3S The DNA of T~, T4, and T6 bacteriophage contain hydroxymethylcytosine instead of cytosine. This compound is destroyed completely by HC104 hydrolysis. In order to preserve this base, 39 pyrimidine nueleotides from these sources should be hydrolyzed with 88 % HCOOH for 30 minutes at 175° . In all these chromatographic procedures the various compounds are identified and followed by means of their ultraviolet absorption spectra. The spectral purity of a given compound is best determined by the ratios of its optical density at various wavelengths. Concentrations of an impurity which are too low to alter the position of the maxima or minima of a given absorption curve may cause significant discrepancies in such ratios. Impurities might also be present which do not absorb ultraviolet light and are thus not dhtected. Although these will not interfere with the 38 For these techniques see Vol. I I I [106-108]. 39 G. R. W y a t t and S. S. Cohen, Nature 170, 1072 (1952).

[26]

PURINES AND PYRIMIDINES

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tion to 38 ° to decompose the hydroxyacetylene diureidocarboxylic acid (II) to allantoin (III) and COs, and aerate the solution for an additional 60 minutes, collecting the evolved COs in Ba(OH)~. This BaC03 represents carbon 6 of the original uric acid. Neutralize the solution to pH 7, make it 0.1 M with respect to Na2C03, and heat it on a boiling water bath for 20 minutes to convert the allantoin to allantoic acid (IV). Make this reaction mixture acid to thymol blue with 10 N HN03, and place it on a boiling water bath for an additional 4 minutes. Cool the solution, neutralize it to pH 7, and add 5 ml. of basic lead acetate to precipitate the glyoxylic acid (VI) as the lead salt. The supernatant fluid contains urea (V). Transfer the precipitate to a centrifuge tube in about 10 ml. of water, and add 1.5 ml. of 4 N H2SO4. Stir this well, and remove the PbSO4. Wash the precipitate, and combine the supernatant fluids. Add 250 to 300 mg. of semicarbazide hydrochloride to this solution, and leave it at 2 ° overnight. Collect the crystalline semicarbazone of glyoxylic acid (VII), wash and recrystallize it, and dry it in vacuo. Dissolve 20 rag. of the semicarbazone in 30 ml. of water, and add 1.5 ml. of 1.5 N KMn04 and 4 ml. of 1 N H2S04. Incubate the mixture at 38 ° in a C02-free atmosphere, and aerate over the evolved COs. The COp collected during the first 7 minutes of the reaction contains equal amounts of the carbazone carbon and the carboxyl carbon of the glyoxylic acid. The specific activity of the BaC08 derived from this is equal to onehalf the specific activity of carbon 5 of the uric acid. The C02 evolved during the next 7 minutes is not collected. The remaining C02 is derived from the aldehyde carbon of the glyoxylic acid and represents carbon 4 of uric acid. Evaporate to dryness the supernatant fluid obtained from the basic lead acetate precipitation, and extract the urea from the residue with absolute ethanol. Evaporate off the ethanol, dissolve the urea in water, acidify the solution, and aerate it with H2S to remove any lead ions which might be present. Neutralize the solution to pH 7 with 1 N NaOH, and add an equal volume of sodium acetate buffer (0.1 M, pH 5.0). Aerate the solution to remove any COs present, add 100 rag. of urease suspended in 3 ml. of acetate buffer, and incubate the mixture at 38 ° for 30 to 45 minutes. Aerate out and collect the COs which represents carbons 2 and 8 of the uric acid. The incubation mixture may then be made alkaline, aerated, and the NH8 collected in acid. This NH3 is derived equally from all four nitrogen atoms of the uric acid. Degradation B. This procedure 48 enables the separation of carbon 2 from carbon 8 and nitrogens 1 and 3 from nitrogens 7 and 9. 4s j . C. Sonne, J. M. Buchanan, and A. M. DeUuva, J. Biol. Chem. 178, 69 (1948).

[9.6]

PURINES AND PYRIMIDINES

637

Dissolve 60 mg. of uric acid in 0.45 ml. of 5.5 N HC1 in a conical 12-ml. centrifuge tube. Add 25 mg. of KClOa over a period of 20 to 30 minutes with continual stirring. Then add 0.45 ml. of water, and let the vessel stand at room temperature for 1 hour. Bubble H2S through the solution for 10 to 15 minutes, and let the vessel stand in the cold overnight. The oxidation converts the uric acid to urea and alloxan (VIII), and the subsequent reduction forms alloxantin (IX) from the alloxan. Collect the precipitate (a mixture of alloxantin and free sulfur) in the cold. Wash it with four 0.5-ml. portions of water, and adjust the combined supernatant fluids to neutrality with 1 N NaOH. Incubate this solution with urease as described above, and collect the CO~, derived from carbon 8 of the uric acid, and the NH3, which represents an equal mixture of nitrogens 7 and 9. Take the alloxantin precipitate up in 2 ml. of water, and heat it on a boiling water bath. Remove the insoluble sulfur, cool the filtrate, add 100 rag. of PbO2, and adjust the mixture to pH 2. Heat the mixture for 20 minutes in a water bath, remove the excess Pb02, and wash the precipitate once with hot water. Treat the combined supernatant fluids, which contain urea, with H2S to remove lead ions, and then degrade the urea as above. The resultant BaCO3 is derived from carbon 2 of uric acid, and the NH3 is derived equally from nitrogens 1 and 3. In practice, it is found that the BaC03 derived from carbon 8 of uric acid is contaminated to the extent of 10 to 15 % with carbonate derived from carbon 2. The reverse contamination does not occur. Degradation C. This procedure 44 gives carbons 4 and 5 and nitrogen 7 as glycine. Therefore the isotope content of nitrogen 7 is obtained directly and that of nitrogen 9 can be calculated by subtracting the value for nitrogen 7 from that obtained for nitrogens 7 and 9 by degradation B. Dissolve 12 rag. of uric acid in 1 ml. of concentrated HC1, and hydrolyze it in a bomb tube at 160° for 18 hours. Aerate off the NHa which is derived from nitrogens 1, 3, and 9 with a water pump at 50 °. Neutralize the solution, and concentrate it to 1 ml. Then add 50 rag. of citrate buffer (pH 2.5) and 50 rag. of ninhydrin, and heat the solution for 8 minutes to convert the glycine amino group to ammonia. 45 Treat the solution with H:S, dilute it to 10 ml., filter, make it alkaline, and aerate off the NH3. This NH3 is derived from nitrogen 7 of uric acid. The isotope content of nitrogen 9 can be independently calculated by subtracting the value for nitrogens 1W3 (degradation B) from the value for nitrogens 1~-3+9 obtained by this degradation. The glycine may also be isolated as hippuric acid for the determina44 C. Wulff, Physiol. Chem. 17, 468 (1892). 45 D. A. MacFadyen, J. Biol. Chem. 155, 507 (1944).

638

TECHNIQUES FOR ISOTOPE STUDIES

[26]

tion of the specific activity of the carbon atoms. This gives an independent value for carbons 4 and 5 of uric acid. Degradation D. This procedure 46,47 separates nitrogens 1 and 7 from nitrogens 3 and 9. Thus, by subtracting the value obtained for nitrogen 7 by degradation C from the value for nitrogens 1 +7, the isotope content of nitrogen 1 can be calculated. Similarly, the isotope content of nitrogen 3 can be calculated by subtracting the value for nitrogen 1 from the value for nitrogens 1~-3 (degradation B) or the value for nitrogen 9 (degradation C) from the value for nitrogens 3~-9 obtained in this degradation. Dissolve 20 mg. of uric acid in 2.5 ml. of water by the addition of 200 rag. of KOH. Then add 0.03 ml. of 30% H~02. Incubate this solution at room temperature for 24 hours with a continual slow aeration. The solution is aerated with acid-washed (NH~-free) air, and the NH3 which is evolved during the reaction is collected in 20 ml. of 2 % boric acid. This NH3 is derived equally from nitrogens 1 and 7. Treat the reaction mixture with a small amount of MnO~ to decompose the excess H~02 and then remove the MnO~ by centrifugation. Cool the supernatant solution, adjust it to pH 5 with 1 ml. of glacial acetic acid, and collect the potassium oxonate (X) which crystallizes overnight. Dissolve this material in the minimal volume of water, add 0.5 ml. of saturated BaCl~, cool, and collect the crystalline barium oxonate. This product can be digested by the Kjeldahl procedure for 6 hours to obtain nitrogens 3 and 9 as NHa.

Cytosine and Uracil (Fig. 10) Cytosine may be deaminated to uracil by the procedure described above for the deamination of the purines. With the aid of two methods, 48 the 4 carbons of uracil may each be obtained separately and the 2 nitrogens together. Degradation A. Dissolve about 20 rag. of uracil (I) in 3 ml. of water with the aid of 0.5 N NaOH and heat. Then add a 5% KMnO4 solution dropwise until the purple color persists. Cool the solution to room temperature, and add a few drops of concentrated H~S04 to precipitate the MnO. Destroy the excess KMn04 with H~O~, remove the precipitate, and wash it with 2 ml. of warm water. Add 1 ml. of 5 N NaOH to the combined supernatant fluids, and heat the solution at 100 ° for 15 minutes. Then acidify the solution to phenol red with 5 N H~SO4, warm it to 100 °, 4s H. Brandenberger, Helv. Chim. Acta 37, 641 (1954). 47 S. C. Hartman, S. B. and M. S. Thesis, Massachusetts Institute of Technology, 1954. ~s U. Lagerkvist~ Acta Chem. ~cand. 7t 114 (1953).

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in vacuo, and, after cooling the flask to 15°, add 0.3 ml. of 100% H2SO4. Warm and shake the flask until the material dissolves completely, recool the flask, add 50 mg. of sodium azide, and warm the flask again until all of this dissolves. Connect the flask to two traps, the first containing 5% KMnO4 in 1 N H2SO4 and the second 0.5 N NaOH, and slowly raise the temperature over a 30-minute period from 38 ° to 60 to 70°. After 30 minutes at the final temperature, aerate the solution with CO~-free air for 10 minutes. The evolved COs is collected in the alkali trap and precipitated as BaCO~ by adding an excess of BaC12. This carbonate is derived from the carboxyl group of the propionate and represents carbon 4 of the pyrimidine ring. Replace the KMnO4 trap with 5 ml. of 0.2 N H~S04, and adjust the original reaction mixture to pH 11 to 12 with 5 N NaOH. Aerate the solution for 15 minutes at 90 to 100°, and collect the ethylamine (VII) in the acid trap. Transfer the ethylamine solution to a small, roundbottomed flask containing 5 ml. of 5% KMnO4. Adjust the solution to pH 8, add an additional 0.5 ml. of 0.5 N NaOH, stopper the flask, and heat it at 90 to 100 ° for 5 minutes. Acidify the solution with H~S04, and steam-distill over the acetic acid. This acetic acid can then be degraded stepwise by the above procedure to obtain carbons 5 and 6 of the pyrimidine ring as BaC03.

Thymine Thymine may be degraded by means of two procedures 5° to obtain the methyl carbon and carbons 2 and 4 separately and carbons 5 and 6 together (Fig. 11). Degradalion A. Add 0.075 ml. of bromine to a suspension of 150 mg. of thymine (I) in 1.5 ml. of water. A rapid reaction ensues with the formation of a precipitate. When the layer of bromine has disappeared, heat the mixture on a steam bath until all the precipitate dissolves. Filter the hot solution. On cooling, 5-bromo-6-hydroxyhydrothymine (II) crystallizes. Dissolve this in a 5 % NaHCO3 solution, and then distill the solution. The distillate will contain acetol (III) and the residue urea. The urea may then be degraded as described above to yield carbon 2 of thymine as B a C Q and nitrogens 1 and 3 as NH3. Treat an aliquot of the distillate with an excess of acetic acid and phenylhydrazine and warm gently to precipitate acetol phenylhydrazone. This may be recrystallized from benzene. The acetol is derived from the methyl carbon and carbons 5 and 6 of thymine. Another aliquot of the acetol solution is reacted with an excess of NaOI at alkaline pH for 30 minutes at room temperature. The iodoform which precipitates is 50 O. Baudish and D. Davidson, J. Biol. Chem. 64, 233 (1925).

642

TECHNIQUES FOR ISOTOPE STUDIES

[26]

washed with water and recrystallized from methanol. It is derived from the methyl carbon of thymine. Degradation B. Add the bromohydroxyhydrothymine to a saturated solution of Ba(OH)~, and heat the mixture on a steam bath for 15 to 20 minutes. BaC03 precipitates. This is derived solely from carbon 4 of thymine. O H2NC--NH~-*

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Orotic Acid Orotic acid may be degraded by the procedure B described for uracil degradation. 48 The product in this case is aspartic acid, instead of ~-alanine. The Dowex 50 column should then be developed with 0.5 N HC1 instead of 2 N HC1. Dissolve the residue obtained after chromatography on Dowex 50 in 1.5 ml. of water, neutralize the solution to pH 7 to 8 with NaOH, and add 0.4 ml. of saturated CuSO4. Adjust the solution to pH 5 to 6 with 0.2 N NaOH, and leave it in the cold for several days. Collect the crystalline copper aspartate. Dissolve this material in water, and aerate the solution with H~S to remove the copper. The aspartic acid may then be degraded by procedures described in Vol. IV [28]. In this way, the specific activity of each of the carbon atoms of orotic acid may be determined. The isotope content of nitrogen 1 of orotic acid may be obtained from the a~partic acid and nitrogen 3 by calculation from the isotope content of the original orotic acid.

[27]

BIOSYNTHESIS OF PROTOPORPHYRIN

643

[27] B i o s y n t h e s i s of P r o t o p o r p h y r i n

By DAVID SHEMIN The procedures described below are some of those which have been used in studies on the biosynthesis of protoporphyrin, the porphyrin moiety of hemoglobin. They include the isolation of hemin, the conversion of hemin to protoporphyrin, the reduction of the latter to mesoporphyrin, the oxidative cleavage of mesoporphyrin to crystalline fragments of the molecule representing particular structures of the porphyrin, and the degradation of the latter compounds to keto acids which on further degradation by standard procedures yield samples of carbon dioxide which arise from carbon atoms which had specific positions in the porphyrin compound. The procedures also include the synthesis of radioactive compounds which are intermediates in the biosynthetic chain and are not readily purchasable. Finally, references are given to the biological preparations used in which studies on the biosynthesis of protoporphyrin can be carried out. Preparation of Hemin ~ A 25-ml. sample of whole blood or washed red cells suspended in saline or cells hemolyzed with water is added dropwise, over a period of about 15 to 20 minutes, to a flask containing 75 ml. of glacial acetic acid and 0.5 ml. of a saturated sodium chloride solution, kept at 95 to 100 °. During this procedure the mixture is continuously and rather vigorously stirred. After addition of the blood sample, 2 to 5 drops of concentrated hydrochloric acid is added and the flask heated on a steam bath for about 1 hour. The formed hemin crystals gathered by centrifugation are washed twice successively with 50% acetic acid, water, alcohol, and ether, and then dried. The yield is approximately 70 to 100 mg. The hemin is purified by recrystallization. A 100-mg. sample of hemin in a 12-ml. centrifuge tube is dissolved in 0.5 ml. of pyridine by rubbing with a stirring rod. After solution, 4 ml. of chloroform is added, and the resultant mixture is filtered through a good grade of paper, by gravity, into a flask containing 15 ml. of a glacial acetic acid solution which has been previously saturated with solid sodium chloride. The filter paper is washed with small amounts of chloroform until it is almost colorless. The combined solution is heated carefully until the temperature reaches H. Fischer, Org. Syntheses 21, 53 (1941).

644

TECHNIQUES FOR ISOTOPE STUDIES

[27]

105 ° . At this time practically all the chloroform has been distilled off. Mter the addition of 2 to 3 drops of concentrated hydrochloric acid, the solution is kept at room temperature for 24 hours to complete crystallization. The crystals are recovered and washed as described above. Preparation of Mesoporphyrin

-Fe Hemin

[H] Protoporphyrin

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A. Preparation of Protoporphyrin

To a three-necked flask, fitted with a reflux condenser and with an efficient stirrer, are added 1 g. of finely powdered hemin and 70 ml. of 98 % formic acid. The stirred mixture is heated to a gentle boil, and while boiling 2.3 g. of powdered iron is added in several portions during a 20- to 30-minute period. The solution becomes deep red in color. After the addition of the iron, the mixture is refluxed for another 15 minutes. The solution is then cooled and filtered through a sintered-glass funnel. The formic acid solution contains protoporphyrin and some mesoporphyrin, as ascertained by determining its absorption spectra3 B. Reduction of Protoporphyrin to Mesoporphyrin

The above formic acid solution is reduced with hydrogen, under atmospheric pressure, with the aid of about 100 to 200 mg. of palladium black catalyst (Baker and Company, Inc., Newark, New Jersey). 3 The uptake of hydrogen is quite rapid, usually complete in about 1 hour. The amount taken up, however, may be less than the calculated amount, since the solution contains varying amounts of mesoporphyrin formed from protoporphyrin during its preparation from hemin with iron and formic acid. After the uptake of hydrogen has ceased, the solution is filtered through a coarse sintered-glass funnel and poured into 5 vol. of a 30% ammonium acetate solution. The precipitate is collected by centrifugation and washed with water. The mesoporphyrin can be purified further by dissolving the material in about 100 ml. of 2% ammonium hydroxide and then causing its reprecipitation by the addition of acetic acid. The mesoporphyrin is washed with water and dried i n vacuo. The yield is about 80 to 90%. 4 H. Fischer and H. Orth, "Die Chemic des Pyrrols," Vol. II, p. 322. Leipzig, 1937. , S. Granick, or. Biol. Chem. 172, 717 (1948). 4 j. Wittenberg and D. Shemin, or. Biol. Chem. 186, 103 (1950).

[27]

BIOSYNTHESIS OF PROTOPORPHYRIN

645

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Hcrnatinic acid

Oxidation

One gram of mesoporphyrin is suspended in 80 ml. of 20% (w/w) of sulfuric acid and stirred with a magnetic stirring bar at room temperature. Over a period of about 30 minutes, 1.8 g. of Cr03 dissolved in a minimum of water is added, and the mixture is stirred for another 17 hours. After this period of time the greenish solution is extracted with ether in a continuous extractor for about 24 hours. The filtered ether solution contains the methylethylmaleimide and the hematinic acid. 5,6

B. Separation of Methylethylmaleimide and Hematinic Acid The above ether solution is extracted once with 40 ml. of a 5 % sodium bicarbonate solution. The separated bicarbonate solution is extracted five times with an equal volume of chloroform. The ether and chloroform solutions containing the methylethylmaleimide are combined. The bicarbonate solution containing the hematinic acid is acidified to about pH2 with 2 N sulfuric acid immediately after the last chloroform extraction. The hematinic acid solution, once acidified, can be stored.

C. Isolation of Methylethylmaleimide The combined ether and chloroform solution is taken to dryness under atmospheric pressure, and the residue is dried in a desiccator under atmospheric pressure. The crude methylethylmaleimide is purified by sublimation at 0.2 to 0.3 mm. pressure, with the bath temperature between 60 and 70° . The melting point of the purified material obtained on resublimation is 66 to 67°; yield, approximately 200 rag.

D. Isolation of Hematinic Acid The acidified aqueous solution containing the hematinic acid is extracted five times with an equal volume of ethyl acetate. The ethyl 5 W. Kiister, Ber. 45, 1935 (1912). 6 H. Fischer, A. Treibs, and G. Hummel, Z. Physiol. Chem. 181i, 33 (1929).

646

TECHNIQUES FOR ISOTOPE STUDIES

[27]

acetate solution is taken to dryness, and the residue, crude hematinic acid, is purified by sublimations at 0.2 to 0.3 ram. pressure, with the bath temperature maintained between 120 and 130°. The material can be further purified by recrystallization from ethyl acetate-petroleum ether or ethyl-ether-petroleum ether; yield, about 300 mg. ; m.p. 115 to 116°. 4,7

E. Decarboxylation of Hematinic Acid to Methylethylmaleimide and Carbon Dioxide The hematinic acid can be decarboxylated by heating the material at 300 ° for 30 minutes under a slow stream of nitrogen. 8 The formed carbon dioxide is collected in barium hydroxide; yield,. 90 to 95%. The black tarry residue is extracted with chloroform, and the filtered solution taken to dryness. The residue is purified by sublimations as described above and by recrystallization from 0.2 % ammoniacal water. The yield of the methylethylmaleimide is 40 to 50%; m.p. 65 to 66 °.

Cleavage of Methylethylmaleimide to Pyruvic and ~-Ketobutyric Acids

0

H

0

HO OH O""'N~"O H

" ~ CO I

+

C00H

I C----O I C00H

To 139 rag. (1 mM.) of methylethylmaleimide dissolved in 10 ml. of water is added 213 rag. (2 X mM.) of sodium chlorate followed by 60 mg. of osmic acid dissolved in 6 ml. of water2 (The precautionary measures, use of a hood and the wearing of goggles, in the handling of osmic acid should be taken.) The mixture, which turns brown, should be permitted to stand at room temperature for 20 hours. After this time the solution is extracted five times with an equal volume of benzene to remove the osmie acid. The conversion to the glycol is quantitative as determined with periodic acid. The tartarimide is then cleaved to pyruvic and a-ketobutyrie acids by means of periodate.10 An exact equivalent of solid sodium metaperiodate (214 mg.) is added. After standing for 20 minutes the solution is extracted with ether for 5 hours in a continuous extractor. The ether H. M. Muir and A. Neuberger, Biochem. J. 47, 97 (1950). 8D. Shemin and S. Kumin, J. Biol. Chem. 198, 827 (1952). 9N. A. Milas and E. M. Terry, J. Am. Chem. ~oc. 47, 1412 (1925). ~0p. Fleury and J. Lange, Compt. rend. 195, 1395 (1932).

[27]

BIOSYNTHESIS OF PROTOPORPHYRIN

647

solution is taken to dryness and the residue dissolved in 1 ml. of 0.5 N HC1 and heated for 1 hour at 80 °. The solution is then mixed with 1 g. of anhydrous silica gel and transferred, with the aid of a small volume of butanol-chloroform solution, to a chromatographic column for the separation of the acids. The pyruvic and ~-ketobutyric acids are separated by partition chromatography on silica gel. H A column 1.6 cm. in diameter and 21 cm. in length containing 10 g. of silica gel is used for 50- to 200-mg. quantities. The stationary phase is 0.5 N sulfuric acid, and the moving phase is a 7 ~ butanol-chloroform solution saturated with 0.5 N sulfuric acid. The f~te of flow through the column, 0.7 ml./min., is adjusted by a head of p~tsure. The effluent from the column is collected in 5-ml. fractions, and each fraction is slurried with water and titrated with 0.01 M sodium hydroxide to the end point of bromothymol blue. Usually the a-ketobutyric acid f~action (yield, 40 to 50%) is obtained between the 40- and 60-ml. fractioas, and the pyruvic acid (yield, 25 to 40%) is obtained in the 90to 120-ml. fractions. The respective keto acids are isolated as their 2,4dinitrophenylhydrazones. Each of the aqueous phases from the titrated effluent of the particular keto acid is combined, concentrated to a small volume, and acidified with HC1. The indicator is removed with DarcoG-60 and an equivalent amount of 2,4-dinitrophenylhydrazine in 2 N hydrochloric acid is added. The pyruvic acid hydrazone is purified by dissolving it in sodium bicarbonate, filtering and reprecipitating with dilute hydrochloric acid, and then by recrystallization from ethanol; m.p. 217 to 218 °. The a-ketobutyric acid hydrazone is purified by dissolving it in half-saturated lithium carbonate, filtering and precipitating with dilute hydrochloric acidi and then by recrystallization by the addition of water to a hot glacial acetic acid solution; m.p. 198 to 200 °.12

Degradation of the Hydrazones of the Keto Acids The hydrazones of the a-ketobutyric and pyruvic acids can be convetted to propionic and acetic acids, respectively, by two convenient methods. In the method of Krebs ~3the hydrazones are oxidized with acid permanganate, whereas in the second method the hydrazones are treated with Ceric sulfate. TM The first method has the disadvantage that the carbo•yl group is diluted with 6 moles of carbon dioxide from the oxida11 F. 1~ D. 13 H. 14 R.

A, Isherwood, Biochem. J. 40, 688 (1946). Shemin and J. Wittenberg, J. Biol. Chem. 192, 315 (1951). A. Krebs, Biochem. J . 32, 108 (1938). H. Corzo and E. L. Tatum, Federation Proc. 12, 470 (1953).

648

TECHNIQUES FOR ISOTOPE STUDIES

[27]

tion of the phenyl group and further the oxidation of the phenyl group is not always quantitative. The second method has the disadvantage that about 10% of the formed carbon dioxide originates from the a-carbon atom, 15and thus the carbon dioxide from the carboxyl group is mixed with some originating from the a-carbon atom. The propionic and acetic acids are recovered by steam distillation. The propionic acid is converted to carbon dioxide and ethylamine by a modification le of the Schmidt reaction. It h a s b e e n demonstrated 17 that by this reaction each carbon atom of the acetic and propionic acids can be analyzed separately. The ethylamine is oxidized by alkaline permanganate to acetic acid. The samples of acetic acid, both from the decarboxylation of the propionic acid and from the pyruvic acid hydrazone, are similarly decarboxylated by the Schmidt reaction. The resulting methylamine is oxidized with alkaline permanganate. The carbon dioxide formed in each step can be collected as barium carbonate. • S y n t h e s i s of Labeled Substrates

Such compounds as glyeine, acetic, succinic, citric, and ~-ketoglutaric acids can be purchased with a particular carbon atom tagged with C 14 A. 5-Aminolevulinic Acid

The choice of the method of synthesis of this intermediate is in part dependent on the position one wishes to have labeled in the molecule. 1. 5-Aminolevulinic Acid-5-C14. TM Ethy! ~-keto-a-carbethoxyadipate was prepared according to the method of Riegel and Lilienfeld19 by the condensation of ethylmagnesiomalonate~° synthesized from 8 g. of diethyl malonate-2-C 14 with 9.3 g. of ~-carbethoxypropionyl chloride synthesized from succinie anhydride. ~1 Preparation of fl-Carbethoxypropionyl Chloride. Under anhydrous conditions 10 g. of succinic anhydride (see below) is refluxed with 10 ml. of absolute ethanol for 1 hour. After this period of time the solution is poured into an evaporating dish and after cooling is placed in a vacuum desiccator over CaCI~ for about 15 hours. The half ester is transferred to a flask fitted with a reflux condenser, 15 mt. of thionyl chloride is added, and the mixture, kept under anhydrous conditions, is kept for 3 hours 15j. C. Wriston, Jr., L. Lack, and D. Shemin, J. Biol. Chem. 215, 603 (1955). 16C. Schuerch, Jr. and E. H. Huntress, J. Am. Chem. Soc. 71, 2233 (1948). 17E. F. Phares, Arch. Biochem. and Biophys. 33, 173 (1951). 18D. Shemin, C. S. Russell, and T. Abramsky, J. Biol. Chem. 215, 613 (1955). 19B. Riegel and W. M. Lilienfeld,J. Am. Chem. Soc. 67, 1273 (1945). g0H. Lund and A. Voigt, Org. Syntheses, Coll. Vol. 2, 594 (1943). ~1U. Eisner, J. A. Elvidge, and R. P. Linstead, J. Chem. Soc. 1950, 2223.

[27]

BIOSYNTHESIS OF PROTOPORPHYRIN

649

at 40 °. The excess thionyl chloride is distilled off and the ~-carbethoxypropionyl chloride is obtained by distillation; b.p. 98°/15 ram. ;48°/1 ram. In a three-necked flask, fitted with a stirrer and reflux condenser and under anhydrous conditions, and containing ethylmagnesiomalonate synthesized from 8 g. of diethyl malonate-2-C 14, in 15 ml. of dry ether, are added dropwise, through an addition funnel, 10.5 g. of 2-carbethoxypropionyl chloride dissolved in 10 ml. of dry ether. The rate of addition should maintain a rather vigorous reflux. After the addition, the mixture (precipitate and solution) is refluxed for 2 hours and bumping avoided by judicious stirring. After this time the solution is cooled, and an ice-cold solution of sulfuric acid (1.5 nil. of concentrated H2SO~ in 30 ml. of water) is added slowly. The mixture is transferred to a separatory funnel and the ether layer washed once with water. The combined water layers are extracted once more with ether, and finally the ether layers are combined. After drying the ether solution over anhydrous sodium sulfate, the ether is removed by distillation and the residue distilled in vacuo. The boiling point of the triester is about 125 to 135 ° at 0.5 ram. pressure. The triester is then hydrolyzed and decarboxylated with concentrated hydrochloric acid. ~ For each gram of the triester 7 ml. of concentrated hydrochloric acid is added, and the lightly stoppered flask is allowed to remain at room temperature (about 25 to 28 °) for 18 to 20 hours. If the hydrolysis is permitted to proceed for a much longer time, the formed /~-keto acid may also decarboxylate. After this period of time the hydrochloric acid is removed by distillation in vacuo, while the outside bath temperature is not permitted to exceed 35 °. A flash evaporator is to be recommended. The crude ~-ketoadipic acid is suspended in about 50 ml. of glacial acetic acid. While the acetic acid suspension is kept at about 18 to 20 °, gaseous ethyl nitrite 22 prepared from sodium nitrite (10% excess) is passed into the suspension. The keto acid dissolves, and the solution is either slightly yellow or slightly brown. After standing at room temperature for about I hour, the acetic acid is removed by distillation in vacuo, and to the oily or semicrystalline residue a cold solution, made of 25 g. of concentrated hydrochloric acid and 25 g. of stannous chloride, is added. The resulting solution is placed in a refrigerator for about 48 hours. After this time the solution is diluted with 10 vol. of water and the tin removed with H~S. The filtered solution is taken to dryness in vacuo, and, after the residue has been dried in a vacuum desiccator, the crystalline product is obtained by crystallization from methanol-ethyl acetate. The material is recrystallized from these solvents; the yield, based on the malonate, is between 35 and 45 %.18 2~ W. L. Semon and V. R. Damerell, Ore. Syntheses Coll. Vol. 2, 204 (1943).

650

TECHNIQUES FOR ISOTOPE STUDIES

[27]

2. ~-Aminolevulinic Acid-l,4 or 2,3-C 14. This procedure is preferable if one wishes to synthesize $-aminolevulinic acid labeled in the succinyl moiety. 18.28 Preparation of Succinic Anhydride. 24 To 5 mM. of labeled succinic acid in a 12-ml. centrifuge tube with a standard taper joint, provided with a condenser and drying tube, 1.1 ml. of reagent-grade, freshly distilled acetyl chloride is added. The mixture is brought to a gentle boil in a water bath. In about an hour's time, solution occurs, after which heating is continued for another hour. After cooling, the stoppered vessel is placed in a refrigerator. The formed crystals are separated by centrifugation and washed three times with 0.5-ml. quantities of ether. The anhydride, dried in a desiccator in vacuo, is white; m.p. 118°; yield, 75 to 80%. 24 The anhydride is converted to ~-carbethoxypropionyl chloride as described above. To an ethereal solution of 1 g. of B-carboethoxypropionyl chloride is added an ethereal solution of diazomethane obtained from 2 g. of N-nitroso-N-methylurea, and the mixture is stirred for 2 hours at room temperature. After this time either dry HC1 gas is bubbled through the solution or an ethereal solution of HC1 is added dropwise until the ~volution of N2 ceases. The solvent is removed by passing a dry stream of nitrogen through the solution, and the residue is dried in vacuQ over NaOH. The oily chloromethyl ketone is treated with 4 ml. of dimethylformamide and 1.24 g. of potassium phthalimide (labeled with 34 .atom % excess N 15 ). T h e resulting orange suspension is stirred vigorously for 1 hour at room temperature and then mixed with 25 ml. of chloroform. The chloroform solution is washed with 50 ml. of water and the washings shaken with 2-ml. portions of chloroform. The combined chloroform solutions are then washed twice with 50-ml. portions of water, once with 25 ml. of 0.2 N sodium hydroxide, and finally four times with water. The chloroform solution is dried by filtration and taken to dryness in vacuo. The crude methyl 5-phthalimidolevulinate is purified by crystallization from methanol, m.p. 96 to 97 °. The phthalimido derivative is then hydrolyzed by boiling for 7 to 10 hours with 6 N hydrochloric acid. After removal of the phthalic acid by filtration, the crude ~-aminolevulinic acid hydrochloride is purified as above. The 5-aminolevulinic acid hydrochloride, m.p. 149 to 151 °, gives a positive ninhydrin test, reduces Benedict's solution in the cold, and on an ascending paper chromatogram has an R I of about 0.42 to 0.44 in a phenol-water mixture. The spot on paper, developed with ninhydrin, is

~3A. Neuberger and J. J. Scott, J. Chem. Soc. 1954, 1820. 24L. F. Fieser and E. L. Martin, Org. Syntheses Coll. Vol. 2, 560 (1943).

[27]

BIOSYNTHESIS OF PROTOPORPHYRIN

651

initially yellow and becomes purple after a few hours. The yield is about 30 % based on the succinic acid.

B. Preparation of Porphobilinogen T h e monopyrrole porphobilinogen m a y be prepared enzymatically from ~-aminolevulinic acid. A fraction from liver ~5 or duck erythrocytes 2~ is incubated with ~-aminolevulinic acid. The presence of porphobilinogen can be demonstrated b y treating a sample with Erlich's reagent, dimethyaminobenzaldehyde. The interference in the color development caused by glutathione, which is in the mixture, can be overcome b y the addition of a small a m o u n t of cuprous oxide. After the period of incubation, the protein is precipitated with trichloroacetic acid, and the porphobilinogen isolated. 27 To the filtrate, adjusted to p H 4 to 5 with a saturated solution of sodium acetate, a 15% solution of mercuric acetate is added until no further precipitation occurs. The precipitate is collected by filtration and washed with a solution of 1.5 % mercuric acetate. The precipitate is then suspended in water and the mercury removed with H~S. The filtrate, adjusted to p H 4 to 5 with 1 N NH4OH, is taken to dryness, and the residue is crystallized by dissolving the material in a minimum a m o u n t of 0.5 N N H 4 0 H and then adding 1 N acetic acid. The slightly pink crystals can be further purified b y repetition of the above crystallization procedure. The pink color is due to the spontaneous conversion of the monopyrrole to porphyrins. In an experiment in which 120 mg. of porphobilinogen was formed, as determined b y the Erlich reagent, 88 mg. were isolated.

Biosynthesis of Heine A s t u d y of the biosynthesis of heme can be carried out in vitro by using avian erythrocytes, 28 a hemolyzed preparation of the red cells, 8,29 or a cell-free extract of the avian cells. 18 Whereas glycine, acetate, succinate, a-ketoglutarate, and citrate are converted into heme in the hemolyzed preparation (permeability is a factor in the whole cell), one must, at present, start with ~-aminolevulinic acid when the cell-free extract is the preparation chosen. 18,3° 25A. Gibson, A. Neuberger, and J. J. Scott, Biochem. J. 68, xli (1954); 6J, 618 (1955). 26R. Schmid and D. Shemin, J. Am. Chem. Soc. 77, 506 (1955). 27G. H. Cookson and C. l~imington, Biochem. J. 57, 476 (1954). 28D. Shemin, I. M. London, and D. Rittenberg, J. Biol. Chem. 183, 757 (1950). 22I. M. London and M. Yamasaki, Federation Proc. 11, 250 (1952). 30D. Shemin, T. Abramsky, and C. S. Russell, J. Am. Chem. Soc. 76, 1204 (1954).

652

TECHNIQUES FOR ISOTOPE STUDIES

[28]

[28] Methods for Chemical Synthesis, Isolation, and Degradation of Labeled Compounds as Applied in Metabolic Studies of Amino Acids and Proteins

By

DAVID M. GREENBERG a n d ~¢~ORTON ROTHSTEIN

I. Methods for Chemical Synthesis of C14-Labeled Amino Acids 1 Introduction The use of isotopically labeled amino acids in biological research has become widespread with the advent of readily available isotopes of carbon, nitrogen, sulfur, iodine, and hydrogen. Almost all the known naturally occurring amino acids have been synthesized, incorporating one or more of these isotopes in the molecule. Because of the innumerable possibilities of multilabeling with combinations of isotopes, this chapter will confine itself to the synthesis and degradation of amino acids labeled solely with C TM. The basic starting material for synthesis of all C'4-containing molecules is BAC1403, available by permit from the Atomic Energy Commission at Oak Ridge, Tennessee. From this material, carbon dioxide-, cyanide-, formaldehyde-, methyl iodide-, and acetate-C TM, the primary building blocks of isotope synthesis, may be prepared. These compounds represent the starting materials for the synthesis of the vast majority of C'4-1abeled amino acids. In cases where their preparation is uneconomical or incon-' venient, they may be purchased from commercial sources. Synthesis of these intermediates will be discussed in detail only where they are an integral part of an experimental procedure for the preparation of a labeled amino acid. However, reference will be given to selected methods for their preparation, where necessary. There are often several different syntheses of a specifically labeled amino acid reported in the literature, not to mention numerous modifications of established procedures. Under these circumstances it becomes difficult to choose any single method as being the best available. This is especially true when one considers that, for many biochemists, the choice of method may depend more on convenience than on yield of product. In the final analysis, where two or more methods exist, the factors of time, cost, availability of elaborate equipment, and chemical yield must be balanced and decided on by the individual undertaking the synthesis. 2,3 Basically, Prepared by Morton Rothstein. 2 The book, "Technique of Organic Chemistry," Vol. VI (Micro and Semi-micro Methods), Interscience Publishers, New York, 1954, contains a great deal of useful

[9-8]

METABOLICSTUDIES OF AMINO ACIDS AND PROTEINS

~53

the author has attempted to select, for each amino acid, that procedure which is most efficient chemically. However, some consideration has been given to simplicity and ease of preparation, and, wherever possible, alternate methods have been outlined.

General Reactions for Synthesis of Amino Acids Containing C 14 The reactions used for synthesizing C14-containiag amino acids cannot be completely categorized. However, the majority of syntheses do fallinto certain general reaction patterns for the various positions of labeling. I. C 0 0 H - C ~4.Treatment of an aldehyde with cyanide-C 14and ( N H 4)~COa to form a hydantoin, which on hydrolysis yields the desired amino acid (Bucherer reaction), is a typical way of producing COOH-C~4-amino acids. (N H4) 2CO3

R C H O -~ KC*N - -

~. R C H

C*~O

I

t

NH

NH

\ /

hydrolysis

RCHC*OOH

I

NH2

C

H

0

3,4-Dihydroxyphenylalanine-l-C14, 4 lysine-l-C14, 5 and tyrosine-l-C1418 have been made by this general method. The reaction is very simple and requires no special equipment. The intermediates and products are all solids and thus can be manipulated with a minimum of trouble. In certain cases, the Strecker reaction can be applied, utilizing ammonia or NH4C1 instead of (NH4)2C03, as in the above, thus yielding the amino acid directly. Alanine-l-C~4, 7 phenylalanine-l-C14,8 leucine_l_C~4,9 and valine1_C~410 are among the labeled amino acids thus prepared. 2. 2-C14-Amino Acids. This type of labeling is generally achieved by condensing the appropriate halide with ethyl acetamidomalonate-2-C 14 information regarding small-scale, synthetic organic chemistry. 3 Both Metro Industries, Long Island 6, New York, and Scientific Glass Apparatus Co. Inc., Bloomfield, New Jersey, market a complete line of small, standard taper glassware which is excellent for carrying out small-scale organic reactions. 4 G. R. Clemo, F. K. Duxbury, and G. A. Swan, J. Chem. Soc. 1952, 3464. 5 Chem. Abstr. 48, 10588b (1954). s R. B. Loftfield, J. Am. Chem. Soc. 72, 2499 (1950). 7 R. B. Loftfield, Nucleonics 1 (3), 54 (1947). s G. O. Henneberry, W. F. Oliver, and B. E. Baker, Can. J. Research 29, 229 (1951). 9 H. Borsook, C. L. Deasy, A. J. Haagen-Smit, G. Keighley, and P. It. Lowy, J. Biol. Chem. 184, 529 (1950). 10 R. E. Selff and B. M. Tolbert, Univ. Calif. Radiation Lab. Rept. 1301 (1951).

654

TECHNIQUES FOR ISOTOPE STUDIES

[9.8]

(or a similar malonic acid derivative), followed by hydrolysis of the product to yield the desired amino acid. RX + CH--(COOR)~--~

I

NHAC

R* hydrolys~ C(COOR)2 RCHCOOH

I

f

NHAc

NH2

By this method, 2-Clt-labeled histidine, 11 lysine, 12 ornithine, 13 homoserine, ~4 and tyrosine 15 have been prepared. The yields from the condensation and hydrolysis steps are good, but unfortunately a rather arduous procedure is required to synthesize ethyl acetamidomalonate-2-C ~4. A good method is reported for the preparation of ethyl cyanoacetamidoacetate-2-C~4.16 Both compounds are available commercially, albeit they are somewhat expensive. 3. COOH or 2-C~4-Amino Acids. Labeling of these carbons can be secured by the a-bromination of a carboxylic acid followed by amination of the product with ammonia. This is, in general, a procedure giving reasonably good yields, since the acid is formed from an alkyl (or alkaryl) halide by carbonation of a Grignard reagent with C~402, or by cyanation with NaC14N or KC14N. •

Mg

RCOOH LihltI~) RCH2OH* --* RCH2Br - - - o o CO2 RCH2COOH --* R C H C O O H N_~ R C H C O O H

J

Br

J

NH2

A carbon 2-labeled amino acid results from reduction of a COOH-C 14acid to the alcohol, conversion to the bromide, and then treatment with Mg and CO2 or directly with KCN. A variation is the reaction of the radioactive bromide with acetamidomalonate to give a 3-C~4-amino acid. Among the C'4-containing amino acids prepared in this fashion are leucine-l-, 2-, and 3-C~4,le,17 alanine-l-, 2-, and 3-C~4,~8 and glycine-1- and 2_C14.19,20

11 G. Wolf, J. Biol. Chem. 200, 637 (1953). 1~H. Borsook, C. L. Deasy, A. J. Haagen-Smit, G. Keighley, and P. H. Lowy, J. Biol. Chem. 187, 839 ~1950). ~3D. B. Sprinson and D. Rittenberg, J. Biol. Chem. 198, 655 (1952). 1~E. P. Painter, J. Am. Chem. Soc. 69, 233 (1947). 16 M. Fields, D. E. Walz, and S. Rothchild, J. Am. Chem. Soc. 73, 1000 (1951). ~6H. Hauptmann, P. T. Adams, and B. M. Tolbert, J. Am. Chem. Soe. 74, 2423 (1952). ~TR. Ostwald, Univ. Calif. Radiation Lab. Repl. 2154 (1953). ~s R. Ostwald, P. T. Adams, and B. M. To!bert, J. Am. Chem. 8oc. 74, 2425 (1952). 19K. Bloch, J. Biol. Chem. 179, 1245 {1949). 20 R. Ostwald, J. Biol. Chem. 175, 207 (1948).

[28]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

655

4. ~-C14H2NH~. This t y p e of labeling is customarily obtained b y the reaction of KC14N with an 00-halide compound followed b y reduction of the resulting nitrile:

Br(CH2)3C(COOEt)2 KCN-4 N C ( C H 2 ) 3 - - C ( C O O E t ) ~

I

[

NHAc

NHAc

H2 PtO= At20

hydrolysis H2NCH2(CH2) a C H C O O H

I NH2 Examples of labeled amino acids prepared in this way are lysine-6-C14, 21 f~-alanine-l-, 2-, and 3-C~4, 22 and ~-aminovaleric acid-~-C~4. 23 A variation of this procedure is found in the preparation of ~-HO-lysine-6-C 1424 where KC14N is added to the appropriate w-aldehyde, yielding a cyanohydrin which on reduction and hydrolysis yields the desired product. 5. Azlactone Reaction. This reaction makes use of the carbon chain of glycine to form the carboxyl and a-carbon of the amino acid being prepared. The reaction has been used to prepare thyroxine-l-C14, 2~ phenylalanine-l,2-C~4, 26 and benzene ring-labeled phenylalanine27 0 • o II • o ArCHO, Ac20 H2NCH~COOH --~ ~ C - - N H C H 2 C O O H AcONa ArCH~C--N

I?oo

P hydrolysis

• o ArCH~CHCOOH NH2

°C--O

II

0

A similar use of glycine to provide the earboxyl and ~-carbon atoms of an amino acid can be made by condensing an aryl aldehyde with h y d a n t o i n (prepared from glyeine). After reduction and hydrolysis of the produeL the desired amino acid is obtained. 2~M. Rothstein and C. J. Claus, J. Am. Chem. Soc. 75, 2981 (1953). 22p. Fritzson and L. Eldjarn, Scand. J. Clin. & Lab. Invest. 4, 385 (1952). ~s M. Rothstein, J. Am. Chem. Soc. 76, 3038 (1954). 34 S. Lindstedt, Acta Chim. Scand. 7, 340 (1953). 25S. C. Wang, J. P. Hummel, and T. Winnick, J. Am. Chem. Soc. 74, 2445 (1952). 26S. Gurin and A. M. Delluva, J. Biol. Chem. 170, 545 (1947) ; M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwieh, "Isotopic Carbon," p. 223. John Wiley & Sons, New York, 1949. 27 A. B. Lerner, J. Biol. Chem. 181, 281 (1949).

656

TECHNIQUES FOR ISOTOPE STUDIES *

o

#

*

-~=o I

ARCH0 -~ C H 2 - - C - ~ 0 --~ A r C H ~ - C

I

I

I

NH

NH

\/

NH

HI e

NH

\/

C

C

0

O

Ir

[28]

H

6----0 hydrolysi~ A r C# H 2 •C H Co O O H J I

ArCH,--CH

f

NH

\

/

NH

NH.o

C

J;

O

This procedure has been utilized for preparing tyrosine-fl-C TM28and 3,4-dihydroxyphenylalanine-fl-C 14.29 6. Acetamidomalonate-Type Condensations. Many labeled amino acids have been prepared by the synthesis of a C~4-containing molecule which will condense with ethyl acetamidomalonate, or a similar compound of this type. Hydrolysis of the product yields the amino acid. R X + CH(COOEt)2 •NaOEt * hydrolysis R* C H C O O H ~ RC(COOEt)2

NHAc

NHAc

NH2

Tryptophan-/~-C14, 3° serine-fl-C14, 31 phenylalanine-4-C14, a2 leucine-3- and 4-C14, 33 glutamic acid-l-C14, 34 aspartic acid-3- and 4-C14,85 and alanine3_C1418 have all been prepared in this way. A l a n i n e =C ~4

DL-Alanine-l-, ~-, and 3-C TM. The method of Ostwald et al.~S gives good yields and has the advantage of using the same procedure for alanine-l-, 2-, or 3-C TM. ~s j. C. Reid and H. B. Jones, J. Biol. Chem. 174, 427 (1948). 29 M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwich, "Isotopic Carbon," pp. 225-227. John Wiley & Sons, New York, 1949. 30 C. tteidelberger, J. Biol. Chem. 179, 139 (1949). 31 M. Levine and It. Tarver, Y. Biol. Chem. 184, 427 (1950). 32 R. Dische and D. Rittenberg, Y. Biol. Chem. 211, 199 (1954). 38 M. J. Coon and S. Gurin, Y. Biol. Chem. 180, 1159 (1949). 84 R. J. Speer, A. Roberts, IV[. Maloney, and H. R. Mahler, J. Am. Chem. Soc. 74, 2444 (1952). 85 R. M. Noller and B. M. Tolbert, Univ. Calif. Radiation Lab. Rept., 2041 (1952).

[28]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

657

* o LiA1H, * o * o * o CO~ C H 3 C O O H - - - - - ~ CH3CH~OH ~ CH3CH2Br--* CH~CH2MgBr-----~ , o ° • o • NHa C H 3 C H 2 C O O H --~ C H 3 C H C O O H ----~ DL-Alanine-l-, 2-, or 3-C 14

I

Br The radioactive ethanol formed by the lithium aluminum hydride reduction of acetic acid was appreciably diluted with nonradioactive ethanol produced by splitting of the solvent (diethyl carbitol) first used for the reduction, thus lowering the specific activity of the product. Cox et al. 3~ reported that the use of t e t r a h y d r o f u r f u r o x y t e t r a h y d r o p y r a n ( T F T P ) as the solvent for lithium aluminum hydride reductions avoided the problem of contamination due to the splitting of solvent ethers. A modification of this procedure for the reduction of acetic acid by Pack and Tolbert 3t is incorporated below into the appropriate sections of the synthesis of alanine-C TM. On the basis of experience with T F T P as the solvent, it can be assumed t h a t the radiochemical yields from the reduction of acetic acid will be at least equal to, if not greater than, those reported originally with diethyl carbitol as solvent, but without concomitant dilution of radioactivity. Procedure: S o d i u m P r o p i o n a t e - l - C ~4. Propionic acid- 1-C 14was prepare d by carbonating 30 millimoles of ethyl magnesium iodide with 20 millimoles of C1402 (from 3.94 g. of BaC1403, 5.02 ~c./mg.)28 The yield was 1.87 g. of sodium propionate-l-C 14 (specific activity 10.4 ~c./mg.) (97.8%). a-Bromopropionic A c i d - l - C TM. D r y sodium propionate (937 mg., 9.87 mc.) from the above preparation was placed in a gas-solid reactor29 The bulb was evacuated to about 10 ~ of pressure and filled with an excess (one-half atmosphere) of dry, purified hydrogen chloride. The solid sodium propionate was then heated gently over a Bunsen burner. After the exchange reaction was complete, the mixture of propionic acid and excess hydrogen chloride was distilled i n vacuo into a large trap cooled with liquid nitrogen. The cooling bath was then changed to a dry ice-isopropyl alcohol mixture, and the excess hydrogen chloride pumped off. (Propionic acid may also be obtained from its salt by use of an apparatus similar to t h a t in Fig. 4--see Leucine-1- and 2-C14.) The propionic acid-l-C ~4, which contained 3 to 5 % water, was distilled 36j. D. Cox, H. S. Turner, and R. J. Warne, J. Chem. Soc. 1980, 3167. 37D. E. Pack and B. M. Tolbert, Univ. Calif. Radiation Lab. Rept. 1957 (1952). 38 M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwich, "Isotopic Carbon," pp. 178-79. John Wiley & Sons, New York, 1949. 39 M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwich, "Isotopic Carbon," p. 162. John Wiley & Sons, New York, 1949.

658

TECHNIQUES FOR ISOTOPE STUDIES

[9.8]

into the bromination vessel 4° which contained 0.04 g. of red phosphorus, 0.02 g. of iodine, and 0.2 ml. of propionyl chloride. The mixture was allowed to reflux on the steam bath for 1/~ hour to destroy the water, 1.5 ml. of bromine was added dropwise, and refluxing was continued for 3 hours. The low-temperature condenser was kept at dry ice-isopropyl alcohol temperature. After the excess bromine was swept out with a slow stream of air, the mixture of a-bromopropionic acid-l-C 14 and a-bromopropionyl bromide1-C 14 was cooled and hydrolyzed by slow addition of 2 ml. of water. Alanine-l-C ~4. The amination was performed in a three-necked flask by slow addition of a-bromopropionic acid-l-C ~4 to a mixture of 6 g. of (NH4) 2CO3 and 15 ml. of concentrated NH4OH. The reaction mixture was kept at 60 ° for 6 hours and then distilled to dryness in vacuo. The crude alanine-l-C ~4 was purified by (1) passage through an ion exchange resin column and (2) high vacuum sublimation. A glass column (30 × 2 cm. outside diameter) filled with 60 ml. of Dowex 50 resin (20 to 40 mesh) was treated by cycling to exhaustion three or four times with 2 N NaOH solution and 2 N HC1, respectively, ending with the acid. The excess acid was washed out thoroughly with water, and 600 to 900 mg. of crude alanine-l-C ~4, dissolved in a minimum amount of water, was added to the column, followed by 500 ml. of 1.5 N NH4OH, followed by 250 ml. of water. The resin was regenerated with 2 N HC1. The water effiuate, NH40H eluate, and HC1 regeneration solution were each evaporated to dryness and were found to contain 3, 88, and 5%, respectively, of the radioactivity put on the resin. In order to remove traces of ammonia, the dry residue of the NH40H eluate was made slightly alkaline with NaOH and evaporated to dryness. After it was redissolved and the pH adjusted to 6.8, the solution was transferred to the lower part of a sublimation apparatus with a 1-cm. sublimation gap, evaporated to dryness, and sublimed at 1 ~ of pressure and 160 to 200 ° for 2 hours with the cold finger at liquid nitrogen temperature. The sublimation residue was redissolved (pH 8.5), adjusted to pH 6.8, dried, and resublimed. The final residue contained 4% of the initial crude alanine activity. The yield of chromatographically and radioautographically pure alanine-l-C 14 was 802 mg. with a specific activity of 9.9 ~c./mg. This represents a radiochemical yield of 80.5% based on sodium propionate-l-C 14 used and a chemical yield of 76 %. Ethanol-l-C14; Preparation of the Hydride Solutions. T F T P (b.p. 124°/14 mm.) was prepared according to the method of Woods and 40 B. M. Tolbert, F. Christenson, F. N. It. Chang, and P. P. T. Sah, J. Org. Chem. 14, 528 (1949).

[9.8]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

659

Kramer 41 from tetrahydrofurfuryl alcohol (TFA) and 2,3-dihydropyran. Lithium aluminum hydride solutions in T F T P were prepared by stirring the hydride (which had been ground in a mortar) with T F T P for 5 to (i hours at 50 to 60 °. The slurry was then filtered through a sintered-glass funnel, and the clear solution was stored in a flask sealed with a serum bottle stopper. The solutions, which were 0.7 to 0.8 M, were analyzed by decomposing an aliquot with butyl carbitol and measuring the volume of the evolved hydrogen. All manipulations were done in a nitrogen atmosphere.

DryN2 Inductionstirrer

~

J

k

TrapT1 FI(~. ]. Appa,rzttus for the reduction of acetic acid-]-C tl to ethanoI-t-C t4.

Sodium acetate-l-C 14 was converted to the free acid by the procedure given above for conversion of sodium propionate-l-C~4 to propionic acid1-C'4. The product was transferred to the dropping funnel of the apparatus shown in Fig. 1 with the aid of 10 to 15 ml. of T F T P . Dry nitrogen was then passed through the system, and trap T~ was immersed in liquid nitrogen. A bath of water at 60 to 70 ° was placed around the reaction flask, and the acid-TFTP mixture was added dropwise to the rapidly stirred hydride solution. The funnel was rinsed with several 1-ml. portions of T F T P . The mixture was then stirred for ]/~ hour longer. Excess hydride was decomposed with TFA, and the ethanol swept into the trap by placing 4, G. F. W o o d s a n d D. N. K r a m e r , J. Am. Chem. Sac. 69, 2246 (1947).

660

TECHNIQUES FOR ISOTOPE STUDIES

[28]

the flask in an oil bath at 150 ° and bubbling nitrogen through the mixture by means of a flitted-glass bubbler tube at a rate of 30 to 40 ml./min, for 5 to 6 hours. Exposed glass between the flask and the trap was wrapped with heating tape and heated to 200 °. The ethanol-l-C TM contained small amounts of T F A and T F T P , but this does not interfere with the subsequent bromination. 4~ Ethyl Bromide-l-C ~4. The ethanol-l-C TM was converted to the bromide by reaction with phosphorous tribromide according to Tolbert et al. 4° Sodium Propionate-2-C TM. The ethyl-l-C TM bromide thus prepared was converted to the Grignard reagent, and this compound was carbonated with inactive carbon dioxide. 43 Alanine-2-C ~4. Sodium propionate-2-C TM (0.935 g., 9.7 millimoles, specific activity 4.66 ~c./mg.) was converted to alanine-2-C ~4 as described for alanine-l-C TM. The yield was 0.689 g. or 67% chemically. The radiochemical yield was 63 %, specific activity 4.08 ~c./mg. Alanine-3-C TM. Acetic acid-2-C 1444 was converted to ethyl bromide2-C ~4 and thence to sodium propionate-3-C TM in the same manner as for propionate-2-C ~4. The propionate was then converted to alanine by bromination and amination. Other Syntheses. Alanine-l-C ~4has been prepared by a simple procedure in 35 % over-all yield (based on cyanide) by the action of labeled cyanide on acetaldehyde in the presence of ammonia :7 CHaCHO + HCN N~ CH3CH~OOH

I

NH2 ~-Alanine-C ~ fl-Alanine-l-, 2-, and 3-C TM. /~-Alanine has been prepared labeled with C TMin each of the three positions by the following sequence of reactions :22 o

C1CH~COOH ~- K C N -~ N C" C°H 2 C* O O H -~ H 2 N C H 2 C H , 5 0 0 H The over-all yield based on KC14N is 44.8%. The authors present only the synthesis of /~-alanine-3-C 14 but have also used chloroaeetic acid labeled in the 1 and 2 positions to obtain the correspondingly labeled 4~B. M. Tolbert, personal communication. 4s S. Gurin and D. W. Wilson, Federation Proc. 1, 114 (1942). 44Acetic acid-2-C ~4is best prepared by reducing C1402 to methanol-C TM in a fashion similar to the reduction of propionic acid reported above,~ and thence to methyl iodide-C 14.This is converted to the Grignard reagent and reacted with inactive carbon dioxide lB. M. Tolbert, J. Biol. Chem. 173, 205 (1948)]. The over-all yield for the conversion of C~40~ to sodium acetate-2-C 14is approximately 60%,

[9.8]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

661

~-alanines. Labeled chloroacetic acid may be prepared in good yield from acetic acid by the procedure of Ostwald 2°,45 (also see Aspartie Acid-3and 4-C 14). Considerable care must be taken to neutralize the excess potassium hydroxide usually found in preparations of KCI*N. Outside of this, the synthesis is straightforward and offers no difficulties. The yield could probably be increased by fractionating the ~-alanine mother liquors by appropriate ion exchange techniques, thus avoiding the dilution with carrier required to obtain a second crop of product. P r o c e d u r e . To the vessel containing 22 mg. of KC14N (1 mc.) and 25 mg. of KOH was added 82.6 mg. of carrier KCN and 5 drops of water to dissolve the contents. One milliliter of partly neutralized monoehloroacetic acid was added dropwise with shaking. The solution of monochloroacetic acid was prepared by dissolving 3.75 g. of the acid in 10 ml. of water, adding 1.56 g. of dried Na2CO~ while heating moderately, and diluting to 25 ml. The excess of free acid was calculated to neutralize 92 % of the free alkali i~ the sample. After 40 minutes on the boiling water bath, the reaction vessel was cooled, 1.90 ml. of 1.124 N HC1 was added, and the solution was evaporated i n vacuo. A few milliliters of dry ether was added, and the mixture again evaporated. The residue was carefully extracted with four portions of dry ether, the extracts being transferred to a specially constructed microhydrogenation vessel where the ether was evaporated off with an air jet (Fig. 2). To the oily residue in the hydrogenation vessel was added 1 drop of water and 2 N KOH (0.9 ml.) to bring the pH to 10 to 11. Four milliliters of 35% aqueous ammonia and approximately 100 mg. of suspended Raney nickel were added, and the mixture was hydrogenated under a pressure of 2 atm. at 20 ° for 17 hours with continuous stirring. The amount of hydrogen absorbed was 99.6% of the theoretical. After hydrogenation, the catalyst was removed by centrifugation, and the supernatant fluid, together with the washings, was evaporated to a small volume i n vacuo. After addRion of 0.145 ml. glacial acetic acid and heating to 50 ° for a few minutes, the evaporation was continued until .~ thick oil remained. This was dissolved in 4 to 5 drops of water, 5 ml. of absolute ethanol was added, and the solution placed in the refrigerator overnight, after seeding with a few crystals of ~-alanine. The first crystallization gave a sticky precipitate. In subsequent crystallizations, however, silky needles of ~-alanine separated out, leaving part of the material as an oily residue. The crystals were separated from the residue by swirling the flask gently, whereupon the suspension of crystals could be decanted and a5 The reported yield of chloroacetic acid-C ~~ from acetic acid-C ~4 is 67% for a single radioactive run. A yield of 85 to 90%, however, is more typical of the procedure.

[28]

663

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

a-Arninoadipic Acid-C ~

DL-a-A minoadipic Acid-6-C 14. a-Aminoadipic acid-6-C 14 has been prepared as follows :2~ HO(CH2)aC(COOEt)2--~ Br(CH2)3C(COOEt)2 + K C N - - ,

I

I

NHAc

NHAc

NC(CH,~)~C(COOEt)2 hydrolysis HOOC(CH2) * 3CHCOOH

I

NHAc

I

NH2

I

The intermediate, I, yields lysine-6-C ~4 on reduction with hydrogen over Pt02 (see Lysine-6-C~4). The procedure for a-aminoadipic acid offers no difficulties. However, the time suggested for the refluxing of the KCI*N with the bromovalerate derivative should be adhered to strictly. Procedure: "y-Acetamido-%,y-dicarbethoxybutyraldehyde. This compound was prepared by the method of Moe and Warner with benzene as the solvent. 47 After neutralization with glacial acetic acid, the mixture was filtered and the benzene removed in vacuo without heating to more than 60 °. A nearly colorless, viscous product resulted. Ethyl a-Acetamido-a-carbethoxy-8-hydroxyvalerate. The aldehyde was reduced and isolated according to Moe and Warner, 47 except that six times the reported amount of catalyst was used. The yield of alcohol was 82% based on crude aldehyde. Ethyl a-Acetamido-o~-carbethoxy-5-bromovalerate. A vigorously stirred solution of 20 g. of the crude hydroxy ester in 90 ml. of dry benzene was cooled to 16° in a water bath, and a solution of 2.8 ml. of phosphorus tribromide in 20 ml. of dry benzene added dropwise over a period of 30 minutes. The mixture was then refluxed for 20 minutes, and the benzene evaporated on a steam bath. The yellow residue was repeatedly extracted with portions of low-boiling petroleum ether. The extracts, after being cooled in a refrigerator, yielded 16.8 g. (68%) of fine white needles. This material was used without purification for the next step. DL-o~-Aminoadipic Acid-6-C 14. To a solution of 2 g. of ethyl-a-acetamido-a-carbethoxy-~-bromovalerate in 20 ml. of ethanol was added 400 mg. of KC~4N in 5.4 ml. of water. An additional 2 ml. of water was used to rinse the last of the cyanide into the reaction flask. The mixture was ,70. A. Moe and D. T. Warner, J. Am. Chem. Soc. 70, 2763 (1948).

664

TECHNIQUES

FOR ISOTOPE

STUDIES

[28]

refluxed for 2.5 hours, distilled to dryness in vacuo, and the residue extracted four times with ether. 48 The ether extracts were evaporated and hydrolyzed for 5 hours with 30 ml. of concentrated HC1. The resulting solution was distilled to dryness under diminished pressure. Addition of water and distillation to dryness was repeated twice, yielding a crystalline residue which was dissolved in a small amount of water and filtered. The volume was made up to about 12 ml. with water, and 15 ml. of ethanol was added. The solution was treated with a slight excess of pyridine, stirred, and placed in the refrigerator overnight. The white, crystalline DL-a-aminoadipic acid-6-C TM was filtered, washed with 50% ethanol until halogen-free, and dried; yield 0.389 g. (41%, not allowing for recovered cyanide). A paper chromatogram (phenol-water) showed one spot only; R I = 0.33. Aspartic

Acid-C

TM

DL-A spartic A cid-3- and ~-C14. Several syntheses of C ~4-1abeled aspartie acid have been reported. They differ only in detail and are based on the condensation of bromo- or chloroacetic acid ester (methyl or ethyl) with acetamido- or formamidomalonate. The product is 3- or 4-C14-aspartate, depending on the labeling in the acetate. The following is the procedure of Noller and Tolbert. 35 The over-all yield is 66% based on acetate-C14: *

°

CH3COOH *

--

*

° OO

C h C1CH2C

H

(C2H5)2SO4

*

o

* C1CH2COOEt

o

C1CH~COOEt -b CH(COOEt)2 NaOEt o* --* EtOOCCH2C(COOEt)2

f

NHAc

P

NHAc hydrolysis o• HOOCCH2C--COOtt

I

NH2Ac One might consider esterifying the chloroacetate with triethyl phosphate rather than diethyl sulfate as directed below, since the former reagent is greatly superior for the esterifieation of sodium acetate. 49 Procedure: Ethyl Chloroacetate-2-C TM. Anhydrous acetic acid was prepared from dry sodium acetate-2-C TM(1.85 g., 22.5 millimole, 13.3 pc./mg.) by the method described for the preparation of propionic acid (see Alanine-C14). The acetic acid was distilled in vacuo into the chlorination apparatus (Fig. 3) which contained 30 rag. of red phosphorus, 15 mg. of iodine, and 0.2 ml. (2.8 millimole) of acetyl chloride. The reaction mixture 45In a similar run for the preparation of lysine, 36% of the starting cyanide-C14was recovered by trapping in dilute alkali the gases given off by the refluxing solution. Use of this trapping procedure would substantially increase the radiochemicalyield. 49G. A. Ropp, J. Am. Chem. Soc. 72, 2299 (1950).

[9.8]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

6()5

was heated to approximately 40 ° to allow any water present to react with the acetyl chloride. Suitable precautions must be observed to accommodate any excess pressure which may develop. The bottom of the reaction tube was cooled in liquid nitrogen until all gaseous products were collected. The liquid nitrogen was then replaced by a dry ice-isopropyl alcohol bath and the excess hydrogen chloride removed i n vacuo.

Freezing mixture

C°ld conden! fingee~

/Reaction tube

I:H FIG. 3. Chlorination apparatus. The frozen mixture was placed on a steam bath where the condenser was filled with dry ice-isopropyl alcohol and opened to the atmosphere through a drying tube. Chlorine gas, passed through a phosphorus pentoxide tower, was bubbled through the reaction mixture at 100° for 2 hours. On cooling, the mixture solidified. The dry ice-isopropyl alcohol mixture was then poured out, most of the liquid chlorine was allowed to escape by evaporation, and the condenser stopcock closed. The reaction tube was immediately immersed in liquid nitrogen to prevent pressure from developing. The chloroacetic acid was distilled into the bottom of the reaction tube by filling the cold finger condenser with boiling water and by gently heating the other exposed parts of the apparatus with a small flame. The condenser and liquid nitrogen both were removed, and 1 ml. of water was added to the frozen mixture, which was then warmed and mixed, the chlorine being allowed to escape. The product was transferred to a 75-ml.

666

TECHNIQUES FOR ISOTOPE STUDIES

[28]

pear-shaped flask with the aid of about 5 ml. of water, immersed in an ice bath, and neutralized with about 20 to 25 ml. of 2 N KOH with phenolphthalein as indicator. The solution was frozen in a dry ice-isopropyl alcohol bath and then broken into small lumps with a spatula. The flask and frozen contents were then placed in a vacuum desiccator and freezedried (36 to 50 hours). Ethyl sulfate (15 ml.) was added to the dry solid, a reflux-distillation head was put in place, and the mixture was refluxed for 30 minutes by heating in an oil bath at 135 °. The temperature of the oil bath was then raised to 170 ° and the distillate collected for a period of about 2 hours. The reaction was judged to be complete when the black residue had acquired a granular appearance or when a blackish material began condensing in the reflux column. At this point, about 1.5 ml. of n-decane was added to the flask through the condenser, and the n-decane distilled out, carrying with it any residual ethyl chloroacetate. When the distillation was completed, the receiving vessel was removed and stoppered to prevent any access of moisture. If the reaction mixture is heated for too long beyond the stopping point described, it may suddenly decompose into a frothy carbonaceous mass. In addition to the desired product, ethyl chloroacetate, and n-decane, the distillate contains some ether, ethyl alcohol, and iodine. These impurities have been found not to interfere with any of the subsequent steps. The yield is 70 to 80 % based on sodium acetate. Aspartic Acid-3-C 14. Diethyl acetamidomalonate (7.32 g., 33.8 millimole, a 100% molar excess) was dissolved in 40 ml. of carefully dried ethanol in a 100-ml. round-bottomed flask fitted with a reflux condenser and protected from the air with a drying tube. To this solution was added 0.51 g. of metallic sodium (22.2 millimole, a 25% molar excess). When the sodium was dissolved, the ethyl chloroacetate was rinsed into the flask with about 5 to 10 ml. of dried ethanol, and the solution was allowed to reflux on the steam bath. After 4 hours, the ethanol was completely removed by distillation. Concentrated HC1 (50 ml.) was then added to the residue and the mixture refluxed for 72 hours. After evaporation to dryness on a steam bath and final drying in vacuo, the crude hydrolyzate was dissolved in a small quantity of water and transferred to a beaker. Copper chloride (7.67 g. of CuC12.2H20, a 33 % molar excess over theoretical total amino acid yield) was then added to the hydrolyzate solution and the total volume brought to 150 ml. This solution was adjusted to pH 5 with 1 N NaOH (approximately 97 ml.) and then placed in the refrigerator for at least 2 days. Approximately 4 ml. of Johns Manville Celite Analytical Filter Aid was then added to the cold mixture. The precipitate was filtered onto a 0.25-cm. bed of Celite and washed with ice water until the filtrate was chloride-free. The precipitate was redispersed in about 150 ml. of water,

668

TECHNIQUES FOR ISOTOPE STUDIES

[28]

The radioactive yield is 16% based on methanol-C 14 but is 47.5% based on the inactive thio compound. This indicates a conversion of methanol-C ~4 to formaldehyde-CTMof about 34%, although the method utilized claims a yield of 55% for this step. 53 The procedure of Murray et al. 54 gives formaldehyde-C~4from methanol-C ~4in 79 % yield, and that of Elwyn and Sprinson 5~ (see Serine-C 14) in 65% yield. Thus, by substituting one of these procedures, one might raise the over-all yield of cystine/~-CTM considerably. Methanol-C ~4may be readily prepared in nearly quantitative yield by the reduction of C~O2 with lithium aluminum hydride27 The procedure is the same as that for the preparation of ethanol-C 14 (see Alanine-C14). Procedure. C~4H~OH (approximately 150 mg., 0.9 me.) was converted to an aqueous solution of C~4H20 5~ (approximately 104 mg. per 5 ml.). This was added to a 10 % excess of ethyl N-benzylthiothiocarbonylaminomalonate (1.30 g.) in pyridine (10 ml.). 5~ The mixture was kept at room temperature overnight, 10 mg. of unlabeled formaldehyde (in dilute aqueous solution) was added, and after a further 7 hours the solvent was evaporated i n vacuo at 60 °. The residue was dried in vacuo overnight over concentrated H2SO4. Anhydrous ether (5 ml.) was added to the viscous oil, and the solution was cooled in an ice-salt bath. Purified thionyl chloride (10 ml.), 56 also previously cooled, was added dropwise to the ether solution at a slow rate. The mixture was allowed to stand in the cold for 1 hour after the addition of the thionyl chloride; as much of the thionyl chloride as possible was then removed by evaporation in vacuo at 20 °. 5 N NaOH in ethanol was then added to the residual oil until the solution was alkaline to litmus. After 20 minutes the alkaline solution was neutralized with 3 h r HC1 and the ethanol removed under reduced pressure. An excess of 3 N HCI was added, and the solution was refluxed for 2 hours. After removal of the HC1 under reduced pressure, water (25 ml.) followed by 1 N NaOH (25 ml.) was added to give a strongly alkaline solution which was allowed to stand for 30 minutes with occasional stirring. After additioa of concentrated HC1 (50 ml.), the solution was refluxed for 24 hours. The HC1 and some benzoyl mercaptan were removed by evaporation in vacuo, water being added to assist in the removal of the last traces. Water (50 ml.) was added, and the solution was twice extracted with ether, the ether layer being removed with a pipet. After evaporation of the aqueous layer, the residue was extracted with ethanol (10 ml.). The insoluble material was filtered off and washed with more ethanol. The filtrate (40 ml.) ~3 H. 54 A. 55 D. 58 E.

R. V. Arnstein, Biochem. J. 49, 439 (1951). Murray, III, C. W. Bells, and A. R. Ronzio, J. Am. Chem. Soc. 74, 2405 (1952}. Elwyn and D. B. Sprinson, J. Biol. Chem. 184, 465 (1950). L. Martin and L. F. Fieser, Org. Syntheses Coll. Vol. 2, 570 (1943).

[28]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

669

was diluted with an equal volume of water, and the solution was oxidized with 0.2 N iodine (required: 10 ml., 52% yield), with starch as an external indicator. Excess iodine was removed with a drop of sodium thiosulfate solution, and the solution was adjusted to pH 5 by adding ammonia (specific gravity 0.880) until alkaline, followed by acetic acid. After 20 hours at 0 ° the cystine-~-C 14 was filtered off and dried (yield 0.2193 g., 47.5 % based on ethyl N-benzylthiothiocarbonylaminomalonate; specific radioactivity 0.63 ~c./mg.). The radioactivity of this material was unchanged after recrystallization from water. G l u t a m i c A c i d - C 14

DL-Glutamic Acid-5-C ~4. DL-Glutamic acid-5-C ~4 has been prepared by the following sequence of reactions in 36 to 42% over-all yield, based on KC14N.56~ O CH~CH2 ~- K C N --~ HOCH2CH2CN -~ C H 2 = C H C N C H 2 = C H C N + CH(COOEt)2 --+ NCCH2CH2C--(COOEt)2

]

NHAc

I

NHAc hydrolysis Glutamic acid-5-C 14

Procedure: Acrylonitrile-l-C 14. KC14N (325 mg., 5 millimoles) in 0.5 ml. of water was added to 1 ml. of a solution of 6.18 g. of 1VIgSO4.TH20 in 10 ml. of water. The solution was cooled to 0 °, and 0.5 g. of ethylene oxide at 0 ° was added. After standing at 0 ° for 2 hours with occasional shaking, the solution was allowed to stand at room temperature for 12 hours. Carbon dioxide was passed into the cooled solution for 30 minutes, the mixture transferred to a centrifuge tube, concentrated under vacuum at 30 °, and the residue extracted three times with 4 ml. and eight times with 2 ml. of ethyl acetate. The combined extracts were dried over calcium chloride. Evaporation of the solvent in vacuum left crude ethylene cyanohydrin. Thirty milligrams of diethylene glycol and 30 mg. of basic 1V[gC03 were added. The vessel was connected to a trap which was cooled in a dry icechloroform mixture and which contained about 80 rag. of finely powdered calcium chloride. A slow stream of nitrogen was passed through the apparatus. The tube connecting the trap and the reaction vessel was surrounded by a jacket, through which isopropyl alcohol vapor was passed. The reaction started shortly after the reaction vessel was placed in a bath at 190°, The yield of material boiling at 76 to 78 ° was ] 60 to 185 rag. (60 to 5~ H. Tiedemann, Biochem. Z. 326, 511 (1955).

670

TECHNIQUES FOR ISOTOPE STUDIES

[28]

70%). (For larger quantities, the ethylene cyanohydrin should be added dropwise to the magnesium carbonate-diethylene glycol mixture.) Glutamic Acid-5-C 14. Thirty-five milligrams of sodium ethylate in 2 ml. of dry ether was added to 1 g. of diethyl formamidomalonate (or 1.1 g. of diethyl acetamidomalonate). The vessel was connected to a high vacuum line, and 180 mg. of acrylonitrile was distilled into the reaction vessel under high vacuum through a tube containing a 2-cm. layer of finely powdered calcium chloride. During the evacuation, both vessels were cooled in liquid nitrogen, after which the liquid was removed from the vessel containing the acrylonitrile. The reaction vessel was shaken for 5 hours in a water bath at 50 ° and then allowed to stand for several hours at room temperature. After acidification with dilute H2SO4 the mixture was extracted with ether and the extracts dried. The ether was removed in vacuo and the residue hydrolyzed with 10 ml. of concentrated HC1 for 6 hours at 116 to 120 °. The solution was then evaporated to dryness under vacuum. Water was added, and the solution evaporated twice more. The residue was dissolved in a minimum of water, the pH adjusted to 3.0 with 0.1 N NaOH, and the crude glutamic acid precipitated with alcohol. In order to remove the glycine formed from unreacted formamidomalonate, the crude product was dissolved in 40 ml. of water and shaken for 30 minutes with 3 g. of Amberlite IR-45 (carbonate form). After centrifugation, the supernatant liquid was shaken twice more with 1 g. of the resin. The resin was washed twice with 10 ml. of water. The glutamic acid was eluted from the resin with 70 ml. of 0.2 N HC1. The eluate was concentrated under vacuum as before, the residue dissolved in 50 ml. of water, the pH adjusted to 3.0 as above, and the glutamic acid crystallized by concentration. The yield was 298 rag. (60%). Other Syntheses. A very similar reaction scheme was reported by Depocas and Bouthillier, 57 but few experimental details were given. Glutamic A c i d - l - C ~4. Carboxy-labeled glutamic acid has been prepared in 47% yield based on cyanide-C ~4 by Speer et al. ~4 The experimental details are available on microfilm; the reaction sequence is as follows: KCN + C1CH~CI-I~OH ------) HOCH2CH~SN--HB-~r BrCH2CH2COOH * 80 %

92 % phthalimido- hydrolysis

malonic ester

---* Glutamic acid-l-C 1~

70-75%

47 % over-all yield

Glutamic Acid-2-C 14. This compound has been prepared by a similrs procedure to that reported above for glutamic acid-5-C 14, but the label 5~F. Depocas and L. P. Bouthillier, Rev. can. biol. 10, 289 (1951).

[28]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

671

originates from ethyl acetamidomalonate-2-C 14. The original procedure is t h a t of Albertson and Archer? s Glutamine-C ~4 DL-Glutamine-C ~4. Pure DL-glutamine-C ~4 m a y be prepared from glutamic acid-C ~4 in 34% yield. The position of the label corresponds to t h a t in the glutamic acid used as starting material. The procedure has been carried out in the authors' laboratory with OL-glutamic acid-2-C ~4 and is a small-scale adaptation of the method of King and K i d d ? 9 The procedure is not suitable for the preparation of L-glutamine-C ~4, as racemization of L-glutamic acid occurs in the course of the synthesis.

0

HOOCCH.~CH2~HCOOH

I

phth~lic A c 2 _ O / ~ N - - N J anhydrid--~ -0

l

~'"l 0

I

II

\ ~--~ 0

0 II • H~NNH~ [I • H2NCCH2CH2CHCOOH ~H~NCCH2CH~CHCOOH 0

I

N

I

NH2

O~-C C~O

<5 Procedure: DL-Phlhalyl Clutamine-2-C 14. DL-Glutamie aeid-2-C 14 (294 mg., 3.6 ~e./mg.), 300 mg. of powdered phthalie anhydride, 1.1 ml. of dry pyridine, and a small boiling chip were placed in a 10-ml. pear-shaped flask equipped with a reflux condenser. The flask was placed in an oil bath at 125 °. As the b a t h temperature was raised to 140 °, smooth boiling commenced, and all but a small residue went into solution. The mixture was refluxed for a total of 2 hours, cooled, and the solvent removed in vacuo at 30 °. A total of 2 ml. of acetic anhydride was added in such a way as to wash down the distilling head. The resulting mixture was boiled for 2 minutes, cooled, and most of the acetic anhydride removed in vacuo. T h e crystalline residue was treated with about 5 ml. of dry ether and stirred thoroughly. The distilling head was washed down with an additional 1 to 2 ml. of ether. This was added to the flask, and the mixture

ss N. F. Albertson and S. Archer, J. Am. Chem. Soe. 67, 2043 (1945). 59 F. E. King and D. A. A. Kidd, J. Chem. Soc. 194% 3315.

[2~]

673

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

was obtained. One further such treatment gave 99 mg. of DL-glutamine2-C 14 (34 % over-all yield), which showed only one spot on paper chromatographs, corresponding exactly with the only spot obtained by radioautographs. No detectable amounts of foreign ninhydrin positive material or pyrrolidone carboxylic acid were present. From the combined mother liquors, including the original ethanol precipitation, approximately 20 mg. of crude DL-glutamine-2-C 14 was recovered by evaporation of the solvent, solution in water, adsorption on a column of Dowex 1, and elution of the glutamine-C ~4 with acetic acid. Glycine-C ~

Glycine-1- and 2-C ~4. Glycine-C t4 is important not only as a labeled amino acid valuable in biochemical research but also as an intermediate in the synthesis of other amino acids of more complex structure. It has been prepared by many workers and in many laboratories. The most generally applied procedure is the following reaction sequence, or some modification thereof: O Zl

O o.

,/I

O

,11

o

C n ~ C - - B r --~ BrCH2C--Br --~ o

*

N~

BrCH2COOH --

o

*

H2NCH.oCOOH 55-60 %

The procedure of Bloch 19 is claimed to be more convenient and to give better yields than similar methods described in the literature. Little handling of radioactive intermediates is necessary, and after the distillation of acetyl bromide all further operations can be carried out in the same flask. The procedure describes only the preparation of glycinc-l-C I4. However, by starting with acetate-2-C '4, the method can readily be extended to the preparation of glycine-2-C '4. The yield of acetate-l-C ~4 from BaC~403 is at least 90%; the yield of acetate-2-C ~4, approximately 60%. Thus the respective over-all yields of glycine-1- and 2-C '4 by this synthesis are approximately 50% and 35%. Procedure. Anhydrous potassium acetate was heated with benzoyl bromide in the presence of benzoic acid to yield acetyl bromide as described by Anker. 8° The acetyl bromide was collected in an ice-cooled flask, and 1.1 moles of bromine per mole of acetyl bromide was added slowly. The mixture was warmed and heated on a steam bath for 11/~ 6~ H. S. Anker, J. Biol. Chem. 176, 1333 (1948).

674

TECHNIQUES FOR ISOTOPE STUDIES

[~8]

hours and then freed of excess bromine and of HBr by a stream of nitrogen. An excess of water was added dropwise to the ice-cooled bromoacetyl bromide. The clear aqueous solution of bromoacctic acid was then added to a volume of concentrated NHs containing 70 moles excess. The solution was kept at room temperature for several hours and then evaporated i n vacuo to dryness. The residue was dissolved in a small volume of water, and the glycine precipitated by addition of 4 vol. of methanol. The yield, after one recrystallization from water-ethanol, was 55 to 60%, based on the potassium acetate used. The radioactivity of the glycine was 100,000 c.p.m, counted as an infinitely thick sample after conversion to BaC03. Other Syntheses. Glycine-C ~4 has been synthesized in 79% yield by the amination of chloroacetic acid-C~4.2°,4~ A completely different synthesis of glycine-2-C ~4 has been reported by Ehrensviird and Stjernholm 6' in which the authors claim superior ease of handling, better yields, and greater speed of preparation. They used C ~a but point out that C ~4 could be readily substituted. *

CNH--C~S + KCN

f

NH¢

basic

load

*

) ¢N~C--CN

LiA1H4

I

carbonate

*

---) cN-~C--CH~NH2

I

NH~

NH¢ Ba(OH) 2

*

HOOCCH~NH~

If a 90% over-all yield e2 of KC14N from BaC14Oa is assumed, the over-all yield for glycine-2-C 14 becomes 45%. Sakami et al. 63 have prepared glycine-l-C ta in 81% yield from NaC~3N " by the following sequence of reactions: 0

O

C

C

II

II

0/

\

NCH:C1 + KCN --~

*

hydroSysm

*

NCH~CN --~ H.oNCH2COOH

/

/

C

rl

C

O

O

II

61 G. E h r e n s v ~ d and R. Stjernholm, Acta Chem. Scand. 3, 971 (1949). 6~ F. L. J. Sixma, H. Hendriks, K. Helle, U. Hollstein, and R. van Ling, Rec. tray chim. 73, 161 (1954). 6s W. Sakami, W. E. Evans, and S. Gurin, J. Am. Chem. Soc. 69, 1110 (1947).

[9.8]

METABOLIC STUDIES OF AMINO ACIDS ANI) PROTEINS

(}75

Borsook et al. 64 report the use of NaC~4N in place of the NaC~aN but give no details. The yield of glyeine-l-C '4 by this method would be about 75% based on carbonate, on assumption of a 90% conversion of BaCt4()~ to NaC'4N. Histidine-C

~4

DL-Histidine-COOH-C1L The following synthetic pathway, 64 based on the preparation of an oxazolone, leads to histidine-COOH-C '4 in ,~ reported yield of 20% from glycine-l-C ~4 (or approximately 14% from BaC~403). The glycine was prepared by the method of Sakami el aI. 6"~ (see Glycine-CX4). O N • il , / % H 2 N C H 2 C O O H --+ q~CNHCH2COOH ÷ O H C - - C CH --~ CH ~--C

N

I

N

I

0

/%

C~CH-- C

\

II

/

C*

CH-

N--Ac

0

N

[I

/%

CH -~ ~C--NH--C--CH

I

N--Ac

I

"

*COOH

i --"

N

II

O

O

II

N

¢C--NH?HCH--~ *COOH

%~ _~ DL_Histidine_COOH_Cl4 -N

Procedure: Hippuric Acid. To 2.11 g. of glycine-l-C 14 dissolved in 22 ml. of 10% NaOH, a mixture of 9.65 ml. of benzoyl chloride and 45 ml. of 10% N a O H was added gradually with stirring. Stirring was continued until all the benzoyl chloride was destroyed. Acidification precipitated hippurie acid, which was purified by repeated extraction with boiling ligroin. Yield, 4.96 g. (98.6%) ; m.p. 183 to 185 ° (uncorrected). 2-Phenyl-4-( l-acetylimidazole-4 (or 5)-methylidene) Oxazolone. Hippuric acid (4.96 g.) was treated with 3.10 g. of imidazoieformaldehyde ~5 and with 3.10 g. of sodium acetate in 12 ml. of acetic anhydride. Yield, 5.74 g. (74%). a-Benzoylamino-fl-imidazole-4 (or 5)-acrylic Acid. Oxazolone (11.95 g.) was boiled with sodium carbonate. 66 Yield of h y d r a t e d acid, 9.81 g. (84%). 6~H. Borsook, C. L. Deasy, A. J. Haagen-Smit, G. Keighley, and P. H. Lowy, J. Biol. Chem. 196, 669 (1952). 6~j. R. Totter and W. J. Darby, Org. Syntheses 24, 64 (I944). ~ F. L. Pyman, J. Chem. Soc. 109, 186 (1916).

676

TECHNIQUES FOR ISOTOPE STUDIES

[28]

Benzoyl-DL-histidine. To 1.50 g. of a-benzoylamino-/~-imidazole-4(or 5)-acrylic acid suspended in 15 ml. of water, 20 g. of 2.15% sodium amalgam was added gradually. The aqueous solution, separated from the mercury, was acidified to pH 5.2. The precipitate was filtered and washed with water; additional crystals were obtained from the concentrated mother liquors. Yield, 51%; m.p. 238 to 239 ° (uncorrected). DL-Histidine Hydrochloride. Benzoyl-DL-histidine (1.95 g.) was refluxed for 4 hours with 100 ml. of 20% HCI. The aqueous phase was dried in vacuo, after removal of the benzoic acid by repeated ether extraction. I)L-Histidine. The free base was obtained either by treatment of the hydrochloride with silver carbonate in the usual way, or by electrodialysis. The latter method gives a purer product and a greater yield. HistidineHC1 (170 rag.) dissolved in 100 ml. of water was placed in a glass cell, in which were suspended two cellophane bags 2.5 cm. wide, each containing a platinum wire mesh electrode and distilled water to the level of the surrounding solution. The electrodes were in series with a 30-watt 11hvolt lamp and connected to a 120-volt direct current. The solution was stirred occasionally with a glass rod; every 30 minutes the electrodialysis was interrupted and the catholyte and anolyte were removed by pipet and replaced by distilled water. After 4 hours the combined catholytes were evaporated to dryness in vacuo. Yield, 97 %. Other Syntheses. The method of Bouthillier and D'Iorio 67 uses the following reaction sequence:

N

CN

I '

N

CH2CIJr

H--cooEt-+ [

NHAc

~--I

N

COOEt I * hydrolysis

I

NHAc DL-Histidine- C O OH- C'4

The method as reported yields histidine in 6% yield based upon NaC'4N. The low yield is in part due to large losses during the preparation of ethyl acetamidocyanoacetate-C 14. By substituting the procedure of Fields et al. ~5 for preparing this compound (see Lysine-2-C ~4) the yield may be trebled, becoming approximately 18 % based on NaC14N. A drawback to the method is the fact that hydrolysis and decarboxylation of the final cyanacetamido derivative leads to partial loss of the radioactive carbon, thus diluting the specific activity of the histidine-C14OOH. DL-Histidine-a-C ~4. This compound has been synthesized by Wolf u as follows, using the method of Bouthillier and D'Iorio G7 (see also 87 L. P. Bouthillier and A. D'Iorio, R e v . can. biol. 9, 382 (1950).

678

TECHNIQUES FOR ISOTOPE STUDIES

[28]

N a C N + S --~ N a S C N

0

II

, 1

NH2

!

l

N

i

N

NH2

C

I

SH

I

N

N

NH~

\,,,~

C ~-Keto6rnithine is prepared from L-histidine and retains its L-configuration. The reaction with sodium thioeyanate-C TM reconstitutes the imidazole ring without raeemization at the a-amino position, ultimately yielding the natural isomer of histidine-2-C TM. The procedure of Borsook et al. ~ is reported in good detail and may be taken as indicative of all the reported procedures. Most of the other reports give only general directions, referring to the original literature for details. Procedure: Sodium Thiocyanate-C 14. NaSC14N was prepared by the following adaptation of the method of Castiglioni. 7~ Fifty-six milligrams of NaC14N and 40 mg. of sulfur were refluxed with 1.7 ml. of acetone for 45 minutes. The acetone was then drawn off and the insoluble residue refluxed again with 20 rag. of sulfur in 1.5 ml. of acetone for 45 minutes. The combined aeetone solutions were evaporated in a 3-ml. centrifuge tube with a stream of nitrogen. The residue, which consisted of NaSCt4N and some sulfur, was dried over H~S04. Titration of the thioeyanate in similar test runs showed that the NaCt4N is converted quantitatively to NaSC14N. L-Histidine-2-C~4-imidazole. With four 0.5-ml. portions of water (centrifuging each time to remove the sulfur) the NaSC'4N was transferred to a flask containing 835 mg. of o~,~-diamino-~,-ketovalerie acid dihydroehloride and 296 rag. of nonisotopie KSCN. The mixture was heated on a steam bath for 1 hour. Then it was poured dropwise and with constant stirring into a 10% solution of I-IgS04 in 5% H2804 and the mixture allowed to stand 20 hours at room temperature. The precipitate was collected, washed three times with water, suspended in 150 ml. ~1 A. Castiglioni, Gazz. chim. ital. 63, 171 (1933).

[2~]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

679

of water, and decomposed with H~S. The filtrate from the HgS was heated to remove H~S and then brought to pH 8 with Ba(OH)2. The BaS04 was filtered and the thiolhistidine solution concentrated in vacuo to about 2 ml. The concentrate was heated on a steam bath for 1 hour after the addition of 8 g. of ferric sulfate and 40 ml. of water. Two hundred milliliters of hot water was added and, while hot, an excess of Ba(OH)2. The precipitate was filtered and extracted with boiling water, and the combined filtrates were brought to pH 4.0 with H2SO4. After the BaS04 was removed, the solution was concentrated to 5 ml., and 550 .mg. of flavianie acid was dissolved in it with heating. The diflavianate was allowed to separate first at room temperature, then at 0 ° overnight. It was washed twice with absolute ethanol and twice with ether (m.p., 240 to 245°; yield, 486 mg.). Then 484 mg. of radioactive and 760 rag. of nonradioactive L-histidine diflavianate were dissolved in 12 ml. of 2 N H2S04 and extracted seven times with warm n-butanol. The aqueous phase was boiled with Norit, filtered, and brought to pH 7.1 with Ba(OH)2. The filtrate from the BaSO4 was concentrated in vacuo, transferred to a small centrifuge tube, and dried in a stream of nitrogen. The crystalline L-histidine was dissolved in 0.5 ml. of hot water and allowed to cool slowly to 0°; 2 to 3 vol. of absolute ethanol was added after the bulk of the crystals had separated. A main crop of 210 mg. of L-histidine-2-C 14imidazole was obtained after washing three times with absolute ethanol and once with ether; m.p. 272 ° (dec.). Leucine-C

14

DL-Leucine-1- and 2-C 14. These amino acids have been prepared by Hauptmann et al2 s according to the following equations:

CH,~

CH3 *

/

CHCH2COOH

CH:~

LiA1H,

~

--~

*

CHCH2CH~OH

/ CH3

CH3 CH3 \ • H6N \ • CHCH2CH2Br ~ C H CH2CH2COOH ° --~ / OH/ CH3

CH3 CH3 *

o

NHa

C H C H ~ C H C O O H -----~ DL-Leucineq- or 2-C 14 CH~

/

I

Br

680

TECHNIQUES FOR ISOTOPE STUDIES

[28]

The reduction of isovaleric acid was carried out by first preparing the cetyl ester and then reducing this over copper chromite catalyst. The one-step reduction with lithium aluminum hydride 7~ was not used in order to avoid contamination with alcohols caused by solvent splitting (see Alanine-C14). However, this difficulty should not arise if ethyl ether is the solvent, since any small amounts of extraneous alcohol formed would be much lower boiling than the main product and could be removed by distillation. The use of lithium aluminum hydride would greatly simplify the procedure (see Leucine-3-C 14, below). The conversion of isoamyl bromide to isocaproic acid is best carried out via the nitrile as indicated in the equations; the yields obtained via carbonation of the Grignard reagent from isoamyl bromide are considerably lower (40% and 55 to 65%, respectively, for the two methods~8). The labeled leucines obtained by the procedure given below were chromatographically and radioautographically pure. Procedure: Isoamyl-l-C it Bromide. Sodium isovalerate-l-C 14 (2.10 g., 16.9 millimoles, 7.09 mc. total activity) was prepared in 96% yield by carbonation of the corresponding alkyl Grignard with C1402 in 96% yield. 73 The salt was added to an ignition tube containing 5.40 g. of cetyl bromide (17.7 millimoles). The tube was sealed and heated with shaking at 280 ° for 11 hours. After reaction, the ignition tube contents were dissolved in ether and filtered. An ether-insoluble, water-soluble residue containing 0.05 inc. of C ~4was left on the filter paper. The filtrate was washed into a 200-ml. stainless steel reaction vessel 74 and warmed to 40 to 50 ° to evaporate most of the ether. After addition of 5.5 g. of copper chromite, 75 the reaction vessel was closed, evacuated, and maintained at a pressure of 50 t~ for 1.5 hours to remove traces of ether. The reaction vessel was filled to a pressure of 170 arm. with hydrogen and heated at 250 ° with shaking for 10 hours. The hydrogen was released through a spiral trap equipped with a sintered disk 4° and cooled with liquid air. The reaction vessel was then evacuated and maintained at reduced pressure overnight while warmed to approximately 80 °, and the distillate was collected in a trap cooled in liquid nitrogen. The alcohol thus obtained was converted to the bromide with phosphorus tribromide. 18 The isoamyl bromide was purified by washing with 72 R. F. Nystrom and W. G. Brown, J. Am. Chem. Soc. 69, 2548 (1947). 73M. Calvin, C. Heidelberger, J. C. Reid, B. M. Tolbert, and P. E. Yankwich, "Isotopic Carbon," pp. 142, 179. John Wiley & Sons, New York, 1949. 74Micro series reaction vessel. American Instrument Co., Silver Springs, Maryland. 7bH. Adkins, Org. Syntheses 2, 144 (1943).

[28]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

081

water and drying over phosphorus pentoxide; yield, 1.99 g., or 73 % based on the isovalerate used. Isocaproate-2-C ~4. The isoamyl bromide (1.88 g., 12.4 millimoles) was distilled into a vessel containing 1.65 g. of K C N (25 millimoles) and 0.2 g. of potassium iodide in 25 ml. of ethanol. T h e reaction mixture was refluxed

FIG. 4. Acid generation unit and bromination vessel with condenser separated. Flask, 60 ml.; centrifuge tube, 15 ml. for 42 hours, cooled, and 4 g. of silver sulfate and 30 ml. of water were added. A distillation head and condenser were attached, and the labeled nitrile was slowly distilled out. T o the distillate, 15 g. of K O H was added, and the solution was allowed to reflux for 24 hours. The mixture was acidified with 50 ml. of 10 N tt2S04, and the acid steam-distilled to give 0.949 g. of sodium isocaproate-2-C 14 with a specific activity of 2.90 pc./mg. (calculated 3.01 pc./mg.) ; yield, 55% based on isoamyl bromide used. DL-Leucine-2-C 14. a. BROMINATION OF ISOCAPROIC ACID-2-C TM. Sodium isocaproate-2-C 14 (0.508 g., 2.90 ~c./mg.) was placed in a Pyrex tube which was connected to a spiral trap (Fig. 4), and the salt and equipment were dried in vacuo at room temperature. The system was disconnected

684

T E C H N I Q U E S FOR ISOTOPE STUDIES

HO(CH2)4CHO

(NH+)~CO3

> HO(CH2)4--CH

*

NaCN

[2~]

C ~ O --,

I

J

NH

NH

\/ C

Jl

O * hydrolysis C--O +

~---0 N_~ H2N(CH~)4CH

Br(CH2) 4--CH

r

I

NH

NH

\/

J

I

NH \

NH /

C

C

0

O DL-Lysine-l-C '4

The latter workers simplified the procedure and claim an improved yield of DL-lysine-l-C '4 (21% over-all, based on cyanide-C'4). Procedure. Two changes were made in the procedure of Gaudry. 77 The solution of ~-hydroxyvaleraldehyde from the hydrolysis of dihydropyran was converted to 5-~-hydroxybutylhydantoin in one step by adding ethanol and heating with (NH4)2CO3 and NaC'4N. Also, 5-6-aminobutylhydantoin was hydrolyzed to lysine by heating with 48 % HBr under a reflux for 24 hours. The radiochemical yield of DL-lysine-l-C 14 was 21% based on NaC'4N. Other Syntheses. Lysine-l-C 14 has been prepared by the carbonation of cyclohexanone with C1402 in liquid ammonia, followed by esterification of the product. Treatment of the resulting methyl-2-ketocyclohexane-1C14-carboxylate by the Schmidt reaction yielded lysine-l-C ~a in 12% over-all yield. 79 In addition, a recent Russian paper reports a simple method for preparing lysine-l-C 14 in a 60% over-all yield. ~ A new and simple procedure gives DL-lysine-l-C t* in 66% yield based on cyanide-C ~4. 7,, The method involves the preparation of DL-a-aminopimelic acid-l-C 14 via the hydantoin, followed by conversion to lysine with hydrazoic acid (Schmidt Reaction).

Et0OC(CH~)4CHO

• Ba(OH)2.) C--O I NH

K&N

- , EtOOC(CH2)+CH (NH,)2CO. I NH

\/ C

Jf

O

[28]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS •

HOOC(CH~)~CHCOOH I

HN3

685

~ DL-Lysine-l-C14

H2SO4

NH2 DL-Lysine-2-C ~4. The following reaction sequence, reported by Fields, et al., '5 offers the best route to the preparation of DL-lysine-2-C ~4. O

H C

COOEt NCH~CH2CH2CH2I -t- *CHCN--~

I

NHAc

\c / O 0

H C

/~~

~"

/ C

COOEt 'hydrolysis N ( C H , ~ ) 4 * C - - C N - - - - - ~ DL-Lysine-2-C ~4

I

NHAc

II

O The yields for the individual reactions are excellent, especially those for tile nitrosation and reduction of ethyl cyanoacetate-2-C 14 to form ethyl acetamidocyanoacetate (90 and 85%, respectively). Special attention has been given to the choice of catalyst and conditions for the latter step. This appears to be by far the best method available for the preparation of ethyl acetamidocyanoacetate-2-C 14. Procedure: Ethyl Cyanoacetate-2-C '4. Conversion of sodium acetate2-C~4 to ethyl cyanoacetate-2-C '4 was achieved by bromination of acetic acid followed by reaction of the product with sodium cyanide and esterification under conditions similar to those described in "Organic Syntheses." s0

79 H. R. V. Arnstein, G. D. Hunter, H. M. Muir, and A. Neuberger, 1962, 1329. 79~M. Rothstein, J. Am. Chen~. Soc. 79, 2009 (1957). so Org. Syntheses, Coll. Vol. 1, 245 (1946), 2nd ed.

J. Chem. Soc.

686

TECHNIQUES FOR ISOTOPE STUDIES

[28]

The labeled compound had a specific activity of 0.30 inc. per millimole. The remaining syntheses were performed with intermediates prepared without isotopic dilution from a sample of labeled ethyl eyanoacetate of this specific activity. Ethyl Isonitrosocyanoacetate-2-C 14. The nitrosation of ethyl cyanoacerate was performed by the method of Snyder and Smith. 81 After acidification with dilute HC1, the reaction mixture was extracted with ether. When the ether had been distilled in vacuo, the oxime remained as a crystalline residue, which, after trituration with benzene, was obtained in 90% yield as colorless crystals; m.p. 131 to 133 °. Ethyl Acetamidocyanoacetate-2-C ~4. Reduction of 2.13 g. of the isonitrosocyanoacetate in 50 ml. of acetic anhydride in the presence of 1.0 g. of platinum-on-charcoal (5 %) was carried out at room temperature and at atmospheric pressure with vigorous shaking. When 2 moles of gas had been absorbed, the rate of reduction diminished markedly and the reaction was interrupted. The catalyst was filtered by suction through a charcoal pad and washed well with isopropyl alcohol. When the solvent had been removed in vacuo at 40 ° and the residue washed with ether, there was obtained 2.17 g. (85%) of product; m.p. 129 to 130 °. N-(~-Iodobutyl)phthalimide. A solution of 13.0 g. of anhydrous sodium iodide and 3.19 g. of N-(~-bromobutyl)phthalimide in 85 ml. of acetone was boiled under reflux overnight. After removal of the acetone in vacuo, the residue was triturated with 50 ml. of water, yielding 3.68 g. (99%). Ethyl P-Cyano-2-acetamido-6-phthalimidohexanoate-2-C 14. Ethyl acetamidocyanoacetate-2-C 14 (851 mg.) was dissolved in 5 ml. of anhydrous ethanol in a 100-ml. round-bottomed, three-necked flask equipped with a mercury-sealed stirrer, a reflux condenser, and a steam-jacketed, pressure-equalizing dropping funnel. To the boiling solution was added a solution of 115 mg. of sodium in 7 ml. of ethanol, followed by 1.68 g. of the iodobutylphthalimide dissolved in 10 ml. of boiling ethanol. The reaction mixture was boiled overnight. After cooling and dilution of the mixture with 15 ml. of water, there was obtained 1.86 g. (80%) of product; m.p. 170 to 171.5 °. DL-Lysine-2-C 14 Monohydrochloride. The crude product, 1.53 g. in l0 ml. of concentrated HC1, was boiled under reflux overnight. After dilution of the reaction mixture with 10 ml. of water and removal of the phthalic acid by filtration, the filtrate was brought to dryness in vacuo. The residual lysine dihydrochloride was dissolved in 10 ml. of boiling 95% ethanol and centrifuged to remove the NH4C1. Treatment of the boiling supernatant solution with 400 rag. of pyridine yielded crude lysine monohydrochloride. On crystallization from 1 ml. of water to which 81 H. L. Snyder and C. W. Smith, J. Am. Chem. Soc. 66, 350 (1944).

[28]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

687

9 ml. of ethanol was added, there was obtained 563 mg. of analytically pure lysine monohydrochloride, dec. 259 to 262 ° (evacuated tube). The filtrate afforded an additional 94 mg. of the pure product, bringing the total yield to 87 %. DL-Lysine-6-Cit. The method of Rothstein and Claus 21 appears to be the simplest method for preparing DL-lysine-6-C14: Br(CH2)3C(COOEt)~ -t- K C N --~ NC(CH2)~C(COOEt)2 NHAc

NHAc H2 hydrolysis , DL-Lysine-6-C 14 PtO2 Ac20

The over-all yield was originally reported to be 21%, but 45% of the cyanide-C 14 was recovered, making the actual radiochemical yield 38%. Since publication of the original paper, it has been found that the complete reduction of the cyano ester could be ascertained by hydrolyzing small aliquots of the reduction medium and testing chromatographically for a-aminoadipic acid. The presence of this compound indicates incomplete reduction. In this manner, the yield of lysine-6-C ~4 was raised to 42%, not taking into account recovered cyanide-C14. 8~ Procedure: Ethyl a-Acetamido-a-carbethoxy-~-bromovalerate. For the preparation of this compound, see a-Aminoadipic acid-6-C ~4. Ethyl a-Acetamido-a-carbethoxy-~-cyano-C~t-valerate. To a solution of 3.25 g. of KC~*N (50 millimoles, 0.19 mc. per millimole) in 49.5 ml. of water and 165 ml. of absolute alcohol was added 16.20 g. (50 millimoles) of ethyl a-acetamido-a-carbethoxy-~-bromovalerate, and the mixture was refluxed for 2.25 hours. The hydrogen cyanide evolved during refluxing and subsequent solvent distillation was absorbed in a trap containing 35 ml. of 1.5 N NaOH (carbonate-free). The reaction flask was placed in a refrigerator overnight. The solvent was then completely removed by distillation under diminished pressure, and the residue extracted with several small portions of ether. The extracts were dried and the ether evaporated, leaving a brown oily product, s~ D~,-bysine-6-C14. The crude cyano compound was dissolved in 60 ml. of acetic anhydride, 0.3 g. of platinum oxide (Adams catalyst) was added, and hydrogenation was carried out at 60 p.s.i, and 50 ° for 12 hours. Then 0.2 g. of PtO~ was added, and the hydrogenation continued for an additional 24 hours. At the end of this time, 50 ml. of ice water was added to 8, L. L. Miller, personal communication. 8aIt may be found more convenient to extract the nitrile directly from the reaction mixture with several portions of ether without previous distillation to dryness.

[28]

METABOLIC S T U D I E S OF A M I N O ACIDS AND P R O T E I N S

689

to 50 °. All the methyl iodide had evaporated within 30 minutes. A nitroprusside test on a small sample of the reaction mixture was positive, so 0.025 ml. of nonisotopic methyl iodide was added to the reaction flask through the side arm. A second 0.025-ml. addition was necessary before the nitroprusside test became negative. The liquid ammonia was then allowed to evaporate slowly, with a stream of nitrogen passing through the solution.

r~

L FIG. 5. Apparatus for the preparation of methionine from methyl iodide. The white, solid residue was dissolved in 15 to 20 ml. of water. Hydriodic acid was added until the solution was acid to litmus but still alkaline to Congo red. Insoluble material was separated by filtration. The filtrate was concentrated i n vacuo to 5 or 10 ml. and then heated to dissolve the crystalline material which had separated. T o the hot solution, 100 ml. of boiling absolute ethanol were added. The mixture was kept at 5 ° overnight. The silvery crystals of L-methionine were collected by filtration, washed with alcohol and ether, and dried. The yield was 860 mg. The material melted with decomposition at 283 °, a value identical with t h a t observed for a pure sample of L-methionine. The optical rotation was [a]2~)2 = 7.75 ° (2-din. tube, c = 0.95, in water). Ornithine-C

~4

DL-Ornithine-2-C 14. This amino acid has been prepared by a procedure similar to t h a t used for the synthesis of DL-lysine-2-C14:~5

690

TECHNIQUES FOR ISOTOPE STUDIES

[28]

O

If C

COOEr

CN

N--CH2CH~CH2I + *CHNHAc--~ PhthN(CH2)3C--COOEt

\c /

I CN

t NHAc

II O

hydrolysis+ DL-Ornithine-2-C TM

Procedure: Ethyl 2-Cyano-2-acetamido-5-phthalimidopentanoate-2-C l ~. N-(~,-Bromopropyl)phthalimide (m.p. 73 to 75°), prepared by the procedure of Rumpf, S6was converted to N-(~,-iodopropyl)phthalimide (m.p. 84 to 86 °) by reaction with sodium iodide in acetone. The reaction of ethyl acetamidocyanoacetate-2-C ~4 and N-(~,-iodopropyl)phthalimide under the conditions used for the preparation of the corresponding derivative of DL-lysine-2-C TM (see DL-Lysine-2-C TM) resulted in a 75% yield of product; m.p. 211 to 213 °. DL-Ornithine-2-C TM Monohydrochloride. Hydrolysis of the phthalimido ester in concentrated HC1 and conversion to the monohydrochloride as described for the preparation of DL-lysine monohydrochloride afforded DL-ornithine monohydrochloride in 79% yield; dec. 215 to 216 ° (evacuated tube). Phenylalanine-C'4 DL-Phenylalanine-COOH-C TM. Carboxy-labeled phenylalanine has been prepared in 28% yield 8 based on BaC~403, as follows: ~bCH2CHO + NaCi4N + NH4C1--* ¢CH2CHC'4OOH

I

NH.,

Procedure: Phenylalanine-COOH-C ~4. The phenylacetaldehyde used in this synthesis was freshly prepared by refluxing 10 g. of 1,1-dimethoxy2-phenylethane with 7 ml. of acidic 50% methanol for 10 minutes. After the solvents were removed by distillation under reduced pressure, the crude phenylacetaldehyde was added to a solution of 0.5575 g. of NH,C1 in 1.7 ml. of water and cooled to 0 °. This was immediately followed by the methanolic solution of NaC~4N (15 ml.). An additional quantity of nonactive N a C N (0.5375 g.) dissolved in 6 ml. of 50% methanol was added very slowly to the reaction mixture, which was then allowed to stand at room temperature for 18 hours. One milliliter of 50% NaOH solution was added to the reactants, and s6 p. Rumpf, B u l l . soc. c h i m . [5] 5, 871 (1938).

[28]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

691

the alcohol removed by distillation in vacuo. The oily layer was separated from the aqueous solution of inorganic salts by extraction with ether. The ethereal solution was acidified with 10 ml. of 30% (by volume) HC1 and distilled into a solution of NaOH. The gummy residue was heated with three 10-ml. portions of 3 N HC1 at the boiling point to extract the phenylalanine from the gummy mass. After the extracts were combined, the solution was evaporated to dryness under reduced pressure. It was found necessary to treat the crude phenylalanine with fuming HC1 at room temperature in order to remove the last traces of gummy impurities. The phenylalanine solution was reduced to 2 ml. in volume; 5 ml. of ethanol was added, and the pH adjusted to 5.96 with alcoholic ammonia. On cooling, phenylalanine separated out; this was then filtered off. The mother liquor was treated with 0.1 g. of Norit A, and the volume reduced to 0.5 ml. of ethanol and cooled to 0% The combined crops of phenylalanine were recrystallized from alcohol, and a yield of 304.7 rag. was obtained. Other Syntheses. Phenylalanine-1- or 2-C 14 can be prepared in 50% over-all yield based on hippuric acid-l- or 2-C '4 by use of the azlactone method. 26 Randomly benzene ring-labeled phenylalanine has been prepared in reasonably good yields by the azlactone method starting from benzene-C~4, 27 and phenylalanine singly labeled in the 4 position of the benzene ring has been prepared. 32 However, in the course of the latter procedure, the material was diluted considerably by the addition of inert intermediates, presumably to facilitate handling, resulting in a final product which did not have a high specific activity.

Hydroxyproline-C14 DL-Hydroxyproline-4-C ~4. The synthesis of this compound is rather long, and the yield quite small (3% over-all from BaC~40.~). However, it is the only published procedure available:87 CH2(COOEt)~ + CICH2CHCH2 --~ C I C H 2 C - - C H 2 - - C - - C O O E t --*

I/

1

0

1

O

C--O

C1

,i

CICH2C--CH2C--COOEt 0

C~---O

NH4OH

+HO-*~N/~--COOI{ H

a~ R. Gianetto and L. P. Bouthillier, Can. J. Biochem. and Physiol. 32, 154 (1954.)

692

TECHNIQUES FOR ISOTOPE STUDIES

[28]

Procedure: Bromoacetic Acid. Anhydrous sodium acetate-2-C TM was transformed into bromoacetic acid by the method of yon Auwers and Bernhardi. 88 The yield of distilled bromoacetic acid was 85 to 90% based on sodium acetate. Sodium Cyanoacetate. The m e t h o d of Scarborough s9 was modified to prepare this substance (see also DL-Lysine-2-C14). Three grams of K C N was dissolved in a little more than the a m o u n t of 3 M N a O H required to neutralize the bromoacetic acid obtained in the preceding step. This solution was added to the bromoacetic acid cooled to 0 °. The reaction mixture was then heated on a water bath for 30 minutes and evaporated to dryness under reduced pressure. Diethyl Malonate. This substance was prepared from sodium cyanoacetate according to the method devised by Christie2 ° The yield, based on the a m o u n t of carbon dioxide employed as radioactive starting material, was 30 to 55%. ~-Chloro-'y-valerolactone-a-carboxylic Ethyl Ester. The sodium derivative of malonic ester was made to react with epichlorohydrin according to the method of T r a u b e and L e h m a n 2 ~ The lactone obtained in a 66% yield had a boiling point of 174 ° at 708 mm. (corrected). a-~-Dichlorovalerolactone. This compound was prepared by the method described b y Leuchs et al. 9~ with slight modification. The two racemic compounds obtained b y chlorination of ~-chloro-~,-valerolactone-acarboxylic ethyl ester were not separated. The fraction distilling from 149 to 152 ° at 8 to 9 mm. (corrected) was collected, a-~-Dichlorovalerolactone was thus obtained in 75 % yield. DL-Hydroxyproline. a-~-Dichlorovalerolactone was made to react with N H 4 0 H according to the method of Leuchs et al., 92 fractional crystallization of the copper salts, followed b y H~S treatment. Purification of nLhydroxyproline was achieved b y crystallization in water-alcohol. The pure product was obtained with an over-all yield of 3 % based on the a m o u n t of labeled carbonate used as starting material. Serine-C 14 (see also Vol. IV [30]) DL-Serine-~-C TM. The synthesis of DL-serine-f~-C TM is straightforward, and the yield of pure amino acid, based on formaldehyde-C TM, is 67%. Formaldehyde-C TM m a y be prepared in 79% yield from methanol-C TM54 (see also Cystine-fl-C TMfor additional comments). s8 K. von Auwers and R. Bernhardi, Bet. 24, 2209 (1891). s9 H. A. Searborough, Proc. Chem. Soc. 80, 306 (1944). 90C. F. Christie, U. S. Patent No. 2,459,144 (1949). 91 W. Traube and E. Lehman, Ber. 84, 1971 (1901). 0zH. Leuchs, M. Giva, and J. F. Brewster, Ber. 45, 1960 (1912).

[9,~]

METABOLIC S T U D I E S OF A M I N O ACIDS AND P R O T E I N S

695

the specific activity of the ester to one-half its starting value. By reusing the acetate recovered from the diazotization step, the chemical yield of amino acid becomes 36% over-all. However, because of the loss of activity, the yield is considerably less than half this value on a radioactive basis. T h e interconversion of the N-acetyl ester of allothreonine and threonine is brought about in quantitative fashion by simple solution in cold thionyl chloride 93 through formation of an oxazoline derivative. Separation of the isomers is carried out on the ion exchange resin Duolite A-4, b u t no details are given. However, a procedure for separating the isomers on Dowex 50 is given below in the section on threonine-l,2-C 14. The acids m a y also be separated by fractionation of their sodium salts in alcohol, 94 although less conveniently. Procedure. Acetyl chloride was prepared from 25 millimoles (representing 1 inc.) of sodium acetate-2-C 1~ according to the method for preparation of acetyl bromide. 6° The fraction boiling at 43 to 56 ° was diluted with 35 ml. of anhydrous ether and added dropwise to 6.9 g. (45.3 millimoles) of ethyl sodium acetoacetate in 100 ml. of ether at - 7 0 °. The precipitated ethyl sodium diacetoacetate was filtered, washed with ether, and dissolved in 160 ml. of absolute ethanol. After addition of 15 g. of NaHCO~ and 30 ml. of water, a solution of phenyldiazonium chloride, obtained from 2.5 nil. of aniline, was added at 10 ° over a period of 20 minutes. The solution was stirred for an additional 20 minutes at l0 °, chilled, and diluted at intervals with 25-ml. portions of cold water until no further precipitation occurred. After removal of the phenylazo ester, the filtrate was concentrated, acidified with H2SOt, and distilled into N a O H to recover radioactive sodium acetate. The latter compound was freed from most of the contaminating NaC1 by extraction with ethanol. The 14.5 millimoles of sodium acetate recovered in this manner was converted into ethyl a-phenylazoacetoacetate as in the first run. The total product was used to prepare a mixture of threonine and allothreonine hydrochlorides 93 (approximately 4:1). The free amino acids were obtained by passage through a column of Duolite A-4; yield, 1.4 g. DL-Threonine-l,2-C it. The method used to obtain threonine-l,2-C ~4 is shown in the following equations: 96 * * pyridine * * CH:~CHO ÷ C H 2 ( C O O H ) 2 - - - - ~ CH3CH = C H C O O H --~ * * Allothreonine~ . . ~14 C H 3 - - C H . . . . . C H C O O H --~ Threonine ~-t,z-t~: OCHa NH~ g6 A. T. Shulgin, O. G. Lien, Jr., E. M. Gal, and D. M. Greenberg, J. Am. Chem. Soc. 79, 2427 (1952).

696

TECHNIQUES FOR ISOTOPE STUDIES

[28]

The actual yield of pure DL-threonine-l,2-C 14 is not stated but is quite small, since 0.58 g. of the N-formyl-O-methyl derivative was obtained from 3.6 g. of crotonic acid-C 14 (9.5%). The former compound on hydrolysis yielded nearly equal amounts of threonine and allothreonine. Procedure. C~4-1abeled crotonic acid (3.6 g.) was converted into a mixture of DL-threonine and DL-allothreonine27 The N-formyl-O-methyl derivative that separated out from the first crystallization (m.p. 140 to 158 °, sintering at 316 °) was worked up, yielding 0.58 g. of a white material (m.p. 213 to 216°). The final yield of recrystallized material was 0.443 g. (m.p. 232 to 243 °) having an activity of 3.1 ~c./mg. The material thus obtained assayed microbiologically as 53 % DL-threonine. Recrystallization of the water-insoluble N-formyl-O-methylthreonine isomer yields a product with a melting point of 164 to 170° (sintering at 147°), which, after hydrolysis and isolation, contains much less of a DL-allothreonine contamination. However, the over-all yield is reduced by 50% as a consequence of this second recrystallization. DL-Allothreonine was isolated from the filtrate remaining after crystallization of the formyl derivatives, and this material was combined with the filtrates from the threonine preparations, and worked up by the chromatographic method described below.

Chromatographic Separation of DL-Threonine and DL-Allothreonine. PREPARATION OF THE COLUMN. A section of Pyrex tubing, 16 mm. by 50 inches, was constricted at one end in such a manner as to have a small bulb 1 cm. in diameter and an outlet tip 4 mm. in diameter. The bulb was loosely packed with glass wool, and another 50-inch section of Pyrex tubing was attached to this by means of a short sleeve of rubber tubing. This column was mounted in position, temporarily closed at the bottom, filled with water. A 6-inch funnel was attached to the top of the column, and sufficient resin to fill the column was added as a slurry in 1.5 N HC1. This was permitted to stand until the resin settled to a constant level (40 hours), after which water was allowed to run through the column by gravity flow until the pH of the effluent water was 6 (universal pH indicator paper). The excess resin and water in the funnel and the upper 10 cm. of the column were removed with a pipet. The funnel and rubber tubing were detached, and, as soon as the resin stirred up by the above operation had settled, the water above the resin was removed. SEPARATION PROCEDURE. The C~4-1abeled DL-allothreonine and concentrated DL-threonine mother liquors as described above were dissolved in 6 ml. of water, forced into the column under 18 cm. pressure, and followed with 5-ml. washings of water, each washing being forced into the 07 H. E. Carter and H. D. West, Org. Syntheses 20, 101 (1940).

[28]

METABOLIC S T U D I E S OF A M I N O ACIDS AND P R O T E I N S

697

column by pressure. Elution with 1.5 N HC1 under 18 cm. pressure with the HC1 reservoir level with the top of the column was then started. This gave an average flow rate of 5 ml./hr. After a forecut of 470 ml. had been collected, a fraction collector designed to collect 15-minute samples was placed under the column and used for the remainder of the separation; seventeen 50-mm. shell vials were used as the receivers. Radioactive peaks were obtained for DLthreonine-C '4 and DL-allothreonine-C~4 at volumes of approximately 600 ml. and 650 ml. of 1.5 N HC1.

TI yptophan-C'4 DL-Tryptophan-fl-C TM. The preparation of tryptophan-/3-C 14 is based on the formation of gramine-C ~4 (3-dimethylaminoindole) starting with formaldehyde-C~4.3° Gramine is then readily converted to tryptophan by condensation with ethyl acetamidomalonate followed by hydrolysis of the product :8~,9s

CH~ -}- CH20* + (CH3),NH ~ ,

--0

.-~] -~JH2N/ CH~

I

I

H

H

?

acetamidomalonate

CH2 - - ( C O O E t ) : hydrolysis ~,,,~\N2

NHAc

] H DL-Tryptophan-f~-C~4 The formaldehyde-C ~4 used in this preparation was obtained from BaC~403 by way of methanol-C 14 in 40 to 60% yield. However, other methods lead to formaldehyde-C '4 in much better yield (see Cystinefl-C~4). For this reason, the preparation of formaldehyde-C~ has been deleted from the procedure given below. Procedure. The formaldehyde-C~t obtained from 25 millimoles of C~O2 (40 to 60% yield), was added to a chilled mixture of 1.42 g. of 44% aqueous dimethylamine and 1.42 g. of glacial acetic acid, and the solution was quickly added to 1.10 g. of indole. Heat was evolved, the indole dissolved, and the mixture allowed to stand at room temperature for 18 hours. The light yellow solution was then added dropwise to an ice-cold solution of 1.42 g. of NaOH in 20 ml. of water, and a white 98N. F. Albertson, S. Archer, and C. M. Surer, J. Am. Chem. Soc. 67, 36 (1945).

[9.8]

METABOLICSTUDIES OF AMINO ACIDS AND PROTEINS

699

Procedure: Indole-2-C 14. Sodium formate-C 14 (total activity 2.75 × l0 s disintegrations per minute, 4.92 rag.) was shaken in a 50-ml. flask with o-toluidine hydrochloride (10.0 mg.) and freshly distilled o-toluidine (2.40 g., 2.22 millimoles) on a steam bath. After about 1 minute, inactive 99% formic acid was added and the mixture heated at 100 ° in an atmosphere of dry nitrogen for 12 hours. The amber-colored liquid was then cooled and treated with 1% HC1 which caused the crystallization of formyl-o-toluidine. This was washed with water and dried in vacuo (222 g., 87.5%). Potassium (1.1 g.) was added to tert-butyl alcohol (20 ml.) in a threenecked 100-ml. flask, and the mixture was warmed in an atmosphere of dry nitrogen until the potassium had all dissolved. The formyl-o-toluidine was then added. The excess tert-butyl alcohol was distilled off, and the residue was heated slowly in a metal bath to 350 to 360 °. After 15 minutes all effervescence had ceased and the reaction mixture was cooled. W a t e r was added and steam passed in to steam-distill the indole-2-C TM which was collected in a cooled receiver as silvery white leaflets (0.608 g., 31.4%). The specific activity was 1.37 X 107 d.p.m, per millimole. Synthesis of tryptophan-2-C 14 from indole-2-C TM was carried out essentially as indicated above for tryptophan-f~-C 14. Other Syntheses. T r y p t o p h a n - l - C la has been prepared in 33% over-all yield from glycine-l-C TM 101 by the following reaction sequence: H 2 N C H 2 C O O H - ~ CH2

C~O +

I

I

NH

NH

\ /

I

C

H

II

0

~ \ - - - -~C H I C N

N[

*

- C~O

]

H

reduction~ hydrolysis T r y p t o p h a n - l - C 13

NH

\ / C

II

O There is no reason why C TM cannot be substituted for C 13, thus leading to t r y p t o p h a n - C O O H - C 14. Tyrosine-C 14

DL-Tyrosine-COOH-C 14. The procedure of Loftfield 6 appears to offer the best means of obtaining DL-tyrosine-l-C 14 (and diiodotyrosine-l-C14) :

10~H. W. Bond, J.

Biol. Chem.

175, 531 (1941).

700

TECHNIQUES FOR ISOTOPE STUDIES

CH30~~--CH2CHSO~Na

+ K~N

[28]

(NH4)2CO3

J

OH CH30~--CH~C

0=0

J

hydrolysis --~ DL-Tyrosine- COOH-C 14

J

NH

NH

\/ C

II

O The reported over-all yield from BaC1403 is 52%.

p-Methoxyphenylacetaldehyde-Sodium Bisulfite Compound. Seven grams of sodium was dissolved in a mixture of 50 ml. of absolute ethanol and 50 ml. of absolute methanol. With stirring and salt-ice cooling, a solution of 36.6 g. of ethyl chloroacetate (0.3 mole) and 40.8 g. of anisaldehyde (0.3 mole) in 70 ml. of anhydrous ether was added at such a rate that the temperature did not rise above 5°. This required about 1 hour. The temperature was then allowed to rise to 20 °. The mixture was poured into 650 ml. of cold water and extracted with three 100-ml. portions of ether. The extract was washed with 300 ml. of water, 300 ml. of 3% NaHCO3 solution, and 300 ml. of water. The washed ether extract was diluted with ether to a volume of 500 ml., chilled to 5°, and then poured into an ice-cold solution of 6.9 g. of sodium in 100 ml. of methanol and 6.0 ml. of water. After standing for 10 minutes, the mixture was filtered with suction. The solid sodium salt was worked into a paste with 50 ml. of ether and 10 ml. of methanol and then sucked to near dryness. Before the sodium-p-methoxyphenylglycidate could dry completely to an intractably hard cake, it was worked into a paste with 40 ml. of ethanol and 30 ml. of water. This was added in small portions to a vigorously stirred solution of 70 g. of NariS03 in 250 ml. of boiling water contained in a 1-1. beaker. Eight milliliters of acetic acid was added, and the clear solution was slowly cooled to 0 °. The p-methoxyphenylacetaldehyde-sodium bisulfite compound, weighing 48.8 g., still contained about 13% sodium bisulfite; over-all yield, 56%. 5-(p-Methoxybenzyl)hydantoin-$-C 14. The HCi4N prepared from 196 mg. of BaC140~ (1.0 millimoIe, 500 uc.) was collected in 1.2 millimoles of sodium methoxide and evaporated to dryness in a 12 X 200-mm. Pyrex tube. To this was added 65 rag. of KCN, 600 mg. of p-methoxyphenylacetaldehyde-s'odium bisulfite compound, 600 rag. of powered (NH4)2CO3, and 3 ml. of 50% ethanol-water. The tube was sealed and heated for 4 hours at 100 °. After the tube was cooled and opened, the

[28]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

701

mixture was gradually warmed to 104 ° to decompose the (NH4)2C03 and remove the ethanol; 2.0 ml. of hot water was added, and the clear solution chilled 2 hours in ice. The crystals were filtered, washed, and dried, 1°2 giving 330 mg. (75 %) of product. Recrystallization from alcohol yielded colorless leaflets of p-methoxybenzylhydantoin, m.p. 176 to 179 °. DL-Tyrosine-COOH-C 14. The above hydantoin (240 rag.) was heated in a sealed tube with 2.0 rnl. of concentrated HC1 for 2 hours, beginning at 140 ° and ending at 160 ° . ]?he tube was cooled in ice, opened, and its contents diluted to 10 ml. with water. The solution was heated to boiling, adjusted with NH~OH and acetic acid to a pH of about 6, filtered hot through charcoal, and then cooled. (Frequently no crystals appear unless the solution is filtered through charcoal even though the solution is clear and colorless.) The tyrosine, m.p. 314 to 315 ° (dec.), weighed 175 rag. (89%). Radioactivity yield from BaC~403, 52%. nL-Tyrosine-o~-C ~. The synthesis of this amino acid has been carried out as follows: ~5

CH~O@--CH2Br

C00Et CN •I ,t -t- C H C N --~ C H 3 0 ~ - - C H 2 C C O O E t L

--

NHAc

I

NHAc

48% H O ~ - - C H 2 5 H C O O H HBr

I

NH~ The yield from ethyl cyanoacetamido acetate-2-C ~4 is 91%. Since the latter compound can be prepared in excellent yield (see Lysine-2-C'4), the over-all synthesis, although lengthy, is quite efficient. Procedure: Ethyl-p-methoxybenzylacetamidocyanoacetate. Ethyl acetamidocyanoacetate-2-C TM (850 mg.) was dissolved in 5 ml. of absolute ethanol in a 100-ml. three-necked flask equipped with a mercury-sealed stirrer, reflux condenser, and dropping funnel. A solution of 119 rag. of sodium in 5 ml. of ethanol was added, and the reaction mixture was cooled to room temperature. From a graduated dropping funnel, 0.79 ml. of p-methoxybenzyl bromide l°a (density 1.41) was added dropwise, with cooling. After 2 hours of stirring at room temperature, the slurry was diluted with 10 ml. of water. The product weighed 1.39 g. (96%), m.p. 169.5 to 170.5 ° . DL-Tyrosine-c~-C 14. The product of the above reaction (1.39 g.) was 10~ The authors used a somewhat complicated apparatus to accomplish the isolation of the crystals by centrifugation. ,03 H. Stephen and C. Weizmann, J. Chem. Soc. 1914, 1152.

702

TECHNIQUES FOR ISOTOPE STUDIES

[28]

boiled under reflux in 15 ml. of 48% HBr for 4 hours. After distillation of the excess acid in vacuo, the residue was dissolved in 5 ml. of water and filtered through a charcoal bed. A slight excess of NH40H was added, and then a slight excess of acetic acid. The tyrosine, washed with water and ethanol, weighed 827 mg. (95%), dec. 303 to 304 ° (evacuated tube). Tyrosine-fl-C 14. The following scheme leads to DL-tyrosine-/3-C 14 in 19% over-all yield based on BaC~403:28 CHO

CH=C q- CH

OCH3

~C / II O

C = O --~

C=O ~

I OCH8

/

5

CH~CHCOOH P, I)

I

OH

Procedure: p-Anisic Acid. A solution of p-methoxyphenylmagnesium bromide was prepared under nitrogen from 9.3 g. (0.050 mole) of magnesium. The reaction was started under ether; then the bulk of the bromide was added over a period of 2 hours as a solution in 40 ml. of 50% ether-benzene mixture. The reaction mixture was re fluxed during the addition, then for 6 hours longer to complete the reaction, which was sluggish. At the end of this time, 50 ml. of ether and 50 ml. of benzene were added. This was necessary to dissolve the rather insoluble Grignard reagent. The concentration of the final solution, determined by titration, was 0.276 M. p-Anisic acid was prepared by carbonating 26 ml. (7.2 millimoles) of the Grignard reagent with C1402 generated from 1.02 (5.18 millimoles) of BaC1403. The purified acid weighed 0.64 g. (81%). p-Anisaldehyde. In a three-necked 30-ml. pear-shaped flask were placed 0.64 g. (4.2 millimoles) of p-anisic acid and 5 ml. of purified thionylchloride. The two side necks were plugged, a reflux condenser was inserted in the middle neck, and the mixture was refluxed for 5 hours. The thionyl chloride was removed in vacuo, and the residue was dissolved in 7 ml. of dry c.p. xylene. To the solution were added 0.003 ml. of quinoline-sulfur poison and 0.05 g. of 5% palladium-barium sulfate catalyst; Rosemund reduction of the acid chloride was then carried out by passing hydrogen through the mixture at reflux temperature. The hydrogen was purified by passage through Fieser's solution and Drierite and was admitted to the reaction flask through a bent capillary which was inserted through one of the side necks and extended below the surface

[28]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

705

procedure, 1°~ which, although not usually considered a good method for the preparation of aliphatic amino acids, gives a surprisingly good yield of valine-2-C '4 (54% based on unreeovered glycine-2-C~4) : CH3

O ,

II

•

It2NCH~COOH--~ ~bC--NHCH2COOH +

\ /

C~-O-~

CH3

CH3 \

/ CH~

, C~C

P C~O ---*

I

!

N

O

%/

CH3 \

/

m

, CHCHCOOH

CH3

I NH2

C

I

¢ Procedure: 2-Phenyl-4-isopropylidine(oxazolone-4-C~4)-5. Glycine-2-C ~4 (120 ~c., 0.76 g., 10.1 millimoles) was converted to hippuric acid-2-C ~4by reaction with benzoyl chloride. The product was ground to a fine powder with 0.6 g. of freshly fused sodium acetate and dissolved in 35 ml. of dry acetone. Acetic anhydride (5 ml.) was added dropwise, and the mixture heated under reflux for 15 hours. The hot solution was poured onto crushed ice and diluted to 250 ml. with water. The precipitate was collected by filtration and dried in vacuo. The oxazolone (m.p. 98 to 100°) weighed 1.16 g. (5.77 millimoles, 57% yield from glycine) and contained 71 ~c. or 59% of the initial radioactivity. The filtrate contained 36 uc. (30% of the glycine activity), of which 28 tLc. (23%) was recovered as 0.27 g. (2.42 millimoles, 23.9%) of glycine hydrochloride. Valine-2-C ~4. The oxazolone was mixed with 2.0 g. of red phosphorus and 12.5 ml. of acetic anhydride. HI (12.5 ml., specific gravity 1.7) was added, and the solution was heated under reflux for 20 hours. The reaction mixture was filtered, and the filtrate evaporated to dryness in vacuo. The residue, dissolved in 100 ml. of 70% ethanol, was poured through a glass column containing 60 ml. of Dowex 50 cation exchange resin (50 to 100 mesh) in the acid form. The amino acid remained quantitatively on the resin; all anions were removed by rinsing with 100 ml. of 70% ethanol followed by 250 ml. of water. The amino acid was eluted from the resin with 250 ml. of 2 N NH40H followed by 250 ml. of water. The eluate was evaporated to dryness on a steam bath i n ~racuo. The resulting valine-2-C ~4 (50 ~c., 0.504 g., 4.3 millimoles) was shown to be free from radioactive or amino acid contaminants ,,5 p. T. Adams a n d B. M. Tolbert, J. Am. Chem. Soc. 74, 6272 (1952).

706

TECHNIQUES FOR ISOTOPE STUDIES

[28]

by two-dimensional paper chromatography and radioautography. The yield was 74.5% by weight from the oxazolone (70.5% radioactivity recovery), or 56 % by weight (54 % of the radioactivity) from unrecovered glycine-2-C ~4. DL- V a l i n e - $ , $ ' - C ~4

*CH3

*CH~

\

\

CHI -}- CH(COOEt)2 --) *CH3

/

I

NHAc

CHC(COOEt)2

/

*CH3

P

NHAc hydrolysis DL_Valine_4,4,_C14

Procedure. Isopropyl-2-2~-C14-iodide (13.2 millimoles, from the reduc-

tion and iodination of acetone-l,3-C 14) containing 11.1 me. was condensed with 26 millimoles of ethyl acetamidomalonate in 20 ml. of tert-butanol, containing 13.2 millimoles of potassium tert-butoxide. The condensation was carried out in a three-necked flask fitted with a stirrer, reflux condenser, and dropping funnel through which the halide was slowly added to the reaction mixture. After the addition, the mixture was refluxed for 24 hours. After hydrolysis and decarboxylation with concentrated HC1, the crude amino acid mixture (8.2 mc., 73% of the initial activity) was found to contain valine and small amounts of radioactive impurities. The product was purified on a Dowex 50 column (2.5 cm. in diameter, 160 cm. long) filled with 700 ml. of resin (199 to 200 mesh) with a capacity of 2100 meq. The impure product was applied, washed with 1500 ml. of water, and then eluted with 1 N HC1 at a flow rate of 20 ml./hr. (0.07 cm./min.). The HC1 fractions containing pure valine were combined and distilled to dryness, the residue dissolved in water, evaporated to dryness on a steam bath, and dried i n vacuo. The dry valine-4-4t-C 14 hydrochloride weighed 1.07 g. (54%) and had a specific activity of 6.0 ~c./mg. Twodimensional paper chromatographic analysis showed the valine to be completely free of radioactive and amino acid contamination. An additional 1.73 inc. of valine (15%) contaminated by a trace (less than 1%) of an unknown amino acid was isolated from the earlier elution fractions. Additional Amino Acids The following C l~-containing amino acids have been separated from the main part of the chapter because of their less general interest. CitruUine-C 14. Citrulline has been prepared labeled in the carbamyl group in good yield. 1°e 106L. H. Smith, Jr., J. Am. Chem. Soc. 77, 6691 (1955).

[28]

707

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

•

(H2NCH~CH2CH~CHCOO)2Cu + K N C O - ~

I

H2S ~---~

NH~. O

ii,

H.2NCNH CH,,CH~CH,.,CH C 0 0H

I

NH~ This method could also be used to obtain a chain-labeled compound by preparing ornithine-C 14 and adding the carbamyl group by the above procedure. DL-3,4-Dihydroxyphenylalanine-C ~4. 3,4-Dihydroxyphenylalanine has been prepared in labeled form with C 14 in both the carboxyl and fl-positions. 4,29 The former compound was prepared in 8.5% yield from KC14N by a method similar to that used for tyrosine-l-C14. 6 3,4-Dihydroxyphenylalanine-~-C'* was prepared by the condensation of the appropriately labeled aldehyde with hydantoin in the usual manner. H y d r o x y l y s m• e - 6 - C 1 4 . This amino acid has been prepared as follows:'~4 OH CCH._,CH.~CH2C--(COOEt)2+H C N ~ N C C H CH2CH2C--(COOEt)2

I

NHAc

I

1

OH

NHAc

H, Hydrolysis * --~ ~ ----~ H~_NCH2CHCH2CH~CHCOOH

I

OH

]

NH2

The over-all yield is 34% based on KC~4N. T h y r o x i n e - l - C ~4. Starting from glycine-l-C ~4, thyroxine-l-C ~4 has been prepared in 53% over-all yield by the method of Wang et al. 25 by way of the azlactone synthesis (see Vol. IV [35]). Homoserine-2-C ~4. This amino acid has been prepared in this laboratory essentially by the method of Painter24 ¢OCH.,CH.,Br -~- CH(COOEt)2--~ ~bOCH2CH2C--(COOEt)2 f

i

NHAc

I

NHAc

HBr --~ DL-Homoserine lactone.HBr

The yield was 38%, exclusive of a large amount of additional material in the mother liquors. Norvaline-3-C 14 and Norleucine-3-C H. These amino acids were prepared in good yield by the action of n-propyl iodide-l-C 14 and n-butyl bromide-l-C 14, respectively, on ethyl acetamidomalonateJ5 The halides

708

TECHNIQUES FOR ISOTOPE STUDIES

[28]

were prepared by reduction of the corresponding acids followed by halogenation of the resulting alcohols. ~-Aminovaleric Acid-~-C ~4. This labeled amino acid has been prepared as follows: 23 BrCH2CH2CH2COOEt -~ K ~ N --* NCCH2CH.~CH2COOEt * H2 -* Hydrolysis

*

H2NCH2CH2CH2CH2COOH

Iodotyrosine-C ~4. Iodination of tyrosine-C ~4leads to the diiodo derivative in 57% yield s (see Vol. IV [35]). II. Methods for Isolation and Degradation of Labeled Compounds 1°7

Methods of Isolation for Radioactive Counting Preparation of Samples of Protein, Amino Acids, and Other Solids In experiments involving study of protein synthesis or the incorporation of amino acids into protein it is essential to isolate the protein of tissues free from lipids and nucleic acid material and to prepare from these samples suitable for radioactive counting. A commonly used procedure for the isolation of mixed protein is as follows :108 An amount of the protein solution containing 10 to 50 rag. of protein is precipitated with 10% trichloroacetic acid (TCA) in the ratio of 1 vol. of protein solution to 4 vol. of TCA. The protein precipitate is resuspended in 10 ml. of 5 % TCA four times, in 8 ml. of 95 % ethanol once, in 8 ml. of 3:1 alcohol-ether mixture for 3 minutes at 60 ° three times, and finally in 10 ml. of ether. During the second TCA wash the tubes are heated for 15 minutes at 90° to eliminate nucleic acid components. 109 After the ether wash the tubes are heated under a bank of infrared lamps to dry the protein to a soft, easily transferred pellet. The protein is again dispersed in 2.5 to 3 mh of U.S.P. ether in a small, loose-fitting Potter-Elvehjem glass homogenizer and poured on a tared aluminum disk (4.5 cm. in diameter in our laboratory) held in a special holder. Three or four volumes of petroleum ether is added so as to mix the liquids thoroughly, and the volatile solvents are then evaporated off. The evaporation is accelerated by means of infrared lamps (pairs of 250-watt lamps in series), care being taken to avoid boiling. This procedure yields a smooth layer of protein on the plate that adheres well to the aluminum disk, provided the weight of the protein is not over about 40 rag. The 107 Prepared by D. M. Greenberg. 1 0 s A. E. Peterson and D. M. Greenberg, J. Biol. Chem. 194, 359 (1952). 10~ W. C. Schneider, J. Biol. Chem. 161, 293 (1945).

710

TECHNIQUES FOR ISOTOPE STUDIES

[28]

under infrared lamps, and the dry amino acid hydrochlorides are inserted into a Geiger-Milller counter (thin end window or gas flow) and counted. Crystallization of Amino Acids and Amino Acid Derivatives. To obtain samples of amino acids of sufficient size for isotopic analysis, recourse is often had to the procedure of diluting or trapping the desired labeled amino acid by the addition of known amounts of the inert amino acid and then to crystallize this or some appropriate derivative of it for purposes of radioactive counting. The majority of the amino acids can be isolated in this manner by the addition of ethanol to provide a solvent mixture in which the desired amino acid is insoluble. Purification is achieved by repeated recrystallization of the amino acid from ethanol-water. An alternative procedure is to convert the amino acid into a derivative suitable for isolation, identification, and isotopic measurement. The most important of these derivatives are given below. Crystalline derivatives giving sharp melting points are obtained by condensing p-toluensulfonyl chloride with certain of the amino acids. 114 These amino acids are alanine, glycine, leucine, isoleucine, methionine, phenylalanine, serine, tyrosine, and valine. The formation of the bright yellow dinitrophenyl derivatives of the amino acids H~ with 2,4-dinitrofluorobenzene 116 appears to be suitable for isolating amino acids for isotopic analysis. These derivatives can be fractionated by adsorption chromatography on silieic acid-Celite columns, ~17or by paper chromatography according to the method of Levy. sIs Arginine can be isolated as the monoflavianate by reaction with flavianic acid (1-naphthol-2,4-dinitro-7-sulfonic acid) and then precipitation of the highly insoluble diflavianate. This is then converted to the better defined monoflavianate. ~19 Histidine can be isolated as the di-3,4dichlorobenzenesulfonate. ~° Serine is usually separated as the salt of p-hydroxy-p'-sulfonic acid azobenzene. TM In general, chromatographic methods have superseded the chemical methods of isolation for most of the amino acids. Qualitative determination of radioactivity in amino acids can be performed by counting the unsubstituted amino acids on the strips of paper chromatograms. H4 E. W. McChesney and W. K. Swann, J. Am. Chem. ~oc. 59~ 1116 (1937). 115 The dinitrophenyl derivatives of certain of the amino acids are light-sensitive. Care should be taken to avoid exposure to bright light in order to prevent decomposition. 116 F. Sanger, Biochem. J. 39, 507 (1945); see Vol. IV [9]. 11~ F. C. Green and L. M. Kay, Anal. Chem. 24~ 726 (1952). ~18 A. L. Levy, Nature 174, 126 (1954); see Vol. IV [10]. 11~ H. B. Vickery, J. Biol. Chem. 1329 325 (1950). 120 H. B. Vickery~ J. Biol. Chem. 143, 77 (1942). 121 M. Bergmann and L. Zervas, Z. physiol. Chem. 162, 282 (1926); 172, 277 (1927).

[~.8]

METABOLIC STUDIES OF AMINO ACIDS AND PROTEINS

711

Degradation of Amino Acids to Determine Isotope Distribution There are a number of general reactions that apply to the degradation of the amino acids as a class and that are suitable for determining the distribution of isotopes among the different carbon atoms in the molecule. These consist of the following: The ninhydrin (or chloramine T) reaction specifically liberates the a-carboxyl group and forms an aldehyde, often volatile, with one less carbon. Periodate reacts with ~-hydroxyamino acids, liberating an aldehyde of the terminal carbons from the ~-carbon on. The Schmidt reaction can be employed to degrade carboxylic acids derived from the amino acids, carbon by carbon, as is true for all straightchain carboxylic acids. Investigators who have reported these procedures usually have reported few details of their methods. In addition, the number of variations is very large. Because of this the standard techniques for these reactions will be reported and mention will merely be made of the schemes employed to degrade the different individual amino acids.

Decarboxylation with Ninhydrin The a-carboxyl groups of the amino acids can be decomposed to CO2 and the isotopic content of the carbon determined. When the isotope is C 14 the COs is usually precipitated as BaCO3 and the radioactivity counted. The reaction that takes place may be represented by the following equation: O

O

II

tl

C.

C C(OH)2 -t- R C H N H 2 C O O H =

/ C

II

O

/ C

II

0 + RCHO + NH3 -t- CO..,

The reaction is highly specific for amino acids. Van Slyke and eoworkers 1~2 state: "The reaction is specific for free amino acids in that it requires the presence, in the free uneonjugated state, of both the earboxyl and the neighboring NH2 or NH-CH2 group." The reaction goes to completion with amino acids having a primary c~-amino group, with ¢~-alanine, 122 D. D. Van Slyke, D. A. M a e F a d y e n , a n d P. B. Hamilton, or. Biol. (2'hem. 141, 671 (1941); 1§0, 251 (1953)