ADVANCES IN PROTEIN CHEMISTRY Volume 17
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ADVANCES IN PROTEIN CHEMISTRY EDITED BY
C. B. ANFINSEN, JR.
M. L. ANSON
Department o f Biological Chemistry
London, England
Harvord Medical School Boston, Massachusetts
KENNETH BAILEY
JOHN T. EDSALL
University o f Cambridge
Biological Laboratories
Cambridge, England
Harvard University Cambridge, Massachusetts
VOLUME 17
1962
ACADEMIC PRESS
New York a n d ond don
COPYRIGHT @ 1962,
BY
ACADEMIC PRESSINC.
ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY B E REPRODUCED I N ANY FORM B Y PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.
ACADEMIC PRESS INC. 111 FIFTHAVENUE NEW YORK3, N . Y.
United Kingdom Edition
Published by ACADEMIC PRESS INC. (LONDON)LTD. Berkeley Square House, London,
W.l
Library o j Congress C’utalog Card Nunibor 44-8863
PRINTED I N THE UNITED STATES OF AMERICA
CONTRIBUTORS TO VOLUME 17 B. KEIL, Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Science, Prague, Czechoslovakia S . M. PARTRIDGE, Low Temperature Research Station, Downing Street, Cambridge, England JERKER PORATH, Institute of Biochemistry, University of Uppsala, Uppsala, Sweden S. J. SINGER, Department of Biology, University of California, San Diego, L a Jolla, California
F.
SORM,Institute
of Organic Chemistry and Biochemistry, Czechoslovalc Academy of Science, Prague, Czechoslovakia
CHARLES TANFORD, Department of Biochemist y, Duke University, Durham, North Carolina
D. B. WETLAUFER, Department of Biochemistry, Indiana University School of Medicine, Indianapolis, Indiana*
* Present
address: Department of Physiological Chemistry, Medical School, IJniversity of Minnesota, Minneapolis 14, Minnesota
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PREFACE W. T. Astbury was one of the great men of protein chemistry; his personal and his scientific influence were profound. This volume opens with a brief tribute t o him by one of us who knew him intimately; its aim is primarily to portray Astbury as a man, and touches only incidentally upon his work. Although the study of proteins in mixed solvents, such as water-alcohol mixtures, has a long history, the systematic use of truly nonaqueous solvents for the study of proteins is relatively new. S. J. Singer, who has been one of the leaders in this field, considers what has been achieved, and points the way to further research, in the first review in this volume. As with the article by Witkop in Volume 16 of the Advances, this is in many respects a preview, rather than a review; a most interesting beginning has been made, but far more extensive researches lie before us. Hydrogen ion titration curves of proteins provide a powerful tool to reveal many aspects of the structures of individual proteins. The characteristic ionization constants of the acidic and basic groups in the amino acids and peptides may be profoundly modified when these groups are incorporated in a protein molecule. An increasing number of proteins have been found in which potentially reactive groups are inaccessible for titration in the native molecule, and become available only after denaturation. Such findings can, in the years ahead, be correlated with detailed knowledge of the three dimensional structure of proteins, as obtained by X-ray diffraction and other methods. The present state of the field is reviewed by Tanford, who has done so much to advance it over the last decade. I n the following review Sorm and Keil grapple with a thorny problem. Are there systematic regularities in the structures of the peptide chains of proteins? Do certain sequence patterns repeat themselves more often than would be expected if we assumed a merely random distribution of amino acid residues in the peptide chains? Is it justifiable, or meaningful, to equate one amino acid residue to another when looking for identical, or analogous, sequence patterns in different peptide chains or in different parts of the same chain? Can significant relations be obtained by comparing two sequences, but reading them in opposite directions, as if a n inversion had occurred in one of them? This review addresses itself to these and other difficult problems. The views of the authors are not likely to win general vii
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or complete assent. The problem of formulating what we mean by a truly random sequence is beset by formidable conceptual and practical difficulties; yet the concept of a random sequence is indispensable for the claim that non-random patterns do appear in the peptide chains of proteins. The mathematical analysis, beginning on page 198, is likely to prove difficuIt reading for most biochemists; indeed even some experts in statistical theory, whom the editors have consulted, have remarked on the difficulty and subtlety of the concepts involved. Many biochemists are likely to challenge the concept of “standard amino acid interchanges,” and the use of inverted sequences for comparison of one portion of a peptide chain with another, as set forth by Sorm and Keil. Their ideas were developed before the new experimental evidence on the coding problem was available, and represent essentially a different domain of thought. This article, like others in the Advances, sets forth the personal point of view of the authors, who have had full scope to express their opinions, after examining the comments of various readers. We expect that it will serve as a valuable stimulus to further inquiry. The use of “molecular sieves” for the separation of molecules of different sizes has acquired extraordinary importance lately. The most widely used, and the most satisfactory, molecular sieves available today are the crosslinked dextrans. The theory and practice of their use is discussed in a brief review by J . Porath, whose work has been so fundamental in the development of this field. Elastin is a protein of widespread distribution and great importance. It has, nevertheless, been relatively neglected by biochemists, who have achieved great triumphs in unraveling the structure of its neighbor, collagen, as discussed by Harrington and von Hippel in Volume 16 of the A d vances. In the fifth review appearing here, s. M. Partridge presents what we believe to be the first extensive review of the chemistry and biochemistry of elastin. Partridge’s treatment shows how much knowledge of elastin has already been achieved, but it will be plain to any reader how much need there is for further research on elastin, and how significant a protein it is for the biologist. The use of ultraviolet absorption spectra in the study of protein structure was reviewed by Beaven and Holiday in Volume 7 of the Advances, ten years ago. There has been a vast outpouring of articles on the subject since. The use of ultraviolet spectra for the exploration of protein structure has ramified in many directions, and it is becoming an increasingly powerful tool for the study of the finer details of protein structure. In the final review of this volume D. B. Wetlaufer surveys the field as it stands today. We are indebted, as always, to the staff of the Academic Press for their
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skillful and devoted work in preparing this volume for publication. We also wish to express our appreciation to Mrs. Katherine Torgeson for preparing the index. C. B. ANFINSEN, JR. M. L. ANSON KENNETH BAILEY JOHN T. EDSALL November, 1962
wI I .LI .4M
THOMAS ASTBURY
WILLIAM THOMAS ASTBURY 1898-1 961 A Personal Tribute By KENNETH BAILEY
“Rill” Astbury was born in that region of North Staffordshire known as the Potteries, an island jungle of pot banks, kilns, brickyards, and steelworks, surrounded by a pleasant and varied countryside. Its people are sturdy and stolid with a facet of brittle humor which defies their harsh environment. He grew up on the fringe of the “Five Towns” and could not claim to be a Potteries man, but there was a great deal in Astbury that was characteristic of the neighborhood. He could well have found a place in the great novels of Arnold Bennett. Astbury was the fourth child of a family of seven. His father, William Edwin, was throughout his life a Potter, providing comfortably for the family by this and other lines of business. Except for the wisdom of his schoolmaster, Astbury might have gone into “the Pots”; instead, he won a scholarship at Longton High School. Here, his interests were shaped by the Headmaster and second master, both chemists, and towards the end of his schooldays he won the only local scholarship available and an exhibition a t Jesus College, Cambridge. He crowned his career at school by becoming Head boy and winning the Duke of Sutherland’s Gold Medal. For all this, he was not what we in a nearby school called a “swot” or crammer. He played cricket, made excursions into amateur dramatics and short-story writing, and was good a t sketching. From his father, both he and his younger brother, Norman, inherited a great love of music-an important feature of Potteries life in those days-and the two brothers ranged over vast fields of “one piano, four hands.” His heroes were Bach, Mozart, Beethoven, and Schubert, and though he loved to argue about it, he would never admit that any other composers approached their towering genius. In later life, as he seemed to undergo a phase of philosophical questioning and perplexity, he turned to the violin and enjoyed home-made music with his son, Bill, and anyone who would oblige at the piano. One of the greatest thrills of his life was to have dined with Yehudi Menuhin. Fifty years ago, few people in the Five Towns were famous in the academic sense, and few had the chance to be. During vacations, Astbury spent much time at the Technical College with A. T. Green, who later xi
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became Director of the British Ceramic Research Association, and he also came to know J. W. Mellor, who was Director of the British R,efractories Research Association and compiler of the monumeutJal ‘Treatise 011 Inorganic and Theoretical Chemistry.” Another contemporary and acquaintance was Reginald Mitchell, whose “Spitfire” did so much to save Britain from the Nazi invasion. After only two terms at Cambridge, he was called up in the First?World War, and his poor medical rating, following appendectomy, caused him to serve with the R.A.M.C. in 1917. He was drafted to Cork and there met his future wife, Frances Gould. This break in his studies did not affect his application, and on his return to Jesus College, he took Firsts in both parts of the Tripos, specializing in Physics in his last year. After Cambridge, he joined Sir William Bragg a t University College, London, and two years later (1923) accompanied him to the Davy-Faraday Laboratory a t the Royal Institution. Here, of course, a new world in mathematical crystallography had opened up, which matched in its exciting prospects the sparkling enthusiasm of Astbury’s own approach to his work and hobbies. He not only had the kindly genius of Bragg to draw upon, but his fellow workers must have set a standard in which mediocrity could find no place. Among these were Kathleen Lonsdale, “Sage” Bernal, Robertson, Shearer, and others. The earliest papers were concerned with classic crystallography-the crystal structure of tartaric acid-and were accepted for publication by the Royal Society. He was led to think of structure analysis in the realm of biology through Bragg himself, who, as he says, “besides being by implication a crystallographer, was by induction a biologist, a molecular biologist.” To illust,rate Bragg’s Faraday Evening lectures on “The Imperfect Crystalisation of Common Things,’’ Astbury provided X-ray photographs of cotton, hair, bone, and sea-hedgehog spines. It could not have been by accident that Bragg had first laid the foundation of X-ray diffraction a t the University of Leeds, and that Astbury, in the light of his cursory exploration of biological structures, should go to Leeds in 1928 as Lecturer in Textile Physics. Bernal has pointed out that only Astbury’s imagination and perseverance could have hoped to disentangle what Astbury himself called “pretty dreadful photographs,” and he attempted it according to the Baconian recipe of torturing his material by boiling, stretching, and giving it a permanent wave. In this, his lack of biological background was a positive asset, in that he did not allow the cellular “junk” to obscure his clear vision. He was nevertheless very much aware of the heterogeneity of his material, and it plays a major part in his detailed interpretations of the complex elastic phenomena of mammalian keratin. The clarity of his approach shines through all his work, and though it led him into error, and
WILLIAM THOMAS ASTBURY
xiii
made some of his reasoning somewhat over-plausible, it did lead to those generalizations of fiber structure on which the more definitive interpretations have necessarily been built. The papers published between 1932 and 1934 reflect the bustling activity of the first years at Leeds. In addition t o the classic paper with H. J. Woods on “X-ray studies of the structure of hair, wool and related fibres” there appeared papers on the molecular structure of feather keratin, gelatin, and on the cellulose fibers in the cell wall of the giant alga Valonia. His only book ‘(Fundamentals of Fibre Structure,” which may still be read with the greatest enjoyment, also appeared at this time. Soon after, he was seeking a biochemist who could help him with experiments on soluble proteins. I was introduced to him by A. C. Chibnall, and though I considered myself a confirmed carbohydrate chemist, I was quickly won over to the idea that proteins were much more exciting and fundamental. Certainly, his impact on a young student could be tremendous, his euphoric evangelizing zeal transforming laboratory routine into a great adventure. So began a collaboration which continued on and off until his death. Although the number of his collaborators was small, his output up to the outbreak of World War I1 was remarkable, all the more because of the diversity of the materials he was thinking about-nucleic acids as well as proteins. This was his most creative and happiest period, and it was a great pleasure for me to share it. We began to “torture” the corpuscular proteins and were able to produce the oriented @-structurefrom denatured edestin. The “cross-p*’ pattern observed with “poached egg white” deeply interested him, and accorded well with his thoughts on long-range elasticity. It turned up again and again, in thermally contracted muscle, Rudall’s epidermin, and bacterial flagella. The occurrence of the a-pattern in myosin and muscle, fibrinogen and epidermin pointed to an ubiquity which is adequately recognized today even in the everyday corpuscuIar proteins. Incidentally, and though it in no way affects the argument, the myosin films from Mylilus and the diffraction patterns both of living and dead Mytilus muscle most probably represented still another a-protein not then defined-the paramyosin of Schmitt and Bear, a type of tropomyosin isolated by the author many years later. It is pertinent to mention in passing the widespread soluble tropomyosin which appears to exist in every type of muscle. If one had to discover a protein to please him, this was it. It not only gave a very pronounced a-pattern, but was capable of aggregating into a linearly fibrous form; further it could be made to form true crystals of which 90 % was salt and water. The crystals which T.-C. Tsao sent him, 1 mm long, “knocked him for six,’’ if one may use his cricketting phraseology. Astbury was a notable lecturer and a notable writer. He took enormous
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pains to maintain an easy, flowing style, even where the arguments were particularly subtle or closely-reasoned, and a t all times there was room for the quip or quotation. He was always so much his natural self that he could communicate his personality to an audience within the first few minutes. At a Faraday Society Meeting he was prevented from beginning his lecture by the conversation of some distinguished scientists in the front row. At last he went, up to them, glared, and in his Staffordshire accent said “Shut oop.)’ He was never too famous not to be flattered by an invitation to lecture, whether to “Industry” or to deliver very special lectures-the Spiers, the Proctor (given both in 1940 and 196l), the Jubilee of the Society of Chemical Industry, the Atkin, and the Mather. His Croonian lecture to the Royal Society adhered to the provision of Croone that it be devoted to “the advancement of natural knowledge in local motion,” via., the structure of biological fibers and the problem of muscle. Astbury had an unusual mixture of enthusiasm, idealism, and, beneath the bluff exterior, quit,e a measure of sentimental feeling. His honesty and loyalty induced in him a degree of sensitivity particularly discernible if he thought some friend or colleague had betrayed some trust or shown some insincerity. One might argue or squabble with him and yet feel compelled to forgive any annoyance hc caused, and on his part he very readily forgave it in others. Whatever difficulties he encountered as head of a department, he was always cheering and cheerful. He had to discard his own elegant a-model, but with characteristic enthusiasm took up the implication of the planar amide group in the PaulingCorey structures, considering it successively in relation to the models for wool, the cross @-pattern,collagen, and feather keratin. His contribution to fiber structure rests not so much on the models themselves as on his pioneer attempts to provide them. Historically, his distinction will seem to lie in the generalizations he made about the types of naturally occurring fibers, the master plans which characterize the a- and p-structures and the connective tissues. He explored their ubiquity arid evolutionary modifications, and laid down the principles on which their complex elastic behavior must be explained. The many honors he received were less important to him personally than as a recognition of the new Science of which he was both Master and Prophet, a world of order and plan, the vast field of Molecular Biology as we know it today. He lived to see it flourish and he lived also to see the crowning triumph of X-ray crystallography in the structures for DNA, myoglobin, and hemoglobin. To me, a privileged friend for more than 25 years, his work and character are inextricably mixed. Time may blur the edges of his personality, but will not obscure the pioneer qualities so evident in his writings that led him forth into his great adventure in the world of fibrous molecules.
CONTENTS CONTRIBUTORS TO VOLUME 1 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PREFACE.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Vii
WILLIAM THOMAS ASTBURY ...........................................
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v
The Properties of Proteins in Nonaqueous Solvents
S. J. SINGER I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 11. Characteristics of Nonaqueous Solvents of Interest. . . . . . . . . . . . . . . . . . 2 5 111. Solubility of Proteins in Nonaqueous Solvents. . . . . . . . . . . . . . . . . . . . . . IV. The Conformations of Protein Molecules in Nonaqueous Solvents. . . . . 10 V. Physical and Chemical Properties of Proteins in Nonaqueous Solvents. . 59 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
The Interpretation of Hydrogen Ion Titration Curves of Proteins CHARLESTANFORU
I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . 11. Dissociation Constants of Appropriate Sm 111. Experimental Titration Data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 IV. Counting of Groups. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 V. Reversibility ; Thermodynamic and Kinetic Analysis. . . . . . . . . . . . . . . . . 90 VI. Semiempirical Thermodynamic Analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . 95 ....................... 121 VII. More Exact Thermodynamic Analysis. . VIII. Volume Changes Accompanying Titratio IX. Miscellaneous Topics. . . . . . . . . . . . . . . . . ....................... 127 X. Results for Individual Proteins, . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 References. . . . . . ........................................... 161 Regularities in the Primary Structure of Proteins F. SORM AND 13. KEIL I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Types of Structural Regularities in Proteins. . . . . . . . . . . . . . . . . . . . . . . . 111. Comparison of Proteins and the Search for Common Types of Structural Regularities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Mathematical Approach to the Evaluation of Structural Similarities between Proteins.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv
167 171 191 198
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V. Relevance of Structural Regularities to Protein Synthesis. . . . . . . . . . . . 201 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Cross-Linked Dextrans as Molecular Sieves
JERKER PORATH
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Principle and Mechanism, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Simple Fractionations Employing Highly Cross-Linked Gels. . . . . . . . . IV. Separation of Peptides and Proteins by Molecular Sieving. . . . . . . . . . . . V. Concluding Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
209 211 211 216 224 224
Elastin S. M. PARTRIDGE I. Introduction. . . . . . . . ........................................ 11. Occurrence and Morp gical Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Physical Properties of Elastin. . . . . . . . . . . . . . . . . . . . . . . . . IV. Isolation and Analytical Characterization of Elastin. . . V. Elastolytic Enzymes.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Chemical Structure of Elastin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . .........................
227 228
,
276 284 297
Ultraviolet Spectra of Proteins and Amino Acids D. B. WETLAUFER
I. Introduction, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 11. Terminology. . . . . . ................................... 111. Experimental. . . . . . . . . . . . . . . . . . ................... ................... IV. The Absorbing Components of Pr V. Spectrophotometric Titrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Difference Spectra and Chromophore Environment. . . . . . . . . . . . . . . . . . 346 .................................... 375 VII. Analytical Applications. . . VIII. Miscellaneous Problems, . . .................................... 380 IX. Summary. ..................................... . . , _ . . 382 References. . . . . . . . . . . . . . . . ........................... 383 Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
THE PROPERTIES OF PROTEINS IN N O N A Q U E O U S SOLVENTS
. .
By S J SINGER Department of Biology. University o f California. Son Diego. La Jolla. California
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Characteristics of Nonaqueous Solvents of Interest ....................... A . General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... B . Classification of Nonaqueous Solvents . . . . . . . . . . . . . . C . Acid-Base Properties of Solvents ..................................... I11. Solubility of Proteins in Nonaqueous Solvents . . . . . . . . . A . Strongly Protic Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Weakly Protic Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Experimental Aspects of Solubility. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . The Conformations of Protein Molecules in Nonaqueous Solvents . . . . . . . . . A . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Factors Involved in Determining Protein Conformations in Solution . . C. Experimental Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Polypeptides in Nonaqueous Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Proteins in Nonaqueous Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Nucleic Acids in Nonaqueous Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G Conclusions . . . . . . . .............................................. V . Physical and Chemica perties of Proteins in Nonaqueous Solvents . . . . A . Quaternary Structure in Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Biochemical Reactions of Proteins in Nonaqueous Solvents and Solvent Mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Protein Solutions a t Low Temperatures ......................... D . Chemical Modification of Proteins in Nonaqueous Solvents . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
1 2 2 4 4 5 5 8 8 10 10 11 23 35 40 53 56 59 59 61 63 64 65
I. INTRODUCTION It is a natural consequence of the paramount importance of water in biological systems. that the solution properties of proteins and other biologically important macromolecules have been almost exclusively studied in aqueous media . It is the properties of these aqueous solutions that must be understood if we seek to understand the behavior of living matter . On the other hand. it can be readily appreciated that much interesting and fundamental information should be obtained by the study of nonaqueous solutions of these substances . In some respects. the situation has its parallel in the field of simple electrolytes . There again. aqueous solutions are of the most immediate concern. but a very considerable insight into the properties of simple electrolytes and their aqueous solutions has come from in1
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S. J. SINGER
vestigations of their nonaqueous solutions (Evers, and Kay, 1960). The accessibility of a wide variety of physical and chemical properties among nonaqueous solvents is the primary reason for their usefulness in such studies. Thus, broad variations in dielectric constant, viscosity, temperature (since many nonaqueous solvents freeze at substantially lower temperatures than water), spectral transparency, solvent molecular volume and structure, relative acidity or basicity, hydrogen bond accepting and donating capacity, chemical inertness-to mention some of the more obvious solvent properties of interest-are at least hypothetically accessible. Prior to the last decade or so, there had been some investigations of proteins in a few nonaqueous solvents, but little systematic exploration of this area was carried out, and the potentialities of these studies were not fully realized. Within the last decade, as a result of a combination of factors, a revival of interest in this field has occurred, and it is safe to predict that this revived interest will be maintained for some time to come. The purpose of this article is to focus attention on this rapidly developing area of biophysical chemistry. The properties of some nonaqueous solvents of interest are examined, some representative recent studies of nonaqueous solutions of proteins and related macromolecules are discussed, and suggestions are derived from these of possible new directions such investigations might profitably explore. No attempt has been made to review thoroughly all the published papers in this area, and no historical survey of earlier studies has been included. There is a substantial older literature dealing with solutions of proteins in mixtures of water and some other solvent, which has not been discussed in this article, because the intent of such studies has generally been to determine the effect of (presumed) relatively small perturbations in the properties of the water solutions produced by the second solvent. Solutions in water or in water-nonaqueous solvent mixtures are discussed primarily as they bear on the corresponding solution behavior in the pure organic solvent. 11. CHAI~ACTEIZISTICS OF NONAQUEOUS SOLVENTS OF INTEREST A . General Considerations Proteins are generally not directly soluble in the common nonpolar solvents, or in the usual polar solvents such as alcohol and acetone. If they were, much more extensive studies of nonaqueous solutions of proteins would probably have been carried out long ago. However, they are directly soluble in strongly protic solvents, as had been discovered early in the history of protein chemistry, and in some polar solvents which have only relatively recently become commercially available. Furthermore, by
PROPERTIES O F PROTEINS IN NONAQTJEOUS SOLVENTS
3
suitable means which are discussed in Section 111, a wide variety of nonaqueous solutions of proteins can be prepared and studied. It is clear that certain general requirements must be met by a nonaqueous solvent, if it is to be useful. Since proteins contain chemically reactive groups, a first requirement of a satisfactory solvent is chemical inertness. That is, the solvent must not cause the rupture or formation of any covalent bonds in the macromolecule other than those with hydrogen atoms. This places a severe restriction on the number of useful nonaqueous solvents, since oxidizing or reducing, alkylating or acylating, etc., solvents are eliminated. This requirement also places some not-so-obvious limitations on solvents which might otherwise appear to be satisfactory. For example, small amounts of residual water may produce scission of peptide linkages in proteins dissolved in acidic or basic solvents. Such residual water may be very difficult to remove from both the solvent and the macromolecule in question. As another example, acidified anhydrous alcohols have been employed as solvents for proteins. Under relatively mild conditions, however, such media serve as excellent esterifying agents for carboxyl groups (Fraenkel-Conrat and Olcott, 1945). In this connection, the compound 2-chloroethanol, which has received much attention recently a s a protein solvent, may contain an appreciable amount of dissolved HC1, and Blout (1960) has reported that the carboxyl groups of some synthetic polypeptides are esterified in this solvent. Another obvious requirement of a nonaqueous solvent is chemical stability under a variety of conditions. Thus, methanol, especially after standing in the presence of air, may contain small amounts of formaldehyde which can react with groups on proteins and nucleic acids. Formamide, N ,N-dimethylformamide, and related compounds, are slowly decomposed by acid or base in the solvent, and the possibility exists that such decomposition may be catalyzed to some extent by a protein dissolved in the solvent. Thus Rees and Singer (1956) found that the apparent osmotic pressure of a solution of insulin in N ,N-dimethylformamide continually increased over a period of a week at 25°C but reached equilibrium at 13.8"C, which might have been due to the slow decomposition of the solvent on the solution side of the osmotic membrane a t the higher temperature. These brief comments are intended to emphasize the critical need to use carefully purified and monitored nonaqueous solvents in any biophysical studies. Investigators, particularly physical chemists, oriented to studies in aqueous solution may tend to overlook the chemical properties of nonaqueous solvents. Useful information about a variety of nonaqueous solvents is collected in
4
S . J. SINGER
several works (Weissberger et aZ., 1955; Scheflan and Jacobs, 1953; Audrieth and Kleinberg, 1953).
B. Classification of Nonaqueous Solvents For the purpose of this article, those polar liquids which have been found to serve as solvents for proteins may be classified into two broad categories, strongly protic and weakly protic. I n the former category are strong anhydrous acids such as formic and dichloroacetic acids, and strong bases, such as liquid ammonia and aliphatic amines. The weakly protic solvents are characterized by being only weak proton donors or acceptors or both (amphiprotism). I n this respect they resemble water. As an operational definition, a weakly protic solvent can be regarded as one whose 1 M solution in water is characterized by 6 < pH < 8. There are some nonaqueous solvents such as pyridine and phenol which may constitute intermediate cases between strongly and weakly protic solvents, but for the purpose of this article these do not have to be separately considered. I n Table I are given a number of solvents which have been used successfully with proteins, together with a summary of some of their physical properties.
C . Acid-Base Properties of Solvents
A protein molecule contains many groups capable of accepting protons from, or donating protons to, suitable solvent molecules. I n general, we are interested in the following equilibria:
+ HX = G i + HzX" HGi + HX = HIG: + X2HX = HgX+ + X-
HGi
(11
(21 (3 1
where HGi is a particular kind of amphiprotic group on a macromolecule and HX is a n amphiprotic solvent. Studies with simple model compounds containing the group Gi in the solvent HX are required to define the equilibrium constants, K1 and K 2, of reactions (1) and (2) respectively, while for reaction ( 3 ) , the autoprotolysis constant, K , , of the solvent is needed. Much of this and related information has been laboriously obtained b y a variety of methods and over a long period of time in the solvent water, and has been correlated with the electric charge properties and titration behavior of proteins in aqueous solution. A similar body of data is required for each nonaqueous solvent whose solution properties are t o be well understood. This informat#ionfor any nonaqueous solvent of interest is almost totally lacking a t the present time. From a qualitative point of view, however, we can make an important
PROPERTIES OF PROTEINS IN NONAQUEOUS SOLVENTS
5
distinction between strongly protic and weakly protic solvents. In a particular weakly protic solvent HX, some of the groups Gi,, are characterized by values of K1 and K 2that are exceedingly small, such that Kl<< K , and K z << K , and reactions (1) and ( 2 ) are essentially completely driven to the left under all conditions. Amide groups and alcoholic OH groups in water solution are examples. In addition, however, there are some groups Gi,, in the same solvent for which K1>> K , and K z << K , , as well as other groups Gi,b for which K z >> K , and K1 << K , . In this solvent, when the concentrations of H2X+and X- are equal, the groups Gi,, are thus largely in the anionic state and the groups Gi,b in the cationic. Carboxyl and amino groups in water solution a t pH 7 , are examples of Gi,, and Gi,b groups, respectively. The protein molecule containing these groups in this solvent will be an ampholyte. It will take on a net positive charge in sufficiently high concentrations of HzX+,a net negative charge at high concentrations of X-, and will exhibit an isoelectric point in between. The situation in a strongly protic solvent, however, is quite different. In a strong acid, for example, a large proportion of the different groups Gi,, , Gi,, , and Gi,b all may be characterized by K z >> K , . An extreme case is the solvent anhydrous H F (Katz, 1954a). Fluoride ion is such a weak base that even aromatic hydrocarbons are appreciably protonated in anhydrous HF solutions (Hammett, 1940, p. 293). Thus, K 2 is of the order of for benzene, and lo-' for hexamethylbenzene, in anhydrous H F (Kilpatrick and Luborsky, 1953). Therefore, all groups of a protein molecule which are as basic or more basic than benzene should be essentially completely protonated in dilute solutions in HF. Not only should RNHz groups be completely in the RNH; form, for example, but carboxyl groups should be protonated to RCOOH;, amide groups to RC+(OH)NHR', alcoholic OH groups to RCHzOHi, etc. The macromolecule should consequently take on a highly cationic character in this solvent. These considerations apply to other anhydrous acids and correspondingly to anhydrous bases to an extent depending on their relative acid or base strengths (Hammett, 1940, Chapter IX). In general, therefore, a protein must be highly protonated in strong acid solvents and highly depleted of protons in strong bases, and will ordinarily not exhibit ampholyte properties in such solvents.
111. SOLUBILITY OF PROTEINS IN NONAQUEOUS SOLVENTS
A . Strongly Protic Solvents A wide spectrum of proteins can be directly dissolved in any one of a number of strongIy protic solvents. This has been demonstrated particuIarly for dichloroacetic acid (Yang and Doty, 1957), hydroffuoric and tri-
TABLEI Properties of Some Pure Nonaqrceous Solvents4 Solvcnt
~1
Strongly protic acids HydroRuoric Formic m-Cresol Phenol Trifluoroacetic Dichloroacetic Acetic Bases Hydrazine Ammonia Ethylenediamine 1,2-propanediarnine Sulfur dioxide Pyridine Weakly protic alcohols Glycerol EtJhylcne glycol Methanol 1 ,2-Propmediol ZChloroethanol Ethanol 1-Propano1 2-Propanol
Dielectric constant
83.60 58.516
11.P5 9. 7S60 8.230*
8.Zzo 6.15¶O 51. 725 2 2 3 3
14.220 12.35'2 12.325
42. 5z3 37.723 32. 6325 32. Oao 25.W 24.3OZ3 20.125 18.345
B.1'.
Refractive
M.P.
Density
index (nD)
Viscosity (CP)
Referencs
19.5 100.7 202.7 181.8 72.4 194 117.7
-83 8.2 12.0 40.9 -15.3 9.7 16.6
0.99184-a 1.2133P3 I .o m 3 0 1.0576" 1.4EMP 1.5585'3 1.0437'5
1.369425 1.543820 1.541841 1.285020 1.370025
0.2406.5
1
1.96626 9.807ao 4. 07646 1.04030
2
113.5 -33.4 116.2 120.5 -10.2 115.6
2 -77.7 11.0 -72.7
1.01415 0.a - ' 0 0.89125 0. ~58425 1.46-1° 0 .9878l5
1.451330 -
1.72.!iZ5 0. 429-lo 0. 8B30
1.261380 1. 117115 0.796116 1.032P5 1.2019~0 0.79366'5 799555 0 . 78095a5
1.473525 1.4331j5 11326625 1.433l20 1.443815 1.359493 1.3835z3 1.374725
290.0 197.8 64.51 188.2 128.6 78.32 97.15
82.40
-41.8
18.2 -12.6 -97.5 I
-67.5 -114.5 -126.2 49.5
O
r
I
1.5067z5
94526 26.0915 0.545"
45.6W 3.913" 1.07825 2.52215 2.85916
2 2
3, 2 4 2
1 1 2
5 1
2 2 2 2 2
2 2 2 2
Amides N -Met hyl acet amide Formamide N ,N-Dimethylacetamide N , N-Dimethylformamide Miscellaneous Propylene carbonate Dimethylsulfoxide Acetonitrile Nitrobenzene Dioxane
178.9a0
204
109.5e5
210.5 165 153 241.7 189 81.6 210.8 101.3
37.82; 36.P 65. I=& 45 37.520
34.8225
2.2125
-61
0.9503a’ 1.129225 0.93662$ 0.944525
1.4468z5 1.43582h 1.42802j
-49.2 18.5 -45.7 5.76 11.80
I. 198725 1.lW~ 0. i71325 1.2082‘6 1.0269%
1.4 m 2 0 1.478721 1.X16= 1.5526’’ 1.q202’5
29.7 2
3. wa”
3.302” 9.610
-
2.55325 1.10027
3.75015 2.165‘8 1.43916
6, 7, 8 2
9, 10, 11 2,9, 10, 12 13, 14 9, 15 2 2 2
Superscripts indicate the temperature in “C at which the data apply. In ref. 2, the dielectric constant of trifluoroacetic acid i s given as 39.5 at 20°C. This value is not acceptable for reasons given in ref. 3. a b
REFERENCES FOR TABLE I -4
1. Audrictb, L. F., and Kleinberg, J. (1953). “Nonaqueoua Solvents.” Wiley, New York. 2. Weissbcrger, h.,Proskauer, E. R., Riddick, J. A., and Toops, E. E., J r , (1955). “Organic Solvents,” 2nd cd. Interscience, New York. 3. Damhauser, W., and Cole, R. H . (1852). J. Am. C h e n . Soc. 74, 6105. 4. “Internat,ional Critical Tables.” (1929). McGraw-Hill, New York. 5. Bcilbron, I., and Bunbury, H. M. (1953). ”Dictionary of Organic Compounds,” revised ed. Oxford Univ. Press, London and New York. 6. Postma, J. C. W., and Arens, J. F. (1956). Rec. trav. chim. 76, 1377. 7. Dawtlon, L. R., Wilhoit, E. n., and Sears, P. G. (1956). J. Am. Chem. SOC.78,1569. 8. Reynolds, W. L., and Weiss, R. H. (1959). J . Am. Chem. SOC.81,1790. 9. “Merck Index.” (1960). Merck, Rahway, New Jersey. 10. Leader, G. B., and Gormley, J. F. (1951). J . Am. Chem. SOC.73,5731. 11. Petersen, R. C. (1960). J , Phys. Chem. 64, 184. 12. Ioffe, B. V. (1955). Zhur. ObshcheiKhim. 26, 902. 13. Kronick, P. L., andFuoss, R. M. (1955). J . Am. Chem. SOC.77,6114. 14. Pepper, W. P. (1958). Ind. Eng. Chem. 60, 767. 15. Douglas, T. B. (1948). J . Am. Chem. Soc. 70, 2001.
8
S. J. SINGER
fluoroacetic acids (Katz, 1954a, b ) , liquid sulfur dioxide (Katz, 1955), ethylenediamine, propylenediamine, and hydrazine ( Rees and Singer, 1956). This spectrum of proteins is often so broad that it includes some that are normally insoluble (such as keratin) as well as those that are soluble, in aqueous media. The broad solubilizing power of strongly protic solvents has generally been assumed to arise primarily from hydrogen bonding to the solute. However, it appears quite probable that a variety of factors is involved, one of which is their capacity to convert proteins into highly deprotonated (in strong bases) or highly protonated (in strong acids) species, as discussed in Section I1,C. The molecular species so formed are then solubilized by their interaction with the polar solvent molecules, It is interesting that the strongly protic liquids that have been found to be effective protein solvents span a broad range of dielectric constants, from 6 for glacial acetic and 12 for ethylenediamine to 52 for hydrazine and 84 for liquid HF. This suggests that the ionizing power of the solvent is not critically involved in solubility, since in media of lower dielectric constant the protein salts must be only weakly dissociated (see Section IV,B,l). In such cases, a strong solvent-solute interaction produced by the formation of hydrogen-bonded ion-pairs (cf. Barrow, 1956) may be most important in solubilizing proteins.
B. Weakly Protic Solvents By contrast to the situation with strongly protic liquids, weakly protic pure liquids are generally incapable of converting proteins into highly protonated or deprotonated species, and at least partly as a result of this, the range of solubility of isoelectric proteins in weakly protic solvents is much more limited. In anticipation of the discussion to be presented in Section IV, it is important to realize that the native conformations of proteins are almost always altered in nonaqueous solvents, and the conformation of any one protein may be different in different solvents. The solubility of a protein might well depend on conformation, since the latter should determine the nature of solute-solvent interactions. Particularly as a result of this additional complication, the solubility of proteins in weakly protic solvents can only be approached in a wholly empirical manner. C. Experimental Aspects of Solubility In preliminary surveys of the solubilities of proteins in nonaqueous solvents, the solvent has generally been added directly to the solid protein. After suitable agitation, those mixtures that appeared homogeneous were classed as solutions. In this manner a number of interesting binary systems have been discovered. Some proteins which are not very soluble in aqueous media are quite soluble in a wide range of suitable nonaqueous
PROPERTIES O F PROTEINS IN NONAQUEOUS SOLVENTS
9
solvents. Certain vegetable proteins, such as zein and gliadin, had been found many years ago to be soluble in 80% ethanol (Robertson, 1918). Swallen and Danehy (1946) discovered that zein was soluble in 27 solvents, some 21 of them being weakly protic. Rees and Singer (1956) reported 15 nonaqueous solvents for zein, 12 of them being additional to those demonstrated by Swallcn and Danehy. It seems clear that the li8t of solvents for zein could be indefinitely extended if desired. Similarly, Rees and Singer (1956) found that insulin could be directly dissolved to the extent of a t least 1 mg/ml in some 13 nonaqueous solvents, closely paralleling the solubility behavior of zein. It is quite probable that in many cases those proteins which are insoluble or difficult to dissolve in aqueous solution, and which are not bound by covalent linkages into some generally insoluble protein matrix, will be soluble in some nonaqueous solvents. For proteins which are relatively water-soluble, the number of weakly protic solvents that have been found by direct solubilization experiments has been significant but nevertheless fairly limited. The indications are, however, that many more systems could be found with some application in the following directions. The lack of any appreciable direct solubility of a protein in a pure nonaqueous solvent may sometimes be a matter of unfavorable rate, rather than free energy, of solution. In any event, in cases where the protein does not dissolve directly, a stable solution can often be prepared in nonaqueous solvent by first dissolving the protein in an aqueous medium and then dialyzing this solution against mixtures successively richer in the nonaqueous component, and finally, against several batches of the pure anhydrous solvent (Geiduschek and Gray, 1956). Thus, dilute solutions of deoxyribonucleic acid (DNA) were prepared by this dialysis procedure in some 11 weakly protic solvents (but not in several others) (Herskovits et al., 1961), whereas direct solubility in such solvents is limited to formamide and ethylene glycol (Rees and Singer, 1956). In a similar manner, Rees and Singer (1956) found that trypsin was directly soluble only in formamide and dimethylsulfoxide, of a number of weakly protic solvents tried, but in a preliminary study (Fleck and Singer, unpublished experiments) stable solutions containing ca. 1 mg of trypsin/ml could also be prepared by dialysis from acidic aqueous solution into methanol, ethanol, ethylene glycol, propylene glycol, and glycolonitrile, but not into acetonitrile or acetone. Furthermore, it should be possible to prepare solutions of proteins by dialysis from one nonaqueous solvent into another, which may be useful with moderately protic solvents which produce fairly acid or alkaline solutions in mixtures with water. A difficulty with the dialysis procedure, however, is that it is relatively lengthy, and the protein may, even at low temperature, undergo slow irreversible changes in the time required.
10
8. J. SINGER
Some proteins that are insoluble in a pure nonaqueous solvent may dissolve much more readily on the addition of a neutral salt (the analog of salting-in in water solutioiis). Thus, Katz ( 1955) found that whereas proteins are generally not directly soluble in pure liquid sulfur dioxide, they are readily soluble in sulfur dioxide containing 6 M NHICNS. Zwitterionic salts (glycine, for example) may he useful in this connection in weakly protic solvents as they are in water (Cohn and Edsall, 1943, p. 617). In a related connection, the solubility of a protein may be considerably greater in a slightly acidified or alkalized weakly protic solvent than in the pure liquid. This would be analogous to the situation which one obtains in aqueous media, in which proteins are generally increasingly soluble the further the pH is from the isoelectric point of the protein, within certain pH limits (Cohn and Edsall, 1943, p. 606). This is probably the reason that 2-chloroethanol is such an excellent solvent for proteins (Doty, 1959). This solvent is not a very stable one, and significant amounts of HCl can be present in it. This may also account for the observation that, although bovine serum albumin is insoluble in pure acetone, methanol, and ethanol, it dissolves in them when trichloroacetic acid ( 1 %) is added (Levine, 1954). On the other hand, the trichloroacetate counterion may itself influence the solubility of the protein salt in the nonaqueous solvents. The presence of counterions bearing hydrocarbon-miscible tails, such as the trichloroacetate anion just mentioned, or tetraalkylammonium cations, instead of the usual inorganic ions, may be a n aid in solubilizing proteins in nonaqueous solvents. Mixed nonaqueous solvent systems are also of great interest and potential versatility as protein solvents, as they are for simple electrolytes (Evers and Kay, 1960). Their systematic use should enormously extend the range of nonaqueous solvent systems and properties. This is already suggested by several studies (cf. Doty et al., 1956; Yang and Doty, 1957). Many solvents not capable of dissolving a given protein may do so in mixtures with a small amount of a nonaqueous good solvent for the particular solute. In another connection, the addition of less than 1 % of one nonaqueous solvent to a solution of a macromolecule in another nonaqueous solvent can profoundly alter the physical chemical properties of the solution (Eirich et al., 1951; Doty et al., 1956).
IV. THECONFORMATIONS OF PROTEIN MOLECULES IN
NONAQUEOUS SOLVENTS
A . Introduction Until fairly recently, the native conformation of a protein molecule has generally been considered to be a characteristic property of the macro-
PROPERTIES OF PROTEINS IN NONAQUEOUS SOLVENTS
11
molecule itself, the numbers and arrangements of its amino acid residues and the covalent and secondary bonds holding the residues together. I n the last decade, however, it has become generally recognized that the solvent water plays an exceedingly important role in determining and stabilizing the characteristic structure that a protein molecule exhibits in an aqueous environment. It is in connection with this problem of the role of solvent in regulating macromolecular structure that nonaqueous solutions of proteins have been most frequently studied in the last few years. In an aqueous protein solution, it is well known that any of a wide variety of treatments, such as an increase of temperature or exposure to pH extremes, leads to denaturation of the protein. Denaturation is generally accompanied by a disorganization and partial randomization of the secondary and tertiary structure of a protein molecule. Therefore, we have been conditioned to think of the native aqueous configuration of a particular protein molecule as the most highly ordered form which its covalent structure permits it to attain. One of the most interesting developments of studies of proteins in nonaqueous solvents, therefore, has been the finding that in certain systems an apparently more highly ordered conformation (greater helical content) of the protein molecule exists than its native aqueous form. On the other hand, there are other nonaqueous protein solutions in which the macromolecular conformation becomes highly disorganized. In order to evaluate these studies, it is first necessary to consider the nature of the interactions, other than covalent bonds, that are important in determining the conformations of protein molecules, and to attempt to assess the effect of a change in solvent on these interactions. A most lucid and informative discussion of macromolecular interactions has been given in an earlier volume of this series by Kauzmann (1959). The following remarks are intended as comments upon and footnotes to the Kauzmann exposition.
B. Factors Involved in Determining Protein ConJormations in Solution 1. Electrostatic Interactions
Two principal kinds of intramolecular electrostatic interactions can operate in protein solutions. One is an attractive type, between closely spaced fixed charges of opposite sign on a protein molecule, to form salt linkages or ion-pair bonds. The other is a longer-range repulsive type, due to the net charge on a protein molecule. I n aqueous solutions, ion-pair bonds, if they exist, do not appear to be of significance in determining the native conformation of a protein molecule (Jacobsen and Linderstrgm-Lang, 1949). On the other hand, repulsive interactions can destabilize the na-
12
6 . J. SINGER
tive conformation in water if the net charge on the protein molecule becomes sufficiently great, as occurs at extremes of pH (Tanford, 1961) or by suitable chemical modification (Habeeb et al, 1959). The compact conformation is then converted to a highly swollen and unfolded conformation. I n what follows, we will largely confine our attention to these repulsive interactions, since in order to discuss the attractive type, detailed information, at present not available, would be required concerning the spatial distribution of charges on the protein molecule. What is the effect on these repulsive electrostatic interactions of changing from an aqueous to a nonaqueous solvent? Does the result contribute to the stabilization, the further ordering, or the disordering of the native aqueous conformation of a protein molecule? It is important to emphasize that. we wish to compare at this point the native conformation of the protein molecule in the aqueous and the nonaqueous solvents. Since, as will be shown subsequently, the native conformation is disrupted in most nonaqueous solvents, it is a hypothetical state not experimentally attainable. Nevertheless, the question just asked can be explored theoretically. This problem is best approached by considering the electrostatic free energy, P, , of a protein molecule due to its net charge and its interaction with an ion atmosphere. For a spherical ion of uniform charge density, according to the Debye-Huckel theory (Cohn and Edsall, 1943, p. 473)
where 2 is the net charge on the protein ion of radius b, t is the unit charge, K is the reciprocal of the thickness of the Debye-Huckel ion atmosphere, and a is the distance of closest approach of the macroion and its counterion, measured between centers. More refined treatments to obtain expressions for Pe are available (Tanford, 1961), but for our purposes Eq. ( 4 ) will suffice. If we consider the value of P , for the native conformation of a protein molecule in different solvents, b is constant and K can be maintained constant by suitable manipulation of electrolyte concentration; hence the effect of changing the solvent can be resolved into the effect on the values of Z and D. The value of 2 is subject to change in two different ways: one due to the influence of the solvent on the acid-base equilibria of the protein; the other due to ion-pairing of the fixed charges on the protein molecule as the dielectric constant is decreased. Ion-pairing may occur between fixed charges of opposite sign on the protein molecule (salt linkages) or it may occur between the fixed charges and small counterions of the electrolyte in the solvent, or the lyate or lyonium ions of the solvent. It will he assumed in what follows, that for the native conformation of the protein in the nonaqueous solvent, as with the native conformation in water, salt linkages between fixed charges are not energetically significant; and that
PROPERTIES OF PROTEINS I N NONAQUEOUS SOLVENTS
13
ion-pairing to counterions, or lyate and lyoriium ions, is the more important factor. The interplay of these factors is difficult to evaluate quantitatively in the general protein-solvent system. Some useful qualitative conclusions, however, may be arrived at in certain extreme cases. One extreme case is that of strongly protic solvents of high dielectric constant. The large value of 2 placed on the protein molecule in such a solvent must substantially increase P , and act to disorder the native aqueous conformation. In view of the discussion in Section II,C, for example, isoionic bovine serum albumin when dissolved in anhydrous HF is potentially capable of binding about 1000 protons per molecule. In aqueous media, the binding of about 10 protons per molecule to the isoionic protein is sufficient to begin the disruption of its native structure (Tanford et al., 1955b). The D value for H F at O"C, 83.6, is close to that of H20. Thus ( P e ) H F / ( P e ) H 2 0 S Z,",/Zfzo . If at one extreme there were no counterion binding to the protein in H F solution (i.e. that Z H F 1000) then (P,)Hp would be about lo4times greater than that value of P, in H 2 0 which corresponds to the onset of electrostatic disordering of the native protein conformation in aqueous solution. Even with a substantial degree of counterion binding in HF, however, ( P , ) H F / ( P e ) ~ , o should still be quite appreciable. Since it is highly unlikely that other kinds of interactions in HF solution would be augmented enough to overcome such a gross destabilizing influence, it can be predicted that the molecules of bovine serum albumin and of other proteins in H F solution are in a highly unfolded state at ordinary ionic strengths. Formic acid ( D = 56.5 at 16OC) and hydrazine (D = 51.7 at 25°C) are two other strongly protic solvents to which similar considerations apply. In strongly protic solvents of dielectric constant less than about 15, however, it is certain that the Z value for a particular protein will be considerably less than the number of protons abstracted from, or donated to, the solvent, as a result of the increase in counterion binding a t low values of D. To investigate the nature of counterion binding, let us compare a particular hypothetical protein in its native conformation in an aqueous and a nonaqueous solvent, S, such that the number of protons bound per protein molecule is the same in both solvents. The fixed charges on the protein molecule in the aqueous solution are taken to be completely ionized; let Zo be the net charge on the molecule in this solution. Let us assume that the different fixed charged groups on the protein molecule behave identically with respect to the binding of univalent counterions in the S solvent; then Zs = a&, where a is the fractional dissociation of the fixed charged groups in S solution. Other things being equal, then, (P,)s/ (Pd)HzO is therefore a2DH,o/Ds, in this situation. The magnitude and variation of CY can be estimated from an analysis of
14
S. J. SINGER
studies of the association behavior of simple electrolytes in solvents of low dielectric constant. It appears both theoretically and experimentally that the equilibrium constant, K , , for the association of a uni-univalent electrolyte to form ion pairs increases exponentially with l/D (Fuoss, 1958; Sadek and FUOSS, 1959; Berns and FUOSS, 1960, 1961). Some typical experimental results for the electrolyte tetrabutylammonium picrate in mixtures of nitrobenzene and CCld (Hirsch and FUOSS, 1960) are shown in Fig. 1. From these data, the fractional dissociation, a, of the electrolyte a t a con-
k
Fro. 1. The variation of the ion-pair association constant with the dielectric constant of the solvent for the electrolyte tetrabutylammonium picrate in nitrobenzene-CClr mixtures (Hirsch and Fuoss, 1960).
centration of M in these solvents has been calculated, with the assumption of ideal solution behavior of the ions. For this system, a2D,,,/D varies with D as shown in Fig. 2. The details of this curve, of course, depend on electrolyte concentration and will be different for different electrolytes, depending on their charge type and interaction with the solvent, etc., but the main features of the function should hold generally. With decreasing D , a2D,,,/D first increases slowly to a maximum and then falls precipitously. The initial part of this variation is due to a decrease in D without any significant occurrence of ion-pairing. As ion-pairing sets in, however, it rapidly becomes the dominant factor because of its exponential variation with 1/D.
PROPERTIES OF PROTEINS IN NONAQUEOUS SOLVENTS
15
For the hypothetical protein described above, it follows that a t equal extents of proton binding to a protein in water and in the solvent 8,and should vary with D at equal values of K in the two solvents, (Fs)B/(Fe)HtO in a manner similar to the function a2D,,,/D shown in Fig. 2. The large net charge on the protein should act to enhance counterion binding over that by a univalent ion at any particular value of D , and hence the maximum in (~,)s/(~,)E,o may shift to considerably larger D values, but otherwise the dependence on D should be much the same as in Fig. 2. If
70
80
FIG.2. The variation with dielectric constant of the function d D a 2 0 / D (see text) for the system of Fig. 1 at an electrolyte concentration of lo-* M.
the valence of the counterions is greater than unity, the maximum should again shift to larger D values. For any real protein, the presence of considerable numbers of different kinds of fixed charges in different local environments on the protein surface might tend to broaden the variation of counterion binding with D. Furthermore, simultaneous changes will generally occur in the degree of proton binding, and hence in 2, as D is varied. It is difficult to take these factors properly into account, but in view of the considerations just given, it seems probable, for the hypothetical native conformation of a real protein in weakly protic solvents, that there should be a range of D values, 80 > D >
16
8. J.
SINGER
-30, in which the binding of univalent counterions is not significant; then (P,)s/(F,)H,~ = P ~ D H , , / P & , D s at constant K , where Ps and PHZO are the numbers of protons bound to the initially isoelectric protein in the solvent S and in HzO, respectively. If Ps > P,,O ,then (F,)s> (p8),,, , and there should be an increase in the tendency to disorder the native conformation, other things being equal. On the other hand, for D < -15, ( F , ) s < (F,),,, in weakly protic solvents, and probably in most strongly protic ones also, and the electrostatic tendency to disorder should be substantially decreased under its magnitude in HzO.
2. Hydrogen Bonds between Peptide Linkages The author subscribes to the opinion expressed by Kauzmann (1959) that it is “unlikely that hydrogen bonds other than those involving peptide linkages can make a major contribution to the (over-all conformational) stability of most native proteins; there are generally relatively few of the other types of groups present, and none of them appear( s) to be especially strong.” In this discussion, therefore, we will confine attention to peptide NH. hydrogen bonds. For such bonds occurring in homogeneous solution, the generalized equilibrium of interest is:
-
a
0
4
NH.. .fj + C-0..
.fj
NH- .O=C + 8.* .S s
(6)
in which S is a solvent capable of acting as both a hydrogen bond donor and acceptor. No specific stoichiometric relations are implied by this equation. Quantitative studies of hydrogen bonding in simple amide systems in solution have as yet been few in number, and these have been carried out mainly in nonpolar solvents. Some thermodynamic data are listed in Table I1 for NH. -O=C hydrogen bonds. Several features of these data are of interest. I n the first place, even in the most nonpolar solvents, which are incapable of forming hydrogen bonds, the value of - AH is quite small, of the order of 3 to 5 kcal/mole of bond. Furthermore, even as weak a hydrogen-bonding solvent as CHC1, markedly diminishes this value, presumably because of competition effects expressed in Eq. (5). Still stronger hydrogen-bonding solvents must tend to drive the equilibrium in Eq. (5) even further to the left, other things being equal. In this connection, some recent measurements of Klotz and Franzen (1960, 1962) of the equilibrium constant, K , for the formation of hydrogenbonded dimers of N-methylacetamide are of particular interest (Table 11). The value of K observed in water solutions of the amide is about that in CC14solution, corresponding to a difference in AFo of about 4.1 kcal/rnole of hydrogen bond. This point of view has led to the realization in recent years that, taken
-
17
PROPERTIES OF PROTEINS I N NONAQUEOUS SOLVENTS
by themselves, intrapeptide hydrogen bonds can confer only marginal stabilization to the native conformation of a protein molecule in aqueous solution (Schellmm, 1955a, b; Kauzmann, 1959, Klotz, 1960). Quantitative information about amide hydrogen bonding is unfortunately very meager in nonaqueous liquids which are protein solvents. In later sections, however, much stress will be placed on the one result (Table 11) that amide hydrogen bonds in dioxane solution are considerably stronger than in water; the difference in AF" for the dimerization of N-methylacetamTABLEI1 Enthalpy of Formation of NH. . .O=C Hydrogen Bonds i n Solutiona -AH
Components
Solvent
1
p-Benzotoluidide Dioxane p-Benzotoluidide N , N-dimethylformamide ii-Valerolact am Acet amide N-Methylacetamide
CCl,
2.3
cc14
3.8
cc14 CHCli CsHe CClr
5.2 1.1 3.5 3.8 4.2b 1.6 0.8b
CHCL Dioxane HzO a
b
(kcal/mole H bond)
O.Ob
Kb
(1 /mole)
4.7 0.52 0.005
After Table 6.2 in Bamford et al. (1956). For the reaction 2A Az , see Klote and Franzen (1962).
ide in the two solvents corresponds to about 2.8 kcal/mole. It would be of great interest to have available corresponding data in a large number of the solvents listed in Table I. Certain semiquantitative considerations are, however, of interest here. The interaction of dilute solutions of amides with other hydrogen bond agents in nonpolar solvents has been studied by infrared spectroscopy by following the effects of these agents on the C-0 and N-H stretching frequencies of the amide (Cannon, 1955; Mizushima et al., 1955). This work is reviewed by Bamford et at. (1956). These studies have shown that the C==O group in amides acts as a strong proton acceptor, whereas the NH group has a relatively weak proton-donating capacity. Thus, intrapeptide hydrogen bonding is the product of a weak donor (NH) interacting with a strong acceptor ( C = O ) , resulting in a moderately weak hydrogen bond (Table 11). Even if a solvent S is a strong proton acceptor, it will not easily displace the strong C = O acceptor from the NH group. Under these
18
S . J. SINGER
circumstances, the proton-donating capacity of a nonaqueous solvent might be expected to be of greater importance than its proton-accepting capacity in determining the state of equilibrium in Eq. (5). This conclusion has several corollaries. In a series of compounds oontaining the OH group, for example, proton-donating capacity can vary widely, as is evident in Table 111. The tendency to disrupt int,rapeptide hydrogen bonding should therefore increase markedly, other things being equal, in the order: alcohols, phenols, carboxylic acids, and finally, substituted carboxylic acids such as trifluoroacetic and dichloroacetic acids. Water and the polyhydric alcohols are difficult to place in this sequence because they can donate and accept more than one proton per molecule to form hydrogen bonds, but these substances probably belong between phenols and carboxylic acids. Experimental data are required, however, to substantiate this. Furthermore, certain nonaqueous solvents such as dioxane, acetonitrile, TABLE111 Enthalpy of Formation of Hydrogen Bonds of OH Compounds with Dioxane in CClr Solutions Compound: benzyl alcohol - A H (kcal/mole H Bond) : 2.1 a
phenol -4.5
o-cresol 5.4
benzoic acid 6.2
Pimentel and McClellan (1960), p. 234.
dimethylsulfoxide, pyridine (and other tertiary amines) , and dimethylformamide, function primarily as hydrogen bond acceptors and have little proton-donating capacity. These solvents should therefore show, roughly speaking, a relatively small tendency to disrupt intrapeptide hydrogen bonds, since they cannot too successfully compete with the peptide NH group for the O=C hydrogen bond. 3. Lyophobic Interactions
The term “lyophobic interactions” is intended to generalize the expression “hydrophobic interactions” to other solvents than water. Hydrophobic interactions have been prominently implicated in determining the native configuration of proteins in aqueous solution. These interactions are actually not of a single relatively well-defined character, as are electrostatic or hydrogen bond interactions, but are rather a set of interactions responsible for the immiscibility of nonpolar substances and water. Proteins contain a substantial proportion of amino acids such as phenylalanine, valine, leucine, etc., with nonpolar side-chain residues. These nonpolar groups should tend, therefore, other factors permitting, to cluster on the
PROPERTIES O F PROTEINS I N NONAQUEOUS SOLVENTS
19
inside of the protein molecule away from the aqueous environment, as a result of these interactions. In the context of this article, lyophobic interactions refer to the forces tending to produce a similar clustering of the nonpolar residues of a protein in a nonaqueous solvent. Other types of lyophobic interactions (as for example between a solvent of low dielectric constant and the ionic groups of a protein molecule) are not included. Hydrophobic interactions have been correlated with unitary free energy changes, A F , , of relatively simpler processes such as (Kauzmann, 1959): Hydrocarbon in nonpolar solvent (mole fraction z) hydrocarbon in water (mole fraction z) (6) Pure liquid hydrocarbon
hydrocarbon in water (mole fraction z)
(7)
It is a most interesting fact that the unfavorably positive values of AF, accompanying these processes are primarily due to negative unitary entropy changes, and not to positive enthalpy changes. This has been interpreted to mean that water molecules become highly ordered around dissolved hydrocarbon molecules (Frank and Evans, 1945), suffering thereby a loss of entropy not compensated for by a favorable enthalpy change. It may be that water is a unique substance in exhibiting this large entropic loss. It has been suggested that similar thermodynamic considerations apply to hydrophobic interactions for protein molecules in aqueous solution (Kauzmann, 1959). If we extend these considerations to nonaqueous solvents, reaction (7) may be written as: Pure liquid hydrocarbon hydrocarbon in nonaqueous solvent (mole fraction z) (7a)
AF, for this process can be determined as follows: if the hydrocarbon is only partially miscible with the solvent, and the saturated solution is sufficiently dilute to be ideal, AF,
=
-RTlnx,
where xa is the mole fraction of hydrocarbon in the saturated solution. If, on the other hand, the hydrocarbon is completely miscible with the solvent, A F , can be obtained from suitable vapor-liquid equilibria data. Consider a solution containing mole fraction x of the hydrocarbon in equilibrium with a vapor, a t total pressure P, containing mole fraction y of the hydrocarbon. For a sufficiently dilute solution, the fugacity f = YP obeys Henry’s law:
f = KX
(8)
20
S. J . SINGER
where K is a constant. AF, for reaction (7a) is then given by: A F , = RT (lnf/fo
- In 2) = Rl’ In K / f 0
(9
where j o is the fugacity of the pure solvent. For the purpose of this article, we have calculated A F , values for the hydrocarbon benzene, using Eq. (8) or (9) depending on the degree of TABLEIV Unitary Free Energy Changes f o r the Transfer of Benzene to Various Solvents“ Temperature (“C)
(kcal/mole)
AFu
Reference
Water Ethylene glycol Formic acid Propylene glycol
18 25 25 25
4.07 1.83 1.45 0.99
1 2 3 2
Methanol Ethanol Isopropanol Acetonitrile CClr
35 45 45 45 40
1.23 0.96 0.88 0.67 0.08
4 5 8 7 8
Solvent
a The first four solvents are only partially miscible with benzene, and A P , was calculated using E q . (8).
REFERENCES FOR TABLE IV 1. Kausmann, W. (1959). Advances i n Protein Chem. 14, 1 . 2. Curme, G. O., and Johnston, F. (1952). “Glycols,” p. 48. Reinhold, New York. 3. “International Critical Tables.” (1929). McGraw-Hill, New York. 4. Scatchard, G., Wood, S. E., and Mochel, J. M. (1946). J . A m . Chem. SOC.68, 1957. 5 . Brown, I., Fock, W., and Smith, F. (1956). Australian J . Chem. 9, 369. 6. Brown, I., Fock, W., and Smith, F. (1956). Australian J . Chern. 9, 364. 7. Brown, I., and Smith, F. (1955). Australian J . Chem. 8. 62. 8. Scatchard, G., Wood, S. E., and Mochel, J. M. (1940). J . A m . Chem. SOC.62, 712.
miscibility of benzene with a nonaqueous solvent. These values are given in Table IV for a number of solvents for which data are available. With Eq. (9) K values were obtained by extrapolation of yP/x to infinite dilution of benzene in the solvent. Unfortunately, data for all these systems are not available at any one temperature, and the A F , values listed in Table IV are not directly comparable. They provide an estimate, however, of the relative magnitude of A F , for different solvents. The process we are in fact most concerned with is:
21
PROPERTIES OF PROTEINS IN NONAQUEOUS SOLVENTS
Hydrocarbon (in water, mole fraction 5) s hydrocarbon (in nonaqueous solvent, mole fraction
5)
(10)
The unitary free energy change of this isothermal transfer process, AF: is given by ( AFu)nonaqueouB solvent - (AFu)water. The calculation of exact values of AF: requires AFu values obtained at the same temperature, but a t least semiquantitative conclusions about AF: follow from the data in Table IV. For all the solvents listed in the table, AFt is negative. This reflects, of course, the fact that benzene is less soluble in water than in any other liquid that can function as a protein solvent. Furthermore, even for those nonaqueous pure solvents in which benzene is only partially soluble a t 25°C (among all the solvents in Table I, this category includes only the weakly protic solvents ethylene glycol, propylene glycol, glycerol, and formamide, and the strongly protic solvents hydrofluoric acid, formic acid, and hydrazine), -AF: is at least 2 kcal/mole. With the majority of the solvents in Table I benzene is completely miscible at 25°C; -AF: must therefore be considerably larger than 2 kcal/mole for any of these solvents. For example, benzene in methanol forms about the most nonideal solution for which the components are still miscible in all proportions at 25"C, yet -AFL is about 3 kcal/mole in this solvent. Most benzenemiscible solvents form benzene solutions that are so much closer to ideal that it is clear that for such solvents -AF; values must cluster between 3.5 and 4.1 kcal/mole. The sum of these AF: , 2 AF: , taken over all the nonpolar residues found in typical protein molecules, can attain very large negative values. If the native conformation of a protein molecule in aqueous solution is indeed in considerable part stabilized by lyophobic interactions, it follows that this stabilization should be substantially if not completely lost on transferring the protein molecule to almost any pure nonaqueous solvent. This destabilization might be expected to be less extensive in those few weakly protic nonaqueous solvents with which hydrocarbons are only partially miscible, such as glycerol, ethylene and propylene glycols, and formamide, than in the other solvents with which hydrocarbons are completely miscible. Furthermore the latter solvents should be very little differentiated under these circumstances, since AF: is so similar for most of them. As is demonstrated subsequently, these expectations are closely realized in fact. It is an oversimplification, of course, to correlate lyophobic interactions in a solvent-protein system solely with the solubility of simple hydrocarbons in the solvent. There are many other kinds of groups besides hydrocarbon ones in protein molecules and the influence of altered solvent environment on such groups must also be considered. In this connection, systematic
22
S. J. SINGER
solubility studies of model compounds in a number of nonaqueous Solvents, especially those described by Cohn and Edsall (1943, Chapter IX) are of value in estimating the free energy changes accompanying the transfer of particular groups from water to nonaqueous solvents. These studies will not be examined in detail, since they are discussed extensively elsewhere (Cohn and Edsall, 1943; Cohn, 1936). They reveal, however, that the nonpolar residues make the most substantial negative contributions to AF; in many solvents; for polar, nonionic residues (those of serine, threonine, tyrosine, etc.), the net contribution is small; while for the peptidelike group, -CH2CONH, it is positive. It is therefore not an entirely unjustified approximation to discuss hydrophobic interactions solely in terms of the nonpolar residues of proteins, which clearly make the largest contribution to them.
4. ConformationaZ Entropy There is a very substantial loss of conformational entropy accompanying the conversion of a protein molecule to its native conformation from a random-coil structure limited only by primary valence bonds. This entropy loss makes a large contribution to the destabilization of the native conformation. However, if the assumption is made that the random-coil form has a similarly large conformational entropy in different solvents, this entropy change should not be much influenced by a change of solvent, and is therefore not considered further at this point. 6. Cooperative Nature of Interactions Having thus attempted to isolate some of the more important types of interactions which determine the structure of proteins in solution, we conclude this discussion by emphasizing the fact that in any real system these interactions are difficult to separate. For one thing, lyophobic and hydrogen bond interactions are generally not independent of one another. A solvent may be very lyophobic for nonpolar groups expressly because the solvent molecules are strongly hydrogen bonded to one another. In such a solvent, therefore, strong solvent-solute hydrogen bonds may also be formed. Furthermore, one type of interaction may profoundly influence the degree to which another participates. For example, it has been shown (see Section IV,B,2) that solute-solute hydrogen bonding such as occurs in the a-helix is relatively weak in aqueous solution. If, however, hydrogen bonds are buried in the interior of a hydrophobic region of a protein molecule, they should become much stronger and thus enhance the stabilizing influence of the hydrophobic interactions. It follows that the helical regions and hydrophobic regions of a protein molecule need not be mutually
PROPERTIES OF PROTEINS I N NONAQUEOUS SOLVENTS
23
exclusive; in fact, there may be considerable coincidence of the two. This is demonstrated directly in the structure of myoglobin (Kendrew et al., 1961). In this protein, all the phenylalanine and methionine side chains are directed inwards in what are clearly hydrophobic regions of the molecule, and of these residues about half are located in helical and half in nonhelical regions. It is therefore difficult to dissociate completely the different kinds of interactions, and to assign to them roles of varying importance in determining macromolecular structure.
C. Experimental Methods At this juncture, it is useful to discuss the experimental methods that are of value in studying and separating the various kinds of interactions in macromolecular systems. A variety of experimental methods have been applied to the determination of protein structure and conformation in solution, and these have been summarized by Kauzmann (1959). In the discussion which follows, emphasis is placed on those methods which have so far been of most use in studies of proteins in nonaqueous solvents, and these remarks should be considered as supplementary to the Kauzmann summary. I . Electrostatic Interactions In order to examine the influence of these interactions, the charge properties of protein molecules have to be investigated in nonaqueous solvents. Titration, electrophoretic, and conductometric experiments could yield important information in this connection, but few studies in nonaqueous solvents have been performed. With respect to titration studies, potentiometric acid-base titrations in nonaqueous media have often been carried out, but these have been mainly concerned with analytical end-point determinations. For protein solutions, titration studies involve the measurement of hydrogen ion activities (see the article by Tanford in this volume). I n this connection, Sage and Singer (1962) have shown that the glass electrode can function as a hydrogen electrode in essentially pure ethylene glycol solutions. A spectrophotometric potentiometric titration was carried out with L-tyrosine ethyl ester in 0.2 M KC1 in ethylene glycol, by means of a glass electrode in conjunction with a Ag-AgC1 reference electrode and a salt bridge containing a saturated solution of LiCl in ethylene glycol. The ionization equilibrium of the phenolic group of tyrosine ethyl ester can be written as:
where K D is the acid dissociation constant in ethylene glycol, and aH+
24
S. J. SINGER
, and uRO- are the activities of hydrogen ion, and the phenolic and phenoxide ion forms of tyrosine ethyl ester, respectively. It was found that the increment in molar extinction coefficient, A€, of a partially titrated solution of tyrosine ethyl ester in ethylene glycol at 296 mM is essentially proportional to the mole fraction of the phenoxide ion species. Thus, if the glass electrode functions as an exact hydrogen electrode in ethylene glycol solution, the following relation should hold :
aROH
RT A ~ T- A6 RT E = constant - - In K , - - In ___ 5 A€ 5
(1 2)
where E is the measured potential, the constant is the sum of the standard half-cell potentials and the (assumed constant) liquid junction potential, 5 is the Faraday, and AeT the molar extinction coefficientof the phenoxide ion species. Therefore, a plot of E against log [(A€, - Ae)/Ae] should give a straight line with a slope of -0.0591 volt at 25”C, as was indeed found (Fig. 3). That the glass electrode can function as a hydrogen electrode may turn out to be true in a variety of other acid- and alkali-stable, weakly protic nonaqueous solvents besides ethylene glycol. This would make it experimentally feasible to investigate the acid-base behavior of simple substances and of proteins in a manner quite analogous to the extensive studies that have been carried out in aqueous solution. It is difficult to define a thermodynamic “pH” scale in a nonaqueous solvent (Gutbezahl and Grunwald, 1953) but an operational “pH” scale can be defined in terms of suitable standard buffers as is done in aqueous media (Bates, 1954). Such studies should be of considerable value in defining the properties of proteins in nonaqueous solvents. It might also be possible to perform electrophoresis experiments in weakly protic solvents that are not readily oxidized or reduced, although little work has been done in this area. As was pointed out by Tiselius (1959) , although the conductance in such media may often be very low, this may be compensated for by the application of high voltages without concomitant large heating effects. Some interesting conductance studies of protein-nonaqueous solvent systems have been carried out. Greenberg and Larson (1935), for example, found that gelatin, casein, and edestin dissolved in glacial formic acid (D = 56.5 at 16°C) showed marked increases of conductivity over that of the solvent, whereas no conductivity increment was observed with the same proteins dissolved in glacial lactic or acetic (D = 6.15 at 20°C) acids. This is in accord with the conclusions reached in Section IV,B,l, that in solvents of low dielectric constant, ion-pairing (in these cases, of the protein cations to lactate and acetate counterions) is essentially complete. Con-
PROPERTIES O F PROTEINS I N NONAQUEOUS SOLVENTS
25
ductivity measurements are easy t o make and should be of considerable utility in estimating the magnitude of electrostatic interactions relative to those existing in aqueous media (see also Herkskovits et al., 1961). From an operational point of view, the influence of electrostatic interactions in a particular protein-solvent system can be greatly diminished, often
-.-
1.0
.8
.6
.4
.2
0
-.2
-A
-.6
-.8
-1.0
A E -~ A € log Ac
FIG.3. Titration data of L-tyrosine ethyl ester HCI in 0.2 2cI KC1-ethylene glycol according to Eq. (11) of text. Line drawn through the d a t a has slope of 0.0591 v . (Sage and Singer, 1962.)
essentially eliminated, in a number of ways. Studies can be made at the isoelectric point of the protein (as revealed by electrophoresis measurements) if the solvent is a weakly protic one. Since 2 = 0 a t this point, P, is a t a minimum. Further, the addition of increasing amounts of supporting electrolyte, by increasing K and by decreasing 2 (through counterion binding), also produces a decrease in I”, [Eq. (4)]. If by these devices, no effect on the conformation of the protein molecule in a particular solvent is observed, then electrostatic interactions are not of primary importance in the system in question.
26
S. J. SINGER
2, Hydrogen Bonds between Peptide Linkages
At least two general classes of intramolecular peptide hydrogen bonds may be recognized, intrachain and interchain bonds. The former type leads to single-chain helical structures of which the a-helix (Pauling et al., 1951) is the only one that has 80 far been shown to exist in protein molecules (Kendrew et al., 1961). Interchain hydrogen bonds can lead to a variety of different structures, among them multichain helices as in collagen, and sheetlike structures as in silk fibroin (cf. Bamford et al., 1956, Chapter IV). Although the a-helix may prove to be the most important of these various structures for proteins, the possibility that other regular hydrogen-bonded structures may exist in specific cases must be kept in mind (Donahue, 1953; Luzzati et al., 1961). Infrared spectra are of considerable interest in the study of peptide hydrogen bonding. (For an extended discussion of this subject, see Bamford et at., 1956, Chapters V and VI.) The formation of the hydrogen bond N-Ha . -0-C weakens the forces acting between the N and H atoms and between the 0 and C atoms, and hence the N-H and 0-C stretching frequencies decrease. On the other hand, the resistance to bending of the N-H and O=C bonds is increased by the hydrogen bond, and the bending frequencies therefore increase. Furthermore, the nature of the peptide hydrogen bonding influences these frequencies. Examination of a large number of polypeptides in thin solid films prepared in a variety of ways has shown that characteristic frequency changes accompany the conversion of intrachain to interchain hydrogen bonds (a-+ p transformation). This is shown in Table V. These changes are conceivably due to a change in the conformation of the polypeptide chain. A difficulty, however, in applying this method to solutions of polypeptides and proteins in nonaqueous solvents is that many solvents of interest are insufficiently transparent in the important regions of the infrared, and much of the work heretofore has therefore been carried out with solid films of these macromolecules. The study of helical structures in solution has been given great impetus in recent years by the discovery that such structures contribute markedly to the optical rotatory properties of polypeptides and proteins (Cohen, 1955; Moffitt, 1956; Moffitt and Yang, 1956). This subject is undergoing very rapid development and continual re-evaluation as more experimental studies are performed. A full discussion of optical rotatory phenomena with these substances is inappropriate in this article, and for further details, the reader is referred to recent reviews (Blout, 1960; Levedahl and James, 1961; Urnes and Doty, 1961). Here a brief summary of the subject must suffice. The equation of Moffitt and Yang (1956), although without adequate theoretical basis (Moffitt et al., 1957), does describe the optical rotatory
27
PROPERTIES OF PROTEINS I N NONAQUEOUS SOLVENTS
properties of a number of helical polypeptides and proteins: [m'],=
In this equation,
Mo _ _3_ . blx na + 2 100
[m']h is
=
ad:
+----b-d
A2 - A:
(A*
2 x,:
the efTective residue rotation at wavelength X,
TABLEV Wave Numbers of Principal Bands i n Polypeptide Infrared Spectra03 b C=O (str.) Polymer
a
(cm-1)
1657 1655 (1652) Poly-L-glutamic acid 1652 Poly-L-lysine hydroiodide 1656 Poly-y -methyl-L-gluta1654 mate Poly-y-benayl-L-gluta1653 mate Poly-L-leucine-I,-phenyl1656 alanine (1 :1) Poly-DL-alanine 1661 1665 Poly -nL-phenylalanine 1661 Poly -DL-leucine (1659) Poly-y-methyl -D r,-glutamate 1662 Poly -7-benzyl-DL-ghtamate 1661 Poly-DL-leucine-DLphenylalanine (1 :1) 1661 Poly -DL-leucine-DL-aamino-n-caprylic acid (2:l) (1659) Poly -L-alanine Poly -L-leucine
NH (def.)
NH (str.)
B
a
(cm-1)
B
(cm-1)
(cm-1)
(cm-')
(cm-')
1635
1545 1545 (15491 1550 1531 1551
1524
3293 3292
3283
1525
3292
3287
B
1628
a
3292
1549 1545 1629
1539 1542 1547 (1551)
1631
1515
3305 3300
1526 1551
3297
1542 1551 (1553)
Figures in parentheses refer t o 1% solutions in nonpolar solvent. Data from Bamford et al. (1956).
which is the specific rotation [.]A corrected for the refractive index, n, of the solvent, and for the average molecular weight, M O, of the amino acid residues; and a0 , bo , and A0 are constants. The constant uo is a composite of a number of contributions to the rotation, but the constant bo appears to be more directly correlated with helical structures, in view of the following considerations.
28
S.
J. SINGER
A number of synthetic polypeptides have been studied by optical rotatory dispersion measurements in the visible arid near ultraviolet, with some of the results shown in Table VI. Almost all of these systems can be fitted to the Moffitt-Yang equation with A 0 = 212 f5 mp. However, the values of bo fall into two groups. In one group (group I, Table VI), the values of bo are all remarkably similar, - 630" f Z O O , and are essentially independent of solvent, provided the solvent does not disrupt the helix (see Section IV,D). In solvents which by other criteria do convert group I polypeptides from the helical to random-coil form, bo generally becomes zero. In the other group (group 11) bo takes on a variety of values ranging from 0" to +611° in solvents in which the polypeptides are helical and shows no characteristic change in solvents in which the macromolecules are randomly coiled. At first sight, these results suggest that the parameter bo is subject to such wide variations for helical polypeptide structures as to be of little use in studies of the helical content of proteins. The situation, however, appears to be more favorable than this. First of all, it is worth noting that the optical rotatory disperions of the free amino acids themselves segregate into the same two groups (Strehm et al., 1961). The structural distinction between these two groups appears to be the nature of the substituent on the &carbon atom of the amino acid. All the monomeric amino acids of the group I polypeptides have a methylene group (or an H-atom in the case of alanine) on the P-carbon, and all of those in group I1 have some other functional group so situated. Now, globular proteins generally contain about 90 % of amino acids of group I and only about 10 % of group 11. In copolymers of group I and group I1amino acids, the group I component is the dominant one (Blout, 1960, 1961) in determining the sign and magnitude of bo . Hence, it is not unreasonable that helical structures in protein molecules might exhibit the characteristics of group I polypeptide helices. Furthermore, those proteins that by other criteria appear to be extensively helical, exhibit bo values of -630" f 30" (Table VI) in their native conformations and values close to zero when denaturated (Cohen and Szent-Gyorgyi, 1957), which corresponds precisely to the behavior of group I polypeptides. Finally, in the case of myoglobin, for which X-ray crystallographic methods show that 77 % of the amino acid residues are in the right-handed a-helical conformation in the crystal, optical rotatory dispersion data obtained in aqueous solution in the ultraviolet from 360 to 240 mp, can be fitted to the Moffitt-Yang equation to give a helical content in reasonable agreement with the observed value (Urnes et aE., 1961). There appears to be, therefore, considerable correspondence between the optical rotatory dispersion parameters of the group I polypeptides and of
TABLE VI Opdical Rotatory Dispersion of Polypeptides and Some Proleins" Helix-forming solvents Macmmoleculc Group I polypept,ides Pol y-L-alanine Poly-y-benzyl -L-glutamate
Solvent
bo
----I~-
Poly-L-glutarnic acid Poly-L-leucine Poly -L-lysine Poly -N-carbobenzoxy-L-Iysine h3 a Copoly-L-lysine-L-glutaniic acidb Group I1 Polypeptides Poly -L-tyrosine Poly-0-acetyl-L-serine Poly -1-benzyl -L-histidine Poly-L-serine Poly-L-histidine.j5 HzO Poly-&benzyl-L-aspartate
-m
Random coil-forming solvents ha _ _ _ _ _ _ - ~
Solveiit
Film Ethylene dichloride Dioxane CIICl, N , N-Dimethylformamidc HzO, pH 4.4 Dichloroacetic acid 0.2 M NaBr in HzO, pH 11.5 N , N-Diinethylformainide 2-Chloroethanol
- 390 0
Dichloroacetic acid Hydrazinc
+%
- 177 0
H20, pH 10.5 Trifluoroacetic acid HzO, pH 6.8
0
Trifluoroacetic acid
+450
0 0 +G11
0.15 M NaCl in HzO, pH 10.85 25:75 Dichloroacetic acid:ethylene dichloride Benzyl alcohol 10 M LiBr in HzO 0.2 M NaCl in HzO, pH 6 CHCl3
-600 -620
HzO, 0.6 M KCl E I 2 0 , O . G M KC1
- 190d 0
-f3B
-630 -625 -6430 -610 -650 -650 -625 -G3G
+sa 0
NU
0
+3%
0 0
0.15 iM NaCl in HzO, pH 12.3 Dichloroacetic acid
Dichloroacetic acid 8 M Urea in Hz0
0.2 M NaCl in HzO, pH 3.7
Proteins Paramyosin Tropomyosin
Data froni Hlout (1SGO). Doty el al. (1958). = NL signifies a nonlinear plot. d Not completely denat,rired (Cohen and Saent-Gyorgyi, 1957).
u
b
HzO, 9.5 M urea H2U, 9.5 M urea
30
S. J. SINGER
certain proteins in their right-handed a-helical, as well as their disordered, conformations.' Optical rotatory dispersion measurements of globular proteins in aqueous solutions yield a range of bo values from around zero to - 300' (Table VII) . It has been suggested that the fraction - bo/630" be used as a measure of the fractional helical content of globular proteins (Cohen and SzentGyorgyi, 1957; Doty, 1959). Several problems associated with this suggestion must be considered. If such globular proteins indeed have only a fraction of their amino acid residues in the helical form, the helical segments may be fairly short and might have atypical optical rotatory properties. It appears reasonable theoretically, however, that even short helices TABLE VII Estimated Per Cent Excess of Right-Handed Helical Contents of Proteins* Protein Tropomyosin Insulin Bovine serum albumin Ovalbumin Lysozyme Pepsin Histone Ribonuclease Globin H
Water solution"
2-Chloroethanol solutione
88 38 46 31 29
110 45 75 85 63 44 72 67 74
31 20 16 15 ~~~
a
~
Data from Doty (1959). (-bo/630) X 100. Average of (-b0/630) X 100 and (a7/650) X 100 (see Doty, 1959).
exhibit the rotations characteristic of infinite ones, and that their contributions within a protein molecule be additive (Zimm et al., 1960). At least the following possibilities must also be considered: ( a ) both right- arid left-handed a-helices may be present in a protein molecule, and partially compensate each other's rotatory contributions; ( b ) helical structures other than the a-helix might be present in certain globular proteins (Bamford 1 Some ambiguity must be attached t o this statement in view of the conclusions reached by Luzzati et al. (1961) on the basis of small angle X-ray scattering studies from dilute solutions of polybenzyl-L-glutamate in dimethylformumide, pyridine, and m-cresol. These authors conclude t h at the conformation of the polypeptide in these solutions is t h at of the 310-helix(Donohue, 1953) and not that of the a-helix. The value of bo = -630" in Eq. (13) was obtained from optical rotatory dispersion measurements with similar solutions, and therefore if the conclusions of Luzzati et al. are correct, this value characterizes the 310-helix. It would then be necessary to determine whether the bo value for the a-helix is significantly different.
PROPERTIES OF PROTEINS IN NONAQUEOUS SOLVENTS
31
et al., 1956, Chapter IV), and have different rotatory properties; and ( c ) structures other than helical ones, such as interpeptide chain structures and asymmetric disulfide groups (Strehm et al., 1961) might affect the rotatory dispersion. Our primary objective in the context of this article is to examine the rotatory dispersion of proteins in different solvents. The qualifications raised in the preceding paragraph, while of significance to the interpretation of the absolute value of bo in any one system, are of less importance when comparing the value of bo for a given protein in different solvents. In this connection, the demonstration that bo for the helical conformation of poly7-benzyl-L-glutamate is unaffected by different solvents (Maffitt and Yang, 1956) (Table VI) is particularly pertinent. In subsequent sections, therefore, the procedure will be followed of estimating the changes in helical contents of a particular protein in different solvents by comparing the values of - bo/630° in the different solvents (Cohen and Szent-Gyorgyi, 1957; Doty, 1959). The alternative proposals made by Yang and Doty (1957) that the helical content of proteins be estimated from values of [aIDor from A, in the equation [a] = k/(X2
- A:)
are subject to considerable ambiguity in nonaqueous solvent systems (Tanford et al., 1960; Tanford and De, 1961). These parameters appear to reflect more than only helical structural changes in protein molecules. It has recently been found (Simmons et d.,1961) that a pronounced Cotton effect exists in solutions of helical polypeptides and proteins in the wavelength region from about 260 mp to 220 mp. A large trough in rotation occurs at 233 mp which is removed when the helix is disrupted. The magnitude of [ ( ~ ] 2 3 ~may therefore be found to serve as an independent measure of the helical contents of proteins. This effect has not yet been extensively applied to proteins in nonaqueous solvents, but it should prove to be of great interest for proteins dissolved in those solvents which are sufficiently transparent in this region of the spectrum. 5. Lyophobically Bonded Regions of Protein Molecules
It is difficult at the present time to prescribe general methods by which effects on lyophobic interactions can be specifically distinguished and measured. In certain cases, pK shifts of ionizable groups may occur when they are situated in the interior of hydrophobic regions of protein molecules, where they are inaccessible to the aqueous phase. On the other hand, pK shifts may be due to a variety of other causes as well, such as hydrogen bonding to other groups, vicinal electrostatic effects, or longrange electrostatic effects (Kauzmann, 1959). Some of the phenolic OH
32
S. J. SINGER
groups of tyrosine residues of proteins undergo large pK shifts, as can be readily observed by spectrophotometric titration experiments (Crammer and Neuberger, 1943). Tyrosine OH groups are of special interest in this context because they are not ionized a t physiological pH, unlike most other titratable groups of protein molecules. They are therefore more likely to be buried (probably hydrogen-bonded) in hydrophobic regions of low dielectric constant within a protein molecule, than are for example, carboxylate, ammonium, or guanidinium groups. Such buried tyrosines should exhibit large pK shifts. The tyrosines of bovine pancreatic ribonuclease ( RNase) appear to be n case in point. Three of the total of six tyrosine residues per RNase inolccule titrate reversibly with a normal pK of about 10, but the other three titrate only a t much higher p H and then irreversibly (Shugar, 1952; Tanford et al., 1955a). These results suggest that the RNase molecule has to undergo a profound structural rearrangement before the three anomalous tyrosines become accessible to titration. Furthermore, the absorption of RNase due to tyrosine residues a t about 280 mp exhibits a hyperchromic effect presumably as a result of the special environment of the three anomalous tyrosines. With several other proteins, such 2,s bovine serum albumin (Tanford and Roberts, 1952), lysozyme (Tanford and Wagner, 1954), and p-lactoglobulin (Tanford and Swanson, 1957), pK shifts of the phenolic OH groups of tyrosine residues are observed, but these are of a qualitatively different nature. Thus, the tyrosines of any one of these proteins cannot be readily differentiated into a normal and an abnormal variety, since the spectrophotometric titration data for these proteins are reversible and fall on single smooth curves, in contrast to the situation with RNase. On the other hand, the tyrosine residues of ovalbumin show comparable behavior to the three abnormal tyrosine groups of RNase (Crammer and Neuberger, 1943). About 2 of the total of 9 tyrosine residues appear to titrate normally, but the remainder are not titrated up to p H 12. At p H 13, these anomalous tyrosines become titratable, and this is accompanied by the irreversible denaturation of the ovalbumin molecule. It is therefore suggested (Tanford et al., 1955a) that at least those tyrosine residues which are distinguished by being titratable only a t p H > -12, and whose titrations are accompanied by an irreversible denaturation of the molecule, be presumed to exist in hydrophobic regions of their respective protein molecules in aqueous solutions. The titration characteristics of these anomalous tyrosine residues in nonaqueous solvents may then be examined to determine whether the hydrophobic regions in which they are presumed to exist persist in the nonaqueous solvent (Sage and Singer, 1958, 1962).
PROPERTIES OF PROTEINS I N NONAQUEOUS SOLVENTS
33
In certain cases, inaccessibility to reagents other than Hf and OH- may also be used to distinguish and to follow the fate of hydrophobic regions of protein molecules. For example, the three normal tyrosines of RNase may be iodinated under conditions which differentiate them from the three abnormal buried tyrosines (Cha and Scheraga, 1961). Another possible example involves methionine residues. It appears probable that the relatively nonpolar methionine residues will often be located in hydrophobic regions of protein molecules, as is explicitly demonstrated in the structure of the myoglobin molecule (Kendrew et al., 1961). It is known that the S of free methionine in aqueous solution reacts readily with iodoacetate to form a sulfonium salt and that the rate of the reaction is essentially independent of pH over a wide range (Gundlach et al., 1959a, b). The four methionine residues of native RNase at pH 5.5, on the other hand, do not react with iodoacetate; however, if the RNase is treated with iodoacetate at pH 2.8, or at pH 5.5 in the presence of 8 M urea, the methionine residues react extensively, and the molecule is irreversibly denatured (Stark et al.. 1961). That is, its activity is then lost, and interestingly, its anomalous tyrosines titrate normally. These results clearly imply that the four methionine residues are buried in the hydrophobic regions of the native molecule, and react with iodoacetate only when they are exposed. This suggests a possibly general method of following the fate of hydrophobic regions of protein molecules dissolved in suitable nonaqueous solvents. Let us assume that, as in the case of RNase, at least some of the methionine residues of a native protein molecule in aqueous solution show little reactivity towards iodoacetate or iodoacetamide. Then if the rates of reaction, under standardized conditions in the nonaqueous solvent, of the methionine S of the protein and of free methionine with these reagents are the same, it may be presumed that these groups on the protein are exposed in the solvent and that the hydrophobic regions are disrupted. The rates of reaction at the S of methionine can readily be followed by n,mino acid analyses (Gundlach et al., 1959a, b ) . The ultraviolet spectra of many proteins exhibit small shifts of absorption maxima to shorter wavelengths and small decreases in extinction coefficient at these maxima when the native conformations are disrupted in aqueous solution (see Beaven and Holiday, 1952; Beaven, 1961; and the article by Wetlaufer in this volume). These effects are presumably due to the special environments in which chromophoric side chains, particularly those of tyrosine and tryptophan, find themselves within the native protein molecule as compared to the free chromophores in aqueous solutions. The special properties of these native environments have been attributed variously to hydrogen bonding, particularly of tyrosine residues (Scheraga and Laskowski, 1957), vicinal electrical effects, including ion-
34
6. J. SINGER
dipole and dipole-dipole interactions (Wetlaufer et al., 1958), and hydrophobic bonding (Yanari and Bovey, 1960). The last named authors by studies of the simple model compounds benzene, indole, and phenol in aqueous and hydrocarbon solvents, adduce evidence that these spectral effects can be associated with the polarizability of the medium surrounding the aromatic residues. On passing from isooctane to water, i.e., from the medium of larger polarizability (higher refractive index) to lower, the spectra of all three model compounds exhibit similar shifts to shorter wavelengths, which are closely analogous to those exhibited in the transformation of native to denatured proteins in aqueous solution. These spectral changes in proteins, Yanari and Bovey conclude, can therefore be adequately accounted for by hydrophobic bonding in the native proteins. In nonaqueous solutions of proteins, the situation may be more complicated. For example, Yanari and Bovey found that relative to isooctane, ethanol and water have opposite effects on the spectra of phenol and indole. By the criterion of polarizability, however, ethanol should be intermediate between isooctane and water. I t is suggested by these authors that differences in hydrogen bonding of ethanol and water to the model compounds are responsible. Since most of the nonaqueous solvents for proteins are hydrogen bond-forming agents, it is not clear what spectral effects to expect in any particular solvent. In view of these results, two observations may be made: ( a ) as a corollary to studies of protein ultraviolet spectra in any particular nonaqueous solvent, the spectral properties of relevant simple compounds in that solvent must be investigated; and (b) any changes in protein spectra produced as a result of modification of the native protein conformat,ionin a particular nonaqueous solvent must be superimposed on changes resulting simply from the replacement of the aqueous environment by the nonaqueous one of generally different polarizability and refractive index. In the extreme case, for example, it may make little or no difference spectrally whether the aromatic chromophores remain internally bound within the protein molecule, or whether they become exposcd to the solvent, and hence no useful information about protein conformations can he expected. More studies have to be made to clarify to what extent spectral changes can be useful in the investigation of proteins in nonaqueous solvents. Many other methods of studying protein structure in solution have been proposed and tested (Kauzmann, 1959). Many of these can be used to establish that structural changes in a protein molecule may have occurred in a particular solvent, but almost all suffer from their inability to discriminate unambiguously between effects produced within helical or hydrophobic regions of protein molecules. The elegant method of the rate of deuterium exchange of proteins in aqueous media (LinderstrGm-Lang,
35
PROPERTIES OF PROTEINS I N NONAQUEOUG SOLVENTS
1955), which might be extended with suitable modifications to nonaqueous solvents such as alcohols and glycols, also falls in this category. Taken together with the results of methods already discussed, however, such measurements might provide useful information in nonaqueous media. The ultimate in structural studies would, of course, involve X-ray crystallographic studies of protein crystals prepared from nonaqueous solvents of the kind that are now being so successfully carried out with certain protein crystals prepared from aqueous media (Kendrew et al., 1961). A priori, there is no reason to exclude the possibility that proteins might be crystallized from pure nonaqueous solvents, although no reports of such attempts have appeared.* This is particularly so in view of the fact that in certain pure solvents, proteins appear to exhibit a more highly ordered (helical) conformation than they do in water solution. With these remarks we now proceed to discuss the experimental studies that have been made to date on the conformational properties of polypeptides and proteins in nonaqueous solvents.
D. Polypeptides in Nonaqueous Solvents Synthetic polypeptides provide very useful model systems for examining some of the problems of protein structure, as has already been discussed in Section IV,C,2. In the course of an important and extensive series of investigations of these compounds by Bamford, Elliott, and their co-workers (summarized in Bamford et al., 1956), it was shown that films of polypeptides cast from different solvents exhibited two different structures, designated a and fi, which were distinguished by two different classes of infrared spectra (Table V). The a-form is now known to be associated with the a-helix, and the 0-form with extended interchain structures. These results suggested that the a + 0 transformation in polypeptides could be achieved by appropriate solvents. This conclusion for these particular experiments, however, has been disputed by Blout and Asadourian (1956), who suggest that the polypeptide samples employed in these experiments were mixtures of a- and /3-structures in the solid state, and that extraction of one or the other form by particular solvent, was involved in these film-casting studies, rather than a true a * &transformation. An interesting study of the influence of solvent on the structure of polyf
2 In many instances (cf. Cohn et al., 1947; King et al., 1956) proteins have been crystallized from mixtures of water and organic solvents, but t o our knowledge, in no case from pure nonaqueous solvents. In this connection, the use of solvent additives t o induce the crystallization of proteins involves the not fully recognized hazard t h a t appreciable conformational changes may be induced in the protein molecules i n such solvents (see Section IV,E).
36
S . J. SINGER
y-benzyl-L-glutamate has been made in the course of a series of studies by Doty, Blout, and co-workers (Doty et al., 1956; Yang and Doty, 1957). The liquids tested as solvents for a high molecular weight (130,000) polypeptide sample could be placed into four categories: (1) nonsolvents; (2) solvents in which extensive aggregation of the polypeptide occurred; (3) solvents in which the polypeptide was molecularly dispersed and in a helical conformation’; and (4)solvents in which the polypeptide was molecularly dispersed and in a disorganized, random-coil conformation. These liquids are listed in Table VIII. The criteria employed to distinguish between helical and random-coil structures were viscometry and anomalous rotatory dispersion. TABLEV I I I Solution Properties of Poly-~-benzyl-~-glutamate~~ Helix-f orming
Nonsolvents
* ~ ~ ~ ~ : “solvents g
Formic acid
Benzene
Acetic acid Propionic acid Formamide
CHCI,
Dioxane Ethylene di chloride
E thylenediamine CCll
N,N-Dimethylformamide m-Cresol Dioxane CH C13-formainide
Random coil-forming solvents Dichloroacetic acid Trifluoroacetic acid Hydrazine
Ethylene dichloride Pyridine
Polymer of 130,000 weight-average molecular weight.
* Data from Doty et al. (1956), and Yang and Doty (1957). The solvents which were found to induce the random-coil conformation are all strongly protic solvents. Their capacity to form strong solventsolute hydrogen bonds is probably primarily responsible for the disruption of the helix. In this particular polypeptide no strongly acidic or basic side chains are present, and it is not likely that the weakly protic ester and amide groups enter significantly into acid-base reactions with these solvents. On the other hand, it would be gratifying t o have unequivocal evidence that this is truly a negligible factor. The possibility should be kept in mind that in a strong acid such as trifluoroacetic acid a small but significant fract,ion of the amide groups might be protonated. While this might not result in serious electrostatic interactions in a medium of as low dielectric constant as that of trifluoroacetic acid, it might by periodically disrupting intrapept>idehydrogen bonding along the helix, contribute substantially to the destabilization of the helix. (See below on the effect of helix length on stability.) Perhaps nuclear magnetic resonance studies 1
Footnote 1 on page 30.
PROPERTIES OF PROTEINS IN NONAQUEOUS SOLVENTS
37
in some of these solvents would be useful in determining the state of the amide group. The helix-forming solvents all appear to be relatively weaker hydrogen bond agents than the random coil-forming solvents, The conclusion seems justified that this nonionic homogeneous polypeptide assumes a helical conformation if no unusually strong solvent-solute hydrogen bonds interfere. The same conclusion is forthcoming from studies of other nonionic polypeptides (Downie et al., 1957, 1959). Two important qualifications of this conclusion must be made. While hydrogen bond interactions may be of primary importance in stabilizing the helical forms of such polypeptides, other factors contribute as well. This was particularly demonstrated by Fasman (1962). A number of different nonionic polypeptides in the helical form in chloroform solution were titrated with dichloroacetic or trifluoroacetic acids to determine the concentration of acid required to convert the polypeptide to the random-coil form, as judged by optical rotatory criteria. It was found that for the polypeptides poly-P-benzyl-L-aspartate, poly-e-carbobenzyloxy-L-lysine, and poly-7-benzyl-L-glutamate, about 7 %, 38 %, and 68 % dichloroacetic acid was required, respectively. For poly-L-methionine, the helical form is partially stable even in 100 % dichloroacetic acid, while poly-L-leucine is completely helical in 100% dichloroacetic acid, and requires a stronger acid, trifluoroacetic acid, to convert it to the random coil. These results reflect the contribution of regular homogcneous arrays of side chains to the stabilization of the helix, which must be included in the broad spectrum of effects termed lyophobic interactions. On the other hand, since the regularity and homogeneity of side chains is a feature of polypeptides not shared by proteins, it is not clear to what extent these results of Fasman's bear directly on the problem of lyophobic interactions in proteins. A second qualification is that the helical conformation in a given solvent is much less stable if the number of residues in the helix is less than a certain critical value, due to end effects (Schellman, 1955 a, b; Blout and Asadourian, 1956; Goodman and Schmitt, 1959). Doty el al. (1958) extended these observations by studying the influence of solvent on the conformation of an ampholytic polypeptide, copoly-Llysine-L-glutamic acid. In trifluoroacetic acid, the C=O stretching frequency is 1635 ern-', and bo in Eq. (11)is zero. The polypeptide is clearly present in the random-coil conformation. In 2-chloroethanol, on the other hand, the C=O stretching frequency is 1655 cm-I (see Table V) and bo is -636") and the polypeptide is in the helical conformation. Again, in trifluoroacetic acid, strong solvent-solute hydrogen bonds, particularly C=O. * .HOOCCF3 bonds (see Section IV,B), most likely play a prominent role in destabilizing the helix. While the copolymer must be present
38
S. J. SINGER
in this solvent with all the e--NHz groups of the lysine residues converted to e-NHt groups, these should certainly be extensively ion-paired to trifluoroacetate counterions in a medium of as low dielectric constant as trifluoroacetic acid (see Section IV,B,l), Repulsive electrostatic interactions in this solvent must therefore be of negligible structural significance. The possible protonation of amide groups in this solvent, however, which was discussed above, should be noted again. I n 2-chloroethanol solution, the C=O. . .HOCHnCH2C1hydrogen bond is considerably weaker than the corresponding C=O. . .HOOCCF, bond, and most likely as a. result of this, the polypeptide assumes the helical form. In aqueous solutions of this copolymer, the conformation is markedly dependent on pH. In acid solution b, = -310; at pH 7 bo = -100; and at pH 12 bo = 0. It is evident that the change in electrostatic interactions with pH is critical in this solvent. In acid solution, where the polypeptide is about 50 % helical, the electrostatic interaction of the E-NH: groups tends to disrupt the helix (poly-L-glutamic acid under these conditions is entirely helical). If the ionic strength is raised in the acid solution, the helical content is increased. At neutral pH, there is little net charge on the molecule since both COO- and NH; groups are present in roughly equal numbers, yet the helical content is only about 15%. In this case, the net increase in short-range electrostatic repulsions between adjacent, like charges (the copolymer being of more-or-less random sequence), and perhaps the loss of side-chain carboxyl-carboxyl hydrogen bonds, probably serve to reduce the helical content below that found in acid solutions. At pH 12, the large net negative charge on the molecule acts to destabilize the helix. Of course, these electrostatic effects are critical because of the large dielectric constant of water and because the electrostatic factors are superimposed on the low intrinsic stability of intrapeptide hydrogen bonds in aqueous solution (Section IV,B,2). In these relatively simple polypeptide systems, therefore, the experimental studies so far performed suggest a t least a crude correlation between the capacity of a solvent to disrupt polypeptide helices and the capacity of a solvent to form strong hydrogen bonds, although in special cases electrostatic and lyophobic interactions also appear to be involved. There is little apparent correlation with any other independent property of the solvents. The polypeptide poly-L-proline is an interesting and special case because of the exclusively imino peptide linkages of the polymer. The properties of this macromolecule and its high and low molecular weight analogs are discussed in the review of Harrington and von Hippel (1961), and will only briefly be mentioned here. The polymer can exist in two forms of markedly different optical rotatory properties, and can be converted from one form
PROPERTIES OF PROTEINS IN NONAQUEOUS SOLVENTS
39
to the other, depending on the solvent. In acidic solvents such as acetic or formic acids form I1 of the polypeptide exists, and if these solutions are diluted with propanol, form I is produced. The former structure is thought to be a left-handed helix with all trans-imide linkages, and the latter a right-handed helix with all cis-imide linkages. For the purpose of this review, there are several significant features of these experiments to be noted. Poly-L-proline does not have any intrapeptide hydrogen bonds because of the absence of peptide H-atoms, yet it exists in apparently only two principal conformations in solution, both highly ordered. Furthermore, the stability of these two conformations is dependent on the solvent, and hence on solute-solvent interactions. The fact that form I1 is stable in acidic solvents suggests that strong hydrogen bonding between the peptide C=O groups and the OH groups of the solvent plays a critical role in stabilizing this conformation, and that the weaker hydrogen bonds formed between the C=O groups and alcohol solvents are inadequate to overcome the effects of solute-solute (lyophobic) interactions favoring form I. [That solute-solute interactions are more prominent in form I is consistent with form I having a shorter helical pitch and lower asymmetry, hence greater compactness, than form I1 (Harrington and von Hippel, 1961).] Another interesting feature is that the interconversion of the two forms of poly-L-proline is catalyzed in both directions by small amounts of strong acid (Steinberg et al., 1960). These authors provide strong evidence that the mechanism of interconversion involves proton binding at the imide linkages of the polymer, thus facilitating cis-truns-isomerisation about the imide linkage. This is of particular pertinence in considering the effects of strongly protic solvents on proteins containing proline imino peptide linkages. Much remains to be done with synthetic polypeptide systems in nonaqueous solvents, particularly weakly protic ones. If indeed the helical conformation of a polypeptide (other than poly-L-proline) in solution is mainly stabilized by intrapeptide hydrogen bonds, this can be used to arrange different nonaqueous solvents in the order of their effectiveness as hydrogen bond-forming agents towards the amide group. A solution of poly-P-benzyl-L-aspartate in the helical configuration in chloroform, for example, could be titrated to determine the volume or mole fractions of different weakly protic nonaqueous solvents required tjo disrupt the helix. This information should correlate with independent studies of the hydrogen bonding of simple amides in the pure nonaqueous solvents. Such differentiation of the effective hydrogen bond-forming capacity of nonaqueous solvents would be very valuable in quantitative studies of helixcoil transformations in polypeptides and in the interpretation of the effects
S. J. SINGER
40
of these solvents on protein conformations as is discussed in the following section.
E . Proteins in Nonaqueous Solvents 1 . Weakly Protic Solvents
Although chronologically, structural studies of proteins in strongly protic solvents preceded those in weakly protic ones, it is advantageous to discuss the latter studies first. Yang and Doty (1957) were the first to obtain clear evidence for conformational changes in protein molecules in solvent mixtures rich in weakly protic liquids. By the criterion of anomalous optical rotatory dispersion, they showed that silk fibroin is extensively helical in a solvent mixture containing 15 % (by volume) of dichloroacetic acid and 85 % ethylene dichloride, whereas it is considerably less helical in a concentrated aqueous LiBr solution. Sage and Singer (1958) found that the three anomalous tyrosine residues of RNase titrated normally in pure ethylene glycol solution, which suggested that the hydrophobic regions of the molecule present in aqueous solution were disrupted in the nonaqueous solvent (Sage and Singer, 1962). More recently, Doty and co-workers (Doty, 1959) have found that a large number of globular proteins are directly soluble in the solvent 2chloroethanol. While the apparently unique solubilizing power of this substance is probably attributable to the HC1 present in the unstable solvent, some very significant measurements of the optical rotatory dispersions of proteins in a “pure” weakly protic nonaqueous solvent were made as a result of this discovery. In all cases studied, proteins exhibited larger, often substantially larger, values of -bo in 2-chloroethanol than in HzO (Table VII). These effects are reversible with change of solvent. Increased helical content in proteins dissolved in such solvents is probably connected with ( a ) decreased hydrogen-bonding capacity of the solvent compared to water, and ( b ) decreased electrostatic repulsive interactions between the fixed charges on the protein molecule in the low dielectric constant solvent as compared to water, due primarily to counterion binding. The suggestion that intrapeptide hydrogen bonding is stronger in 2-chloroethanol than in water is consistent with the arguments advanced in Section IV,B,2; with the related experimental results obtained with the copolymer copoly-L-lysine-L-glutamic acid in HzO and in 2-chloroethanol (Doty et aZ., 1958) described in Section IV,D; and with results in other solvents discussed later in this section. Nevertheless, despite intrinsically weaker intrapeptide hydrogen bonding (and presumably also weaker side-chain hydrogen bonding) in water
PROPERTIES OF PROTEINS IN NONAQUEOUS SOLVENTS
41
solutions than in 2-chloroethanol, and despite increased electrostatic repulsive interactions, globular proteins, instead of assuming a random-coil conformation in water solution, exhibit highly folded, compact, and relatively stable structures in HzO. It appears necessary to conclude that some interactions other than, and in addition to, hydrogen bonding must be involved in stabilizing the native conformations. The interactions involved must be hydrophobic interactions. In the absence of the results in 2-chloroethanol, it might have been argued that hydrogen bonding in all its various forms is the major kind of interaction responsible for stabilizing the native conformatlion of globular proteins, but that the low apparent helical content of some of these proteins in H2O is the result of either of two possibilities: ( a ) the presence of comparable amounts of both left- and right-handed helices; or ( b ) the presence of a preponderance of interpeptide (and therefore not helix-forming) hydrogen bonds as compared to intrapeptide ones. If either of these possibilities were actually realized, however, it would be difficult t o explain why a nonaqueous solvent such as 2-chloroethanol should so profoundly favor the formation of right-handed a-helices, since, other things being equal, there is no reason why the solvent should discriminate so markedly between one kind of N-He . .O=C hydrogen bond and another. Reference to Table VII indicates that with some globular proteins as much as a fourfold increase in - b0/630, and hence by inference in net right-handed helical content, is found in 2-chloroethanol as compared to H2O. It follows then that hydrophobic interactions must play a vital role in stabilizing the native configurations of many proteins in aqueous solution. Corresponding increases in -bo are observed upon the addition of similar amounts of other weakly protic, hydrocarbon-miscible, nonaqueous solvents t o protein solutions in H2O (Tanford et al., 1960; Tanford and De, 1961). Among the solvents so far examined which exhibit this effect are dioxane, ethanol, dimethylformamide, N-methylpropionamide, and 1-propanol. There is therefore nothing unique about 2-chloroethanol in this respect except its solubilizing capacity for proteins (Section II1,C) which is not nearly as extensively exhibited by the other pure solvents. An example of the studies of Tanford and co-workers is given in Fig. 4. The addition of a number of nonaqueous solvents to an aqueous solution of p-lactoglobulin a t pIl 3.0, ionic strength 0.02, results in a gradual increase in -bo ; the parameters - [m’]and -a0 in Eq. (13), however, first show an increase and then a decrease as the solvent mixture is enriched in the nonaqueous component. The maximum values of -[m’] and -a0 occur at a solvent composition at which only a small change in bo is found. These results suggest that at least two successive conformational changes are produced on the addition of the nonaqueous solvent to the aqueous
42
8. J. SINGER
solution. Depicted in Fig. 5 , from Weber and Tanford (1959), is one possible schematic representation of these changes. The first change might involve an unfolding of the protein molecule with little change in helical content; the second might then involve a refolding of the molecule into right-handed &-helicalregions. Related experimental results, except for the absence of optical rotatory dispersion data, had been obtained
mi organic sdvmt/100ml solution FIG.4. The effect of solvent additives on the optical rotatory properties of 8lactoglobulin at pH 3, ionic strength 0.02. D = dioxane, E = ethanol, F = formamide, G = ethylene glycol, U = urea. For the last additive, the abscissa is given in grams/100 ml of solution (Tanford et al., 1960, 1962; Tanford and De, 1961).
earlier by Bresler and Frenkel (Bresler, 1958) upon addition of dioxane and chloroethanol to aqueous solutions of bovine serum albumin. It is particularly interesting that the solvents used in these studies are not greatly differentiated by their capacities to alter the conformation of native proteins, and that among them is the solvent dioxane. For reasons expounded in Section IV,B,2, it is fairly certain that intrapeptide hydrogen bonds are stronger in dioxane than in water solution. The transformation from the native conformation to the unfolded state (Fig. 5A ---f B) cannot therefore be caused by the rupture of intrapeptide hydrogen bonds. It is
PROPERTIES O F PROTEING I N NONAQUEOUS SOLVENTS
43
much more reasonable to assign primary responsibility for the transformation to a reduction in lyophobic interactions in the mixed solvent as compared to pure HzO (hydrocarbons being more soluble in the former than in the latter), accompanied by only a slight reduction in the hydrogenbonding capacity of pure HzO. The subsequent transformation from the unfolded to the extensively helical conformation (Fig. 5B -+C) might then be explained as primarily the result of substantially further reducing the hydrogen-bonding capacity of the solvent so as to permit much more extensive intrapeptide hydrogen bonding to take place. On the other hand, it is possible that changes in electrostatic interactions are significantly involved in the conformational transitions that have just
A
C FIG.5. Schematic representations of a protein molecule in several possible conformational states: (A) its native conformation in aqueous solution; (B) a n unfolded conformation retaining the net helical content of the native form but with hydrophobic regions disrupted; and (C) an extensively helical conformation.
been described. These experiments of Tanford and his co-workers were performed with protein molecules carrying their maximum positive charge, in order t o maintain the value of Z constant upon addition of a weakly protic solvent. The addition of a nonaqueous solvent such as dimethylformamide to HzO lowers the dielectric constant of the solvent. For reasons discussed at length in Section IV,B,l, the electrostatic free energy, P, [Eq. (4)],of the native aqueous conformation of a protein molecule should, at constant 2, first increase and then decrease as the concentration of the nonaqueous component of the solvent mixture is increased (Fig. 2). This should favor the formation of an unfolded structure for a protein molecule in mixtures of a nonaqueous solvent and HzO as compared to the two pure solvents. Such unfolding might therefore be induced in a protein molecule in a mixed solvent if the molecule were already on the verge
44
S . J. SINGER
of becoming unfolded in H20, as is the case with many proteins bearing large net charges in aqueous solution. The value of D corresponding to the expected maximum value of P , is difficult to predict, particularly in a mixed solvent, in which, because of the unequal interactions of the solvent components with the protein molecule, the macroscopic value of D and its effective value might differ considerably. It is interesting in this connection that in a mixture of HzO and 2-chloroethanol containing 80 mole % HzO, in which an unfolded state of the protein RNase was detected by viscosity measurements (Weber and Tanford, 1959), a tenfold change in the ionic strength of the solvent mixture has a profound effect on the intrinsic viscosity of the protein, whereas ionic strength has no such effect in either of the pure solvents. At the highest ionic strength employed (0.2 M ) , the intrinsic viscosity is no larger than that in pure 2-chloroethanol. This dependence on ionic strength might be explained (Weber and Tanford, 1959) as a polyelectrolyte effect, i.e., the protein molecule in the 80 mole % HzO solvent mixture may remain in an unfolded state a t all ionic strengths, but the molecular domain occupied by the unfolded chains may increase with decreasing ionic strength as a result of electrostatic repulsions. On the other hand, the dependence of the intrinsic viscosity on ionic strength might also be explained as reflecting the existence of a folded conformation on the verge of destabilization, resulting in the partial conversion from the folded to an unfolded conformation with decrease in ionic strength. More information is required to distinguish between these possibilities. Furthermore, electrostatic interactions may be involved in the transformation of the unfolded protein to the highly helical conformation (Fig. 5B --+ C) in solvents rich in the nonaqueous component. In such solvents, the effective dielectric constant is probably small enough to induce extensive ion-pairing, thereby substantially reducing the electrostatic free energy contribution to the helical conformation below that existing in water, and making a more extensively helical conformation more favorable. Furthcr studies are required to evaluate the relative roles played by changes in lyophobic, hydrogen bonding, and electrostatic interactions in these transformations. A solvent which has been found to be of great interest in connection with protein conformation studies is ethylene glycol. Sage and Singer (1958, 1962) have investigated in some detail the properties of RNase in pure ethylene glycol, containing added neutral electrolyte. They examined the ultraviolet absorption spectrum, the ionization behavior of the tyrosine residues by spectrophotometric titratJion experiments, and the optical rotatory dispersion of the system. These authors found that the hyperchromicity which characterizes the
45
PROPERTIES OF PROTEINS I N NONAQUEOUS SOLVENTS
three anomalous tyrosines of the RNase molecule in water (Tanford et al., 1955a) is lost in ethylene glycol solution. The data in Table IX show that at the absorption maxima near 280 mp, the ratio of molar extinction coefficients of RNase and tyrosine ethyl ester is 6.1 in ethylene glycol, whereas it is 6.9 in water. While part of this reduction might be due solely to solvent effects independent of conformational considerations (see Section IV,C,3), the essentially complete loss of hyperchromicity nevertheless suggests that the three anomalous tyrosines of the RNase molecule which are considered to he buried in hydrophobic regions of the molecule in aqueous solution (Tanford et al., 1955a), are exposed to the solvent in ethylene glycol. Furthermore, the spectrophotometric titration of the tyrosine residues of RNase in ethylene glycol, carried out with a glass electrode, showed that all six tyrosines titrate on a normal sigmoid curve TABLEIX Ultraviolet Spectral Properties of Tyrosine Ethyl Ester and Ribonucleaseo Compound
Solvent
Absorption maximum (mp)
Molar extinction coefficient
Tyrosine e t h y l ester
0.2 M KC1-water 0.2 M KC1-ethylene glycol
275 277
1400 1850
Ribonuclease
0.2 M KC1-water 0.2 M KC1-ethylene glycol
277 275
9700 11400
Sage and Singer (1962).
and reversibly in this solvent whereas they titrate with an inflected curve and irreversibly in aqueous solution (Fig. 6). This also indicates that the tyrosine residues are accessible to solvent and that the hydrophobic regions of the protein molecule present in water are disrupted in ethylene glycol. The value of -bo in Eq. (13), however, was found to be 92", close to the value 105" for neutral aqueous solutions of the protein (Weber and Tanford, 1959). This suggests that the net helical content of the molecule is not significantly changed on transfer from water to ethylene glycol solution, which is in marked contrast to the situation in pure 2-chloroethanol, for example (Doty, 1959). Taken together these results can be interpreted to indicate that the RNase molecule in pure ethylene glycol is in the intermediate conformational state depicted in Fig. 5B. Presumably, the hydrogen-bonding capacity of ethylene glycol and of water, with respect to the peptide group, is sufficiently similar so that intrapeptide hydrogen bonding occurs to about the same extent in the two solvents. This does not necessarily mean,
46
S. J. SINGER
however, that the helical regions of the RNase molecule which are present in water solution persist in ethylene glycol ; helices originally present in hydrophobic regions of the molecule (see Section IV,B,5) in aqueous solution might become disrupted and new helical regions might be formed to an equivalent extent in ethylene glycol. On the other hand, that lyophobic int,eractionswith the nonpolar residues of the protein are weaker in ethylene 6
7
8
9
10
II
12
13
I
I
I
I
I
I
I
I
50
0
200
150
100
-50
-100
-150
pH
-200 mv(HzO)
6 0
;5 E
t t
In4 w
z
v)
g 3 t LL 0
a2 w m
2Z I 0 200
I50
loo
50
0
-50
-100
-150
-200
MILLIVOLTS (GLYCOL)
FIG.6. Spectrophotometric titrationa a t 296 rnfi of RNase in 0.2 M KCl in HzU (dashed curve) and in KC1 in ethylene glycol (solid curve and data). The upper mv and pH scales refer to the HZ0 titration and the lower scale to the ethylene glycol. Forward and back titration data in ethylene glycol show that the titration curve is reversible, unlike that in HzO. (Sage and Singer, 1958, 1962.)
glycol than in water is reasonable in view of the greater solubility of hydrocarbons in ethylene glycol than in water (Curme and Johnston, 1952) (see Table IV). While such lyophobic interactions must be weaker in ethylene glycol solution than in water, they should be considerably stronger in ethylene glycol, which is still only slightly miscible with hydrocarbons, than in nonaqueous solvents such as ethanol or dioxane, which are completely miscible with hydrocarbons at 25°C (see Section IV,B,3). It is therefore
PROPERTIES OF PROTEINS IN NONAQUEOUS SOLVENTS
47
of great interest that Tanford et al. (1962) have recently found with mixtures of ethylene glycol and water, that a very large volume fraction of ethylene glycol is required to effect any conformational changes in p-lactoglobulin; a much larger volume fraction than is required of any other weakly protic nonaqueous solvent examined (Fig. 4). This is completely in accord with the hypothesis that hydrophobic interactions are importantly involved in maintaining the native conformation of p-lactoglobulin in water. Formamide is another nonaqueous solvent only partially miscible with hydrocarbons, It is therefore apparently contradictory to the hypothesis just stated, that formamide was found to be only slightly less effective than ethanol or dioxane as a denaturant for p-lactoglobulin (Tanford and De, 1961) (Fig. 4). However, formamide has an unusually large dielectric constant, and it is possible that the low ionic strength (0.02 M ) employed in these studies was inadequate to minimize electrostatic repulsions within the highly charged p-lactoglobulin molecule. At larger ionic strengths, a more profound difference between formamide and hydrocarbon-miscible nonaqueous solvents might be observed in such experiments (Herskovits. 1962). The effectiveness of urea as a denaturant (Fig. 4) may be mainly related to its strong hydrogen bond-forming capacity; on the other hand, there is evidence available to suggest that urea also acts t o weaken hydrophobic interactions (Kauzmann, 1959; Bruning and Holtzer, 1961) and the effectiveness of urea may therefore be due to a combination of these two factors. In view of these results, it should be profitable to investigate a number of related polyhydroxy solvents other than ethylene glycol as protein solvents. For example, if lyophobic interactions are so important to the maintenance of the native conformations of proteins, the efficacy as protein denaturants of the following three solvents should be in the order propylene glycol > ethylene glycol > glycerol, since this is the order of their decreasing capacity to dissolve hydrocarbons (Table IV). The capacities of these compounds to alter the extent of intrapeptide hydrogen bonding per se, however, should be very similar to one another and to water. Anhydrous mixtures of ethylene glycol or glycerol with still more highly polyhydric alcohols, such as pentaerythritol, might have even less tendency to alter the native conformations of protein molecules. 2. Strongly Protic Solvents
There is ample evidence that in strongly protic solvents, proteins generally exist in an unfolded configuration, resembling roughly the randomcoil form of polypeptides. Thus, Ambrose and Elliott (1951) showed that insulin treated with formic acid and cast as a film exhibits a shift in the maximum of the C=O infrared stretching frequency from 1657 cm-I for
8. J. SINGER
48
the native protein to about 1637 cm-' (see Section IV,C,2). Similarly films of silk fibroin cast from dichloroacetic acid solution show a C=O peak at 1630 cm-' whereas with films cast from concentrated aqueous LiBr solutions, the peak is at 1660 cm-' (Ambrose et al., 1951). Katz (1955) found that solutions of globular proteins dissolved in anhydrous SO2 (containing 2.5 M KI) exhibit a shift of the 1550 cm-l NH deformation band of the native proteins to the vicinity of 1515 cm-'. These shifts in infrared spectra are probably all to be interpreted (see Table V) as having TABLEX Ultracentrifugal Properties of Proteins in Strongly Protic Solventsa Sedimentation constants') Protein
Nonaqueous solvent
Nonaqueous solvent
Water
Zein
Ethylenediamine 0.05 M NaNOa-ethylenediamine
1.1 1.2
2.20
B-Lactoglobulin
Ethylenediamine 0.2 M NaC1-hydrasine
1.1 1.0
3.4
Bovine serum albumin
Ethylenediamine 0.2 M NaC1-hydrazine
4.8, l . F d
4.9
1.6
Fibrin
Ethylenediamine
1.9
9.0"
Bovine ?-globulin
0.2 M NaCl-hydrasine
1.61
8.0
Rees and Singer (1956). Corrected t o the viscosity and density of water at 25°C. c As determined in 60% ethanol. d Two peaks i n sedimentation pattern. * Sedimentation constant for fibrinogen. f Protein underwent decomposition with tiine.
resulted from the disruption of intrapeptide hydrogen bonds and their replacement by hydrogen bonds of other types. Rees and Singer (1956) found that a variety of proteins in solution in ethylenediamine or hydrazine exhibited single well-defined peaks in the ultracentrifuge, but with markedly reduced corrected sedimentation coefficients (Table X), and, in 6ome cases, considerably increased specific viscosities, than in their respective aqueous solutions. These hydrodynamic properties are to be expected with unfolded macromolecules in solution. In terms of the discussion previously presented, these results may be
PROPERTIES OF PROTEINS I N NONAQUEOUS SOLVENTS
49
rationalized as follows. For strongly protic solvents, three factors may be involved in the disruption of the native structures of protcin molecules and their conversion to highly unfolded conformations: (a) The strong hydrogen bonds formed between the solvent arid the protein solute, particularly between the solvent as donor and the amide C=O groups of the protein as acceptor. This factor has already been discussed in connection with synthetic polypeptides, Section IV,D. (b) A marked decrease, compared to water, in lyophobic interactions towards the nonpolar residues of the protein. Of the common strongly protic liquids, only hydrofluoric acid, formic acid, and hydrazine are not completely miscible with simple hydrocarbons, while the others, such as ethylenediamine, dichloroacetic and trifluoroacetic acids, are completely miscible with them. Even in the cases of hydrofluoric acid, formic acid, and hydrazine, the solubility of hydrocarbons is much greater than in water. Thus, a saturated solution of benzene in formic acid at 25°C contains 0.088 mole fraction of benzene, compared to 0.00035 mole fraction of benzene in water (see Section IV,B,3 and Table IV). (c) An increase, compared to water, in intramolecular electrostatic interactions within protein molecules in the case of strongly protic solvents of high dielectric constant (> -40), such as hydrofluoric acid, formic acid, and hydrazine. Protein molecules are highly ionized as a result of the abstraction of protons from the acidic solvent or the donation of protons to the basic solvent. On the other hand, in strongly protic solvents of low dielectric constant ( < -15), such as dichloroacetic and trifluoroacetic acids and ethylenediamine, these ionic charges on protein molecules must certainly be extensively paired to solvent counterions (see Section IV,B,l), and electrostatic repulsive interactions must be of negligible importance in destabilizing the native conformation. It has often been stated that the capacity of strongly protic solvents to disrupt native protein conformations is attributable to strong solventsolute hydrogen bonding. It is clear, however, that this is an inadequate description of the situation. In the solvent formic acid, for example, any one of the three factors just mentioned might contribute enough negative free energy to the unfolded conformation of the protein molecule to cause, by itself, the disruption of the native conformation. It is meaningless, therefore, to attribute the disruption to any single one of these factors. Again, the decrease in lyophobic interactions in trifluoroacetic acid must be of comparable magnitude to their decrease in ethanol, since both solvents are completely miscible with simple hydrocarbons. If this factor makes an apparently sufficiently large free energy contribution to disrupt native protein conformations in ethanol solutions it must, by itself, be capable of doing the same in trifluoroacetic acid. On the other
50
S. J. SINGER
hand, since trifluoroacetic acid is such an effective helix-disrupting solvent for synthetic polypeptides (see Section IV,D), it is also quite likely that the formation of strong solvent-solute hydrogen bonds in this solvent by itself also makes a sufficiently large free energy contribution to disorder native protein conformations. From these considerations, it is evident that studies of protein structure in strongly protic solvents can reveal little if any additional information beyond what has been, and is to be, achieved in weakly protic solvents. Aside from the additional complications introduced by extensive acid-base reactions in such solvents, they are such powerful protein denaturants as t o be indistinguishably effective in this property. 3. Reversibilit y of Conformational Changes
It has been demonstrated in preceding sections that almost all nonaqueous solvents for proteins can induce one or more types of conformational changes in protein molecules. For several reasons it is important t o inquire whether and to what extent these changes are reversible. In the first place, it has been tacitly assumed in previous discussion that a particular nonaqueous solvent is chemically inert towards protein molecules, except for proton exchanges, and that its effects on protein structure involve changes solely in the noncovalent bonding in the protein molecule. I t has already been pointed out, however, in Section II,A, that this assumption may not be fulfilled in particular cases, and a demonstration of reversibility of solvent effects is required to justify it. I n the second place, the complete reversibility of conformational changes may be very important in connection with several practical questions, such as the usefulness of nonaqueous solvents in protein extraction and isolation procedures, and the preparation of chemically modified proteins by reactions carried out in nonaqueous solvents. The latter problem is discussed in more detail in Section V,E. Furthermore, the problem of reversibility has its own intrinsic interest in pursuing the question of whether the native conformation of a protein is thermodynamically or kinetically stable: is the native conformation in aqueous solution completely determined by its primary valence structure, or is it a metastable one “frozen in” during the process of biosynthesis (cf. Anfinsen, 196l)? The criteria used to assess reversibility must be adequately sensitive for the purposes at hand particularly if it is necessary to detect subtle irreversible changes in the native conformation. This is a subject itself worthy of extensive treatment. It is, however, not feasible to discuss it at length in the context of this article. Suffice it to say that it is desirable to examine the conformational state of the recovered protein by several methods rather than by only one; that among the more sensitive criteria
PROPERTIES OF PROTEINS IN NONAQUEOUS SOLVENTS
51
on the physical side are optical rotatory properties, ultraviolet absorption spectra, and their susceptibility to change from stresses such as temperature, pH, and solvent changes; and on the chemical side, the enzymatic and antigenic properties of the protein. The last property, although rarely employed, is particularly useful because of its generality and high degree of sensitivity to conformational changes in proteins. Consideration should also be given to the procedure employed to reverse conformational changes induced in nonaqueous solvents. It is conceivable, for example, that although the recovery of the native conformation of a protein may be favored thermodynamically, the reversal procedure may introduce kinetic barriers to its attainment, That is, a conformation other than the native one may be made metastable in the process of recovery. The reversal procedure must therefore be gradual enough to permit equilibrium to be attained at each stage. Furthermore, different proteins may differ considerably in the degree to which their conformations can be reversibly altered. It has been pointed out (Foss and Schellman, 1959), for example, that the apparent stability of ribonuclease to various treatments is not due to any inherent stability of its native conformation, but rather to its extraordinary degree of reversibility. It has generally been found in those systems investigated so far that the gross conformational changes induced in proteins by weakly protic solvents are at least largely reversible (Sage and Singer, 1958; Doty, 1959; Weber and Tanford, 1959). Inagami and Sturtevant (in Rees and Singer, 1956) showed that trypsin could be recovered from solution in formamide with retention of about 95 % of its enzymatic activity, while from dimethylsulfoxide solution, about 93 % was retained in the presence of 0.05 M CaClz (Inagami and Sturtevant, 1960). These observations were essentially confirmed and extended by Vratsanos el al. (1958). Following up these studies, Fleck and Singer (unpublished experiments) found that solutions of trypsin could be obtained in a number of weakly protic solvents by a dialysis procedure (see Section III,C), and the activities recoverable from these solvents into aqueous solution are shown in Table XI. No attempt was made to maximize these recoveries. Sage and Singer (1958, 1962) showed that ribonuclease could not only be recovered from neutral ethylene glycol into aqueous solution with essentially full retention of enzymatic activity, but that this was so even after all six of its tyrosine residues had been converted to the phenoxide ion form in ethylene glycol. This is in contrast to the situation in water solutions of this protein, in which the titration of more than three of the six tyrosines results in an essentially instantaneous irreversible loss of enzymatic activity (Sela and Anfinsen, 1957). This suggests the interesting possibility that the irreversible transition that occurs in aqueous solutions
52
S. J. SINGER
a t strongly alkaline pH is due to some scission or rearrangement of disulfide or other primary valence bonds (Ryle and Sanger, 1955; Zahn and Osterloh, 1955) within the RNase molecule which is retarded in ethylene glycol solution. This serves to emphasize the possibility that has been raised frequently in the past, that irreversible denaturation of proteins in aqueous media may often be the direct result of changes in primary covalent structure in the protein molecule, and not of irreversible changes in noncovalent binding and conformation. I n many weakly protic nonaqueous solvents, conformational changes can be induced under conditions that are milder and less likely to produce covalent bond alterations than the conditions required to effect similar conformational changes in aqueous media. With proteins dissolved in strongly protie solvents studies of reversiTABLEX I Recovery of Enzymatic Activity of Trypsin from Various Nonaqueous Solvents into Aqueous Media" Solvent: Per cent activity recovered :
methanol ethanol ethylene propylene glycologlycol glycol nitrile 72
94
82
84
72
hydrazine
0
G. Fleck and S. ,I. Singer, unpublished experiments. Enzymatic activity was measured by the rate of alkali consumption by the hydrolysis of benzoylarginine ethyl ester a t p H 8 in a phosphate-NaC1 buffer (Inagami and Sturtevant, 1960). (1
bility have been less extensive, but it has been found that the conformational changes are often apparently not reversible. Thus about half of the proteins recovered from solution in HF were insoluble in water (Katz, 1954a). Rees and Singer (1956) found that bovine serum albumin in hydrazine and ethylenediamine, and y-globulin in hydrazine, could not be recovered in soluble form into aqueous solution. Furthermore, evidence was obtained that degradation of the y-globulin molecule occurred in the hydrazine solutions over a period of hours. These observations taken together suggest that, whatever may be the detailed reasons for the observed irreversibility in strongly protic solvent,s, the possibility of covalent bond alterations must be seriously considered. Much work remains to be done in correlating the extent and the nature of conformational changes produced in proteins dissolved in chemically inert nonaqueous solvents and the precise degree to which these changes are reversible. In this connection the distinction must be made, of course, between the reversibility of conformational changes within covalently bound subunits of protein molecules, and the reversibility of the structural
53
PROPERTIES O F PROTEINS IN NONAQUEOUS SOLVENTS
integration of the subunits into the whole protein molecule. This question is taken up in Section V,A.
F. Nucleic Acids in Nonaqueous Solvents In recent years, increasing interest has been attached to the properties of nucleic acids in nonaqueous solvents. It is outside the purview of this article to review all the studies that have so far been carried out with such systems; certain observations have been made, however, which bear directly on the interpretation of the behavior of proteins in nonaqueous TABLEXI1 Properties of Salmon D N A in Water, Ethanol, and Methanol" DNA sample C Property
0.2 M NaC1HzO
Molecular weight X 5.9 10-6 Radius of gyration, A 2700 Sedimentation con21 stant, ~ 2 0 . ~ Intrinsic viscosity, dl/ 57 gm Extinction coefficient 6550 per mole P
b c
Ethanol
DNA sdmple H-I1 Redialyzedb 0.2 M NaC1H2O
0.2 M
hIeth-
Ng$-
anol
Redialyzed" 0.2 M NaClHzO
6.0-7.0
5.7
7.0
8.2
7.0
680-900 90-100
1780 -
2900 26
1560 41,40
2250
1.3-1.5
43
68
24
65
6300
9020
7070
9350 f 270
-
-
Herskovits et al. (1961). Dialyzed into 99.5% ethanol and back into aqueous salt solution. Dialyzed into 99.5y0 methanol and back into aqueous salt solution.
solvents, and these will be discussed. From the viewpoint of protein structure and behavior, nucleic acids are simpler molecules t n deal with, since their subunits are fewer in number, and at least in thc case of the deoxyribonucleic acids (DNA), their structures are comparahely regular and uniform. Of particular interest is a series of studies (Geiduschek and Gray, 1956; Herskovits et al., 1961; Geiduschek and Herskovits, 1961; Herskovits, 1962) concerning the effect of different solvents on the structural properties of DNA. In ethanol and methanol solutions, the helical conformation of DNA is disrupted, as is indicated by gross changes in ultraviolet absorption, light scattering, and hydrodynamic properties of the solutions compared to those in water (Table XII). Under the proper conditions these changes are substantially reversible. Conductivity measurements
54
S . J. SXNGER
(Coates and Jordan, 1960; Herskovits et al., 1961) suggest that the phosphate groups of the DNA molecule are extensively paired to counterions in methanol solution, such that a2/D (Section IV,B,l) is only roughly onesixth of its value in water. This applies to the disrupted conformation of the DNA molecule as it exists in methanol solutions, but it may be assumed that a 2 / D is not greatly different for the hypothetical native helical conformation in methanol. It can be concluded, therefore, that there is a net reduction in electrostatic repulsive interactions, and in electrostatic free energy, for the native conformation of DNA in methanol compared to water. Similar considerations apply in ethanol, whose dielectric constant is still smaller than that of methanol. Therefore, electrostatic factors alone would tend to stabilize the helical Conformation in these nonaqueous solvents, and in spite of this, the structure is disrupted. To what factors, then, can this destabilization be attributed? I t is widely held that the most important intramolecular interactions stabilizing the Watson-Crick twin-helical conformation of the DNA molecule are the hydrogen bonds formed between purine-pyrimidine base pairs. If this were so, however, it is difficult to explain why the helix should be disrupted in ethanol and methanol solutions and be stable in water. We have discussed in Section IV,E the observations (Doty, 1959; Tanford et al., 1960) that indicate that intramolecular hydrogen bonding in protein molecules is considerably more favored in 2-chloroethanol and ethanol as compared to water. Such solvents should similarly substantially promote internucleotide hydrogen bonding compared to water. [There is much other evidence from a variety of experiments in aqueous DNA solutions which is also difficult to reconcile with the hypothesis that the helical conformation is primarily stabilized by internal hydrogen bonding (Sturtevant et al., 1958) .] On the other hand, as has been pointed out in previous sections, lyophobic interactions towards nonpolar residues are substantially reduced in ethanol, methanol, and many other nonaqueous solvents, below their strength in water. The increase in free energy of the heIical conformation in methanol compared to water which results from this reduction in hydrophobic interactions is apparently sufficiently great to overcome the free energy decreases resulting from changes in hydrogen bonding and electrostatic interactions in methanol compared to water. It follows (Herskovits et al., 1961) that hydrophobic interactions must be vital to the maintenance of the native conformation of DNA in aqueous solution. It is important to realize that the Watson-Crick twin-helical structure of DNA not only permits the maximum number of intramolecular hydrogen bonds to form but is also a structure in which the most intimate clustering
PROPERTIES OF PROTEINS I N NONAQUEOUS SOLVENTS
55
of the aromatic residues of DNA is achieved, and which thereby permits the maximum number of solvent-solvent interactions to occur in a given DNA solution. From the conclusion that hydrophobic interactions are of great importance in stabilizing the native structure of DNA in water, it follows that a wide variety of weakly protic nonaqueous solvents should disrupt this structure, since in all of these, lyophobic interactions should be substantially decreased compared to water. This has been found to be true (Helmkamp and Ts’o, 1961; Herskovits et al., 1961; Herskovits, 1962). The addition of each of the following solvents to an aqueous solution of DNA produced a collapse of the DNA helical conformation, with the numbers in parentheses indicating the mole per cent of nonaqueous solvent at the midpoint of the denaturation process; dimethylformamide (23), dimethylsulfoxide (27), formamide (57), methanol (79), and ethylene glycol (80) (Herskovits, 1962). The smaller the mole per cent of nonaqueous solvent required, the more effective is the solvent; hence the order given is that of decreasing effectiveness as DNA denaturants. In tlhese solvent mixtures the concentration of added electrolyte was 5 X M . This decreasing order of effectiveness is similar to that found by Tanford and co-workers in studies of protein denaturation (see Section IV,E); there is a correlation between this order and the decreasing tendency of the pure nonaqueous solvents to dissolve hydrocarbons (increasing AFu ;Table IV) and with an increasing tendency to interfere with the formation of intrapeptide (and hence, presumably, also internucleotide) hydrogen bonds. Clearly this is essentially the order to be expected if hydrophobic interactions in aqueous solutions are of primary importance in stabilizing the helical conformation of DNA. Of considerable further interest is the finding of Marmur and Ts’o (1961) that the denaturation of DNA in formamide-water mixtures results in the separation of the two polynucleotide strands. An NI4Nl6hybrid Escherichia coli DNA was treated with 95 % formamide a t 37°C for 15 min at an ionic strength of 0.01, the formamide was then removed by dialysis, and the DNA was centrifuged in a CsCl density gradient. The presence of two bands of density different from that of the original hybrid indicates that strand separation occurred. It is of considerable practical interest that these conditions are much milder than those required to achieve strand separation in aqueous solution. Whether strand separation occurs also in solutions of DNA in other nonaqueous solvents is not yet determined. Ribonucleic acids (RNA) have been studied in formamide and dimethylsulfoxide (Helmkamp and Ts’o, 1961), and their secondary structures in these solvents were found also to be disrupted.
56
6. J. SINGER
G. Conclusions To summarize the major results discussed in this section: (a)The native conformations of globular protein molecules are in a rather delicately balanced equilibrium with the solvent water. Any of a wide variety of weakIy protic or strongly protic nonaqueous solvents is capable of radically altering these native conformations. Depending on the solvent, the structure of a protein can be made more highly disordered (random-coil form) or more highly ordered (more helical) than the native aqueous form. (b) Most of the weakly protic nonaqueous solvents appear to induce a two-stage transformation of the native conformation of at least certain globular proteins. Addition of such a solvent to an aqueous protein solution first causes an unfolding of the protein molecule; with further enrichment of the nonaqueous solvent component, the molecule refolds to a conformation with larger helical content than the native. A few solvents, such as forniamide and ethylene glycol, appear to induce the first transformation, but only incompletely, if at all, the second. Much higher concentrations of ethylene glycol than of most nonaqueous solvents studied so far are required to effect even the first of these transformations. (c) The effects of nonaqueous solvents on the stability of the twinhelical conformation of DNA are strikingly parallel to their effects on the native conformations of proteins. These and other observations can be quite satisfactorily explained on the hypothesis that hydrophobic interactions play a major role in stabilizing the native conformation of proteins and DNA in aqueous solution. The marked reduction in lyophobic interactions in almost any nonaqueous solvent compared to water must be critically involved in the conformational changes observed. Although adequate data are lacking, it is evident qualitatively at least that many of these solvents are poorer competitors than water for interpeptide and internucleotide hydrogen bonds. Dioxane is a clear example, since it is only a moderately strong hydrogen bond acceptor, and not a hydrogen bond donor at all. If hydrogen bonds were the only, or the most important, interaction stabilizing these native structures, the addition of dioxane should only make them more stable rather than less. This is not to say that intramolecular hydrogen bonds are unimportant in determining these structures, but rather that they do not appear to be the major source of the free energy required for the purpose. An increasing number of investigators have recently come to believe that the following generalized scheme of interactions accounts for the properties of globular protein and DNA molecules in aqueous solutions. From results such as have been discussed in previous sections, and from a considerable amount of other kinds of evidence (Kauzmann, 1959), it is con-
PROPERTIES OF PROTEINS I N NONAQUEOUS SOLVENTS
57
sidered likely that hydrophobic interactions make the largest single contribution to stabilizing the native conformations of these macromolecules in aqueous solution. On the other hand, hydrophobic interactions are too nonspecific by nature to lead of themselves to unique conformations for these molecules. Intramolecular hydrogen bonds, which by themselves are too weak in aqueous solutions to lead to conformational stability, are nevertheless much more specific, and the cooperation of thcse two interactions results in stable specific structures in aqueous solution.
CREE ENEROY
FIG.7. Schematic diagram of the contributions of various types of interactions t o the free energy of the native conformations of a hypothetical protein or a DNA molecule in aqueous solution. F a , F,: , F H B , and F H I represent tjhe free energy contributions of conformational entropy, electrostatic interactions, hydrogen bonding, and hydrophobic interactions, respectively. The magnitude of F E may vary considerably with the p H and ionic strength of the aqueous solution.
The situation that may obtain is very schematically illustrated in Fig. 7. The free energy contributions to the native conformation of a macromolecule in aqueous solution due to the loss of conformational entropy, to electrostatic repulsive interactions, to hydrogen bonding, and to hydrophobic interactions, are assumed to be separable, and are denoted respectively by F s , F , , F H B, and F H I; the sum of these four terms, F, is negative. In the absence of hydrogen bonding, the free energy of the native conforF - F,, , is, according to this scheme, still negative. In the mation F - F H I is positive and absence of hydrophobic interactions, however, therefore unfavorable. While hydrophobic interactions are thus depicted
58
8. J. SINGER
as providing the major stabilizating influence favoring the native conformation, the contribution of hydrogen bonding makes a specific conformation substantially more likely than others. In almost all nonaqueous solvents, F H I is apparently so markedly increased that even a decrease in F H B is inadequate to compensate and retain F as negative. In order to determine to what, extent these speculations have validity, it is necessary to be able to evaluate more quantitatively the relative contributions of these interactions to the free energies of protein and nucleic acid molecules in water and nonaqueous solvents. For this purpose, a substantial body of quantitative data is required concerning the properties of suitable model compounds in a variety of solvents, including their solubilities, acid-base dissociation constants, and thermodynamics of hydrogen bond formation. The dearth of pertinent data on hydrogen bonds in solvents of interest is particularly frustrating to even a semiquantitative evaluation of the scheme presented in Fig. 7. Although mixtures of water and nonaqueous liquids have been most frequently studied as protein solvents up to the present time, it should be realized that from a quantitative point of view, such solutions are enormously complicated systems. It is well known that the effective microscopic properties of such mixed solvents can be vastly different from their macroscopic properties, and can vary with the solute, because of selective attraction of one of the components of the solvent by the solute. Pure nonaqueous solvents are likely to be more useful for systematic and quantitative studies. The conclusions derived from these conformational studies have many possible important chemical and physiological consequences. From a chemical point of view, for example, the use of nonaqueous solvents as additives in the isolation and purification of proteins, or in their crystallization: may be accomplished with less damage to the proteins concerned if proper recognition is given to possible conformational changes produced in the proteins by the nonaqueous solvent. One important physiological problem which is pertinent involves the fact that the cellular environment of many proteins contains high concentrations of lipid components. In formed bodies such as mitochondria and chloroplasts, proteins are intimately and functionally associated with lipid substances. This is also true of proteins in a wide variety of cellular membranes. The current view is that these proteins are embedded in a lipid matrix; the only structural specification usually made is that the polar ends of the lipid molecules be oriented towards the protein (Danielli, 1951;Sjostrand and Rhodin, 1953). It must be realized, however, that the gross conformations of these proteins in situ might be determined by this association with a nonaqueous
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environment, at least in part, and the functional properties of these proteins might therefore be critically dependent on this environment. One possible consequence of this is that such proteins extracted into aqueous media, and freed from associated lipid, might have entirely different conformations from their native ones, and as a result be functionally altered or inactive. This may be an important factor in interpreting the experiments of Jurtshuk et al. (1961), who found that the mitochondria1 enzyme 8-hydroxybutyric apodehydrogenase, after extraction into aqueous media, has an absolute requirement for the phospholipid, lecithin, in addition to that for DPN, the specific electron acceptor.
V. PHYSICAL AND CHEMICAL PROPERTIES OF PROTEINS IN NONAQUEOUS SOLVENTS Although most recent studies of proteins in nonaqueous solvents, and the major part of this article, have been devoted to problems of protein molecular conformations, there are many other aspects of these systems which might profitably be investigated. A few of these aspects are discussed briefly in this section. A , Quaternary Structure in Proteins The term quaternary structure was introduced by Bernal (1958) to denote the kinds of organized structures obtained by the noncovalent interaction of macromolecular subunits in aqueous solution where the subunits themselves are internally covalently bound. Such structures may be composed of closely similar or identical subunits (homogeneous type), as in the cases of hemoglobin (Field and O’Brien, 1955), P-lactoglobulin (Townend et al., 1961), insulin (Harfeneist and Craig, 1952), glutamic dehydrogenase (Frieden, 1959), and many other proteins; or they may contain unlike subunits (heterogeneous type) as in the case of the protein and ribonucleic acid subunits of tobacco mosaic virus. The noncovalent bonds between the subunits can vary greatly in strength from one case to another, and may be due to any combination of a wide spectrum of forces including electrostatic, hydrogen bonding, and hydrophobic interactions. It is therefore difficult to generalize about the properties of such structures. Nonaqueous solvents may provide a useful approach to the study of quaternary structure. For structures of the homogeneous type, an important problem is the determination of the true minimal subunit size, and a suitable nonaqueous solvent or solvent mixture may induce dissociation of subunits to a much greater extent than is possible in aqueous media. A solvent such an anhydrous formic acid, for example, might be quite generally effective in this regard, assuming that a given protein can be dissolved in it, and that no significant changes in covalent bonding within
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the subunits are produced in the process. For reasons detailed in Section IV,E,2, in a strongly protic solvent of high dielectric constant such as formic acid, the native conformation of a subunit is highly likely to be converted t,o a random-coil form, thereby disrupting the native quaternary structure of thc protein in the process. The highly charged subunits should then be kept dispersed by electrostatic repulsive interactions in the high dielectric constant medium and a molecular weight determination in the strongly prot,ic solvent should reveal the average subunit size. Conformational changes within protein subunits are also produced, as we have seen earlier, in strongly protic solvents of low or high dielectric constant, and in weakly protic solvents as well. It is therefore likely that quaternary structure will also be disrupted in these solvents. However, nonspecific aggregation of the structurally altered subunits is more generally likely t o occur in such solvents than in strongly protic solvents of high dielectric constant; such aggregation would interfere with the determination of the minimal subunit size. Thus, ribonuclease was found to be extensively aggregated in essentially anhydrous ethylene glycol solution (Sage and Singer, 1962). On the other hand, in certain favorable instances, dissociation and essentially complete subunit dispersion may occur in a weakly protic solvent. For example, in dilute solutions of insulin in dimethylformamide and dimethylacetamide (Rees and Singer, 1955, 1956) the protein is largely dissociated into the true minimal subunit containing one A and one B chain. Related to this is the observation by Fredericq (1957) that insulin is essentially completely dissociated in 40 % dioxanewater. Nonaqueous solvents may also be useful in investigations of the nature of the noncovalent binding between subunits. If hydrophobic interactions, far example, were significantly involved in a particular association system in aqueous solution, the addition of weakly protic, nonaqueous solvents should produce dissociation, with the order of effectiveness of the solvents being that described in Sections IV,B,3 and IV,E,l. Since the noncovalent binding between subunits might be expected ordinarily to be weaker than the noncovalent binding maintaining the native conformation within the subunits themselves, the dissociation might be produced a t such small volume fractions of the nonaqueous solvent that significant conformational changes might be avoided. Otherwise, such conformational changes would interfere with the interpretation of the results. The appearance of conformational changes could be monitored by optical rotatory dispersion measurements. One system that would be of interest to investigate in this respect is the protein of tobacco mosaic virus, whose subunits have been shown b y Lauffer et al. (1958) t o undergo an endothermic association in aqueous solution.
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If hydrophobic interactions are involved in this association, a s has been suggested by Kauzmann (1959) on the basis of this observation, it should be sensitive to the introduction of small amounts of the appropriate weakly protic nonaqueous solvents. The endothermic association of sickle cell anemia hemoglobin (Murayama, 1956) is another case to investigate. B. Biochemical Reactions of Proteins in Nonaqueous Solvents and Solvent Mixtures It is very likely that nonaqueous solvents and mixtures can be of use in the elucidation of the mechanisms of biochemical reactions carried out by proteins, such as enzymatic catalysis and antigen-antibody interactions. With the rapidly accumulating knowledge of the effect of nonaqueous solvents on the conformation of proteins to guide us, it should be possible to carry out increasingly informative experiments in such directions. 1. Enzymatic Reactions
A number of studies have been made on the kinetics of enzyme-catalyzed hydrolysis reactions in aqueous media containing nonaqueous solvents in varying proportions. One of the most careful and in te r e s h g of these was performed by Inagami and Sturtevant (1960) on the trypsin-catalyzed hydrolysis of benzoyl-L-arginine ethyl ester (BAEE) in dioxane-water mixtures. They found, quite remarkably, that the maximum rate, T,,,, , of hydrolysis does not vary greatly with increasing dioxane concentration, and that even in 88 volume % dioxane, rmax is 68 % of that iii water itself at the same pH. On the other hand, the apparent MichtLelis-Menten constant, Km(app) , which was assumed to be the dissociation constant of the enzyme-substrate complex, increases markedly with increasing dioxane is 1300 times its value in water concentration until in 88 % dioxane, Kmtapp) at pH 8, and 5500 times at pH 8.6. These authors suggested that this apparent increase in the dissociation of the enzyme-substrate complex with increasing dioxane concentration could reasonably well be accounted for by the increase, with decreasing dielectric constant of the solvent, in thc electrostatic repulsion of the critical positively charged guanidinium group of the substrate and the net positively charged enzyme, but thcy recognized that this dissociation could also be due to conformational changes in the protein produced by the high concentration of dioxane. No conformational studies were made, however. The fact that rmaxdid not change greatly despite the increased dissociation of the enzyme substrate complex they attributed to the compensating factor that water acts as an inhibitor a t the enzymatic site, and that on increasing the dioxane concentration and decreasing the water concentration of the solvent, the water is dissociated from the site.
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The conformation of the trypsin molecule, or at least of its active site, is apparently unusually stable to a decrease in lyophobic interactions with the solvent, since several other globular proteins undergo marked structural changes at much smaller dioxane concentrations than 88% as was discussed in Section IV,E,l. The structural studies discussed in that sFtion suggest also that formamide, in which pure solvent trypsin is directly soluble (Rees and Singer, 1956), might profitably be substituted for dioxane in the experiments of Inagami and Sturtevant, since it is a less effective protein denaturant and is also inert chemically. Ethylene glycol or glycerol, on the other hand, may participate directly in transfer reactions catalyzed by the enzyme, as in the case of the chymotrypsin-catalyzed transfer of alcohols to acyl groups (Balls and Wood, 1956; MacDonald and Balls, 1956). These compounds would therefore not function as chemically inert solvent additives. This brings us to the possibility that enzymes might exhibit catalytic activity in pure nonaqueous solvents. Many enzymes classed as hydrolases, for example, can catalyze transfer reactions that do not involve water as a stoichiometric participant. Chymotrypsin has already been mentioned in this regard. RNase is another case: in aqueous solutions containing low concentrations of alcohols, it catalyzes the reaction between cyclic mononucleotides and the alcohols to form alkyl phosphate esters (Heppel and Whitfeld, 1955; Heppel et al., 1955). It would be of interest, for example, t o determine in an anhydrous solvent in which RNase undergoes little or no detectable conformational change (perhaps this might be glycerol or a glycerol-sugar mixture) whether such transfer reactions to the solvent or other suitable receptor could be detected. Other enzyme-substrate systems might also be amenable to such experiments.
2. Antigen-Antibody Reactions A considerable variety of chemical groups can function as antigenic determinants, and a number of different kinds of forces have been implicated (Pauling et al., 1943) in the formation of the relatively weak noncovalent bond between a particular antigen and its specific antibody. Along with the recognition of the importance of hydrophobic interactions in determining the conformation of proteins, the possible significance of these interactions in the reactions between one protein and another, or between a protein and a small molecule, has been discussed (Kauzmann, 1959). In connection with this possibility in antigen-antibody systems, the study of the effects of nonaqueous solvents may provide some useful information. A very suggestive observation (Grant, 1959) has been made that 20% dioxane strongly reduces the amount of precipitate formed in several pro-
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tein antigen-antibody systems. The effect is reversible, since removal of the dioxane by dialysis results in extensive specific precipitation. On the other hand Tanford et al. (1962) have found that 60 % ethylene glycol does not inhibit precipitation in Ouchterlony diffusion experiments with a different antigen-antibody system. Precipitating antigen-antibody systems are too complex to afford unambiguous interpretation of these solvent effects. Aside from conformational changes that might be induced in the antigen and antibody proteins by the solvent, the solubility of antigen-antibody aggregates might be altered; these factors could obscure the effect of solvent on the formation of the antigen-antibody bond per se. Systematic studies of the reactions of simple haptens and their purified specific antibodies, by equilibrium dialysis, or preferably some more rapid method of measurement, should be informative about the importance of hydrophobic interactions in any particular antigen-antibody reaction. Such studies must of course include measurements on the effect of solvents on the conformation of the antibody protein molecules. The reversible dissociation of antigen-antibody bonds by nonaqueous solvents such as dioxane may prove of considerable practical use in procedures for the isolation and purification of specific antibodies (cf. Singer et al., 1960).
C . Protein Solutions at Low Temperatures The solubility of proteins in nonaqueous solvents makes it possible to study them in homogeneous solution at temperatures below the freezing point of aqueous solutions, since many pure nonaqueous solvents have freezing points well below 0°C (Table I). Freezing temperatures can be lowered even further by the use of solvent mixtures. This opens a new dimension in protein chemistry the significance of which can only dimly be appreciated at the present time. Several possible problems of interest may be mentioned, however. The equilibrium state of any process involving an enthalpy change must be affected by temperature. The conformational transitions in proteins discussed in Section IV,E are a case in point. The transition from the native to the denatured form in a nonaqueous solvent is usually an endothermic process, and a decrease in temperature will favor the native form. In such cases, it is possible that the disruption of the native conformation in a given protein-solvent system, which observed at room temperatures, may be reversed at sufficiently low temperatures. [On the other hand, particularly in mixed solvents, the transition from an ordered to a disordered state may be an exothemic process (Doty and Yang, 1956; Foss and Schellman, 1959), and the reverse effect of temperature may be ob-
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served in these cases.] One may, therefore, be able to obtain solutions of protcins in their native conformations in nonaqueous solvents morc readily a t temperatures well below 0°C than a t 25°C. Indeed this consideration was fundamental to the development of the techniques for fractionation of proteins in ethanol-water mixtures a t low temperatures by Cohn el al. (1946). It would be of particular interest to study the rates of enzymatic and antigen-antibody reactions at low temperatures, where they might be appreciably slower and more readily measured than in aqueous solutions at room temperature. These and other conformational considerations discussed in this article are of obvious practical importance in the preservation of biological structures a t low temperatures (Smith, 1954). It is also possible that at low temperatures, optical and electronic processes in protein systems may be more readily investigated (Freed, 1958; Freed et al., 1958).
D. Chemical Modijication of Proteins in Nonaqueous Solvents The chemical modification of proteins in nonaqueous solvents is already of considerable importance, but its potential significance is even much greater. Among those reactions which so far have been carried out systematically with proteins in nonaqueous solvents are : esterification of carboxyl groups of proteins in anhydrous alcohols containing either HC1 (Praenkel-Conrat and Olcott, 1945) or thionyl chloride (Bello, 1956); and acetylation of amino and hydroxyl groups with acetic anhydride (Vratsanos el al., 1958: BelIo and Vinograd, 1956). It is clear that many modification reactions may be found to proceed readily in nonaqueous solvents which occur in poor yield or not at all in aqueous media, either because of low solubility of the required reagents, or interference by the water. In the context of this article, we wish only to emphasize that the conformation of a protein molecule is dependent on the solvent. The rates of modification reactions at particular groups on a protein molecule will therefore differ in a particular nonaqueous solvent compared to aqueous media not only through a generalized solvent effect, but also indirectly through the effect of solvent on the relative accessibility of the protein groups tjo the reagent. Furthermore, if groups which are normally buried in the interior of the native conformation of a protein molecule in aqueous solution, become available and are modified in a nonaqueous solvent, the conformation may be irreversibly altered. That is, on return of the modified protein to an aqueous medium, the native conformation may not be recovered because of interference from the modifying groups.
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ACKNOWLEDGMENTS The author’s experience with the subject of this article was obtained with support from grant A-2441 from the National Institutes of Health, U. S. Public Health Service. The author is indebted to Drs. Charles Tanford, E. Peter Geiduschek, and T. T. Herskovits for the opportunity to see their manuscripts prior to publication, and to these individuals and Dr. Bruno Zimm for many helpful discussions. He is also very grateful to several colleagues, who, observing signs of distress at various occasions during the writing of this article, quickly supplied generous quantities of a mixture of water and the appropriate nonaqueous solvent.
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Shugar, D. (1952). Biochem. J . 62, 142. Simmons, N . S., Cohen, C., Szent-Gyorgyi, A. G., Wetlaufer, D. B., and Blout, E. R. (1961). J . Am. Chem. SOC.83, 4766. Singer, S. J . , Fothergill, J. E., and Shainoff, J. R. (1960). J . Am. Chem. SOC.82,5G5. Sjostrand, F. S., and Rhodin, J. (1953). Exptl. Cell Research 4, 426. Smith, A. U. (1954). In “Biological Applications of Freezing and Drying” (R. J . C. Harris, ed.), pp. 1-62. Academic Press, New York. Stark, G. R., Stein, W. H., and Moore, S. (1961). J . Biol. Chem. 236, 436. Steinberg, I. Z., Harrington, W. F., Berger, A . , Sela, M., and Katchalski, E. (1960). J . Am. Chem. SOC.82, 5263. Strehm, J., Krishna-Prasad, Y. S. R . , and Schellman, J. A. (1961). Tetrahedron 13, 176. Sturtevant, J. M., Rice, S. A., and Geiduschek, E. P. (1958). Discussions Faraday SOC.26, 138. Swallen, L. C . , and Danehy, J. P. (1946). In “Colloid Chemistry” (J. Alexander, ed.), Vol. VI, p. 1140. Reinhold, New York. Tanford, C. (1961). “Physical Chemistry of Macromolecules,” Chapter 8. Wiley, New York. Tanford, C., and De, P. K. (1961). J . Biol. Chem. 236, 1711. Tanford, C., and Roberts, G. L. (1952). J . Am. Chem. SOC.74. 2509. Tanford, C., and Swanson, S. A. (1957). J . A m . Chem. SOC.79, 3297. Tanford, C., and Wagner, M. L. (1954). J . A m . Chem. SOC.76, 3331. Tanford, C., Hauenstein, J. D., and Rands, D. G . (1955a). J . A m . Chetn. SOC.77, 6409. Tanford, C., Swanson, S. A . , and Shore, W. S. (1955b). J . Am. Chem. SOC.77,6414. Tanford, C., De, P. K., and Taggart, V. G. (1960). J . A m . Chem. SOC.82, 6028. Tanford, C., Buckley, C. E., 111, De, P. K., and Lively, E. P. (1962). J . Biol. Chem. 237, 1168. Tiselius, A. (1959). In “Electrophoresis” (M. Bier, ed.), p. xix. Academic Press, New York. Townend, R., Kiddy, C. A., and Timasheff, S. N. (1961). J . AWLChem. SOC.89, 1419. Urnes, P. J., and Doty, P. (1961). Advances i n Protein Chem. 16, 401. Urnes, P. J . , Imshori, K., and Doty, P. (1961). Proc. Natl. Acad. Sci. U . S . 47,1635. Vratsanos, R. M., Bier,M., and Nord, F. F. (1958). Arch. Biochem. Riophys. 77,216. Weber, R . E., and Tanford, C. (1959). J . A m . Chem. SOC.81, 3255. Weissberger, A , , Proskauer, E. S., Riddick, J . A., and Toops, E. E., J r . (1055). “Organic Solvents,” 2nd ed. Interscience, New York. Wetlaufer, D. B., Edsall, J. T., and Hollingworth, B. R. (1958). J . Biol. Cheni. 233, 1421. Yanari, S., and Bovey, F. A. (1960). J. Biol. Chenz. 236, 2818. Yang, J. T., and Doty, P. (1957). J . A m . Chem. SOC.79, 761. Zahn, H., and Osterloh, F. (1955). Makromol. Chem. 16, 183. Zimm, B. H . , Doty, P., and Iso, K. (1960). Proc. Natl. Acad. Sci. U.S. 46, 1601.
THE INTERPRETATION OF HYDROGEN ION TITRATION CURVES OF PROTEINS BY CHARLES TANFORD Department of Biochemistry. Duke University. Durham. North Carolina
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . ............................ I1. Dissociation Constants of Appropriate 1 Molecules . . . . . . . . . . . . . . . . . I11. Experimental Titration D a t a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Electrometria Titration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Reference Points . . . . . . . . . . ............................ C . Spectrophotometric Titration for Phenolic Groups . . . . . . . . . . . . . . . . . . D . Other Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Effect of Solvent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV . Counting of Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................... A . Counting Procedure . . . . . . . . . . . . . . . . . . . . . . . . .................... B . Difference Counting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Rcversibility; Thermodynamic and Kinetic Analysis . . . . . . . . . . . . . . . . . . . .
A . Reversibility and Time-Dependence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................... ; . . . . . . . . . . . . . . . . C . Kinetic Analys ....................................... c Analysis . . . . . . . . . . . . . . . . . . . . . 7 . . . . . . VI . Semiempirical The A . The Equation of LinderstrZm-Lang . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B Empirical Procedure., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . The Electrostatic Interaction Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Intrinsic pK’s and Their Interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Heats and Entropies of Dissociation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V I I More Exact Thermodynamic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII . Volume Changes Accompanying Titration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I X . Miscellaneous Topics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Binding of Ions Other Than Hydrogen Ions . . . . . . . . . . . . . . . . . . . . . . . . B . The Isoionic Point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Charge Fluctuations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X . Results for Individual Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Chymotrypsinogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Chymotrypsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Collagen Fibrils. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D . Conalbumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . a-Corticotropin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . Cytochrome c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . Fetuin . . . . . . . . . . . . . . . . . . . . .................................. H . Fibrinogen and Fibrin., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . Gelatin . . . . . . . . . . . . . . . . . . . ................................... J Hemoglobin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
.
.
69
70 71 76 76 78 80 80 82 82 82 85 90 90
95 99 111 119 121 124 127 127 128 130 131 131 133 133 133
138 138 139 139
70
CHARLES TANFORD
K. Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L. @-Lactoglobulin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. Lysozyme.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N. Myoglobin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0. Myosin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P. Ovalbumin.. . . . . . . . . . . . . . . . . ................... Q . Papain.. . . . . . . . . . . . . . . . . . . . . .................................. It. Paramyosin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. Pepsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T. Peroxidase.. . . . . . ............................................. 6.Ribonuclease., , . . ............................................. V. Serum Albumin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . W. Thyroglobulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Trypsinogen, . . . . . . . . . . . . . . . ................................. Y. Trypsin., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
142 144 147 149
153 153 154 154 154 156 160 160 161 181
I. INTRODUCTION In recent years several reviews have been published on the subject of hydrogen ion titration curves of proteins. Among these there are good general introductions to the subject, which include some description of experimental procedures (Tanford, 1955a; Kenchington, 1960); a review by Linderstrgm-Lang and Nielsen (1959), which gives a lucid introduction to the theoretical treatment of protein titration curves; and a review by Steinhardt and Zaiser (1955), which emphasizes anomalous behavior. A review by Jacobsen et al. (1957) is devoted to use of the pH-stat. Apart from treating that subject in some detail, it contains experimental procedures of general utility in the determination of titration data. More complete developments of the subject may be found in textbooks (Edsall and Wyman, 1958; Tanford, 1961a). The definitive treatment of the mathematical theory, for polyelectrolytes in general, and specifically including proteins, is that of Rice and Nagasawa (1961). The existence of these earlier reviews makes it possible for the present treatment to be limited in scope. It will be sufficient to touch only superficially on experimental techniques and on the theoretical derivation of equations The major objective will be, as the title of the paper implies, to show what one can learn from titration curves that is of general interest to protein chemistry. From this point of view, titration curves do not represent just another way of physically characterizing a protein molecule. More than most other physicochemical methods which are in common use, titration studies tend to emphasize individual differences among proteins, and this is reflected in the organization of this paper. There is a large section, entitled “Results for Individual Proteins,” which contains the many features of
HYDROGEN ION TITRATION CURVES OF PROTEIK
71
titration curves which are unique to individual proteins, and which cannot be described in general terms applicable to all proteins.
11. DISSOCIATION CONSTANTS OF APPROPRIATE SMALL MOLECULES In most common proteins, one out of every three or four amino acid residues contains a titratable acidic or basic group. The number of titratable groups per molecule thus ranges from about 20 to about 250 for the many common proteins with molecular weights below 100,000, and it is even larger for proteins with very high molecular weight. The titratable groups which occur in greatest abundance are the carboxyl groups of glutamic acid and aspartic acid side chains; the amino groups of lysine side chains; the guanidyl groups of arginine side chains; the imidazole groups of histidine side chains; and the phenolic groups of tyrosine side chains. Less frequent are the thiol groups of cysteine side chains and the phosphoric acid groups of phosphoserine or phosphothreonine side chains. Heme proteins contain titratable carboxyl groups attached to the heme, and may also have acidic water molecules attached to the heme iron atom. Acidic water molecules may also be attached to other metalloproteins. Glycoproteins, flavoproteins, and nucleoproteins contain additional titratable groups as part of their non-protein conjugates. Finally, the terminal amino and carboxyl groups of nearly all polypeptide chains are in the free titratable form. The structures of the more common titratable groups are shown in Fig. 1. (Other parts of the protein molecule, such as the peptide group, also possess acidic or basic properties, but they are not titrated within the range of pH 1.5 to 12, within which titration studies are usually confined. Protein molecules tend to become degraded outside this range of pH.) We want to know, before we examine the titration curves of proteins, a t what pH these groups might be expected to become converted from their acidic to their basic form. The simplest initial assumption is that proteins will not behave differently in this respect than do other organic molecules. With this assumption we would expect all effects on the acidic properties, except the effects of electrostatic charge, to be short-range effects. As a first approximation, the pK of the carboxyl group of glutamic acid (apart from the effect of electrostatic charge) could be equated with the pK of acetic acid (4.76) or propionic acid (4.87). To obtain a better estimate we can correct for the fact that the glutamic acid side chain in proteins is in fact under the influence of the polar NH and GO group attached to the third carbon atom from the COOH group. The expected pK might then be set equal to the pK of a compound containing polar groups similarly located, such as monoalkyl glutarate (pK = 4.55). Within an uncertainty of about 0.1 or 0.2 the same expected pK is usually obtained in this way,
72
CHARLES TANFORD
regardless of which of several alternative model compounds is chosen. Within an uncertainty of this order the effect of ionic strength within reasonable limits is also negligible, so that, in seeking data of this kind for appropriate model compounds, we need not confine ourselves to experimental data which permit evaluation of the effect of ionic strength.
CH-R
a-Amino group
I
$H
Lo I
CH-CH, I
co
OH
Aspartic acid
I
NH CH-CH,-CH,-CH,-CH,-NH: I
I
Lysine
b I
AH I CH-CH,-CH,-COOH
Glutamic acid
I
NH
Q
I
I
CH--CH~-CH2-CH,--NH-C I
co I Histidine
i
NH I CH CH,- SH
-
ko 1
’Qrosine
1
I
y CH-CH,--COOH A0
a
. .
Cysteine
Arginine
\
NH,
I
NH 0 I II CH-CH,-0-P-OH I A0 OH I
Phosphoserine
i
NH I CH-R’ AOOH
-Carbowl group
(1
FIG.1. Fortnullts for the most irnportant titratable groups of protein molecules. The model compounds listed in Table I were chosen to reseinble thexc groups it8 closely as possible.
The relation between structure and acidity of organic compounds has been the subject of much study. Those aspects which are of interest in connection with protein titration curves have been reviewed in definitive manner by Edsall and Wyman (1958) and by Edsall (1943), and the reader is referred to these reviews for a discussion of the theoretical and empirical principles which are involved. For the present purpose it is sufficient to extract the data which will lead to the “expected” pK values of the titratable groups of proteins, and this has been done in Table I.
HYDROGEN ION TITRATION CURVES OF PROTEIN
73
TABLEI Dissociation Conslants of Model Compounds i n Aqueous Solution at or Near 26'Cn Compound
pK observed
Correction required pK corrected
Compounds resembling a-COOH group CHa-CO-CHe-COOH 3.58 3.6 CHa-CO-NH-CHz-COOH 3.60 3.6 Tetraalanine 3.32* Electrostatic" 3.5 Cysteine Microscopic constantsd 3.8 Glutamic acid Microscopic constantsd 4.3 Tyrosine Microscopic constantsd 4.3 Compounds resembling 8-COOH groiip of aspartic acid side chain CHs-CO- (CHe)e-COOH 4.59 4.6 ROOC-(CHz)z-COOH 4.52 4.5 4.5 HOOC- (CHz)2-COOH 4.24 Statistical factore Compounds resembling Y-COOH group of glutamic acid side chain ROOC- (CHZ)a-COOH 4.55' 4.6 4.7 HOOC- (CHZ)3-COOH 4.36 Statistical factors 4.6 Glutamic acid Microscopic constantsd Compound resembling porphyrin COOH groups C sH 6- (CHI)2-COOH 4.66 4.7 Compound resembling imidaaole group of histidine side chain Poly-histidine 6.150 6.15h Compounds resembling wNH: group HeN-CO-CHZ-NH$ 7.93 7.9 ROOC-CHZ-NH: 7.7 7.7 Leucine ester 7.6 7.63 7.8 Glycylglycine ester 7.75 7.8 Tetraalanine 7.966 ElectrostaticC 7.0 Die thy1 glut amate 7.04 Cysteine 6.8 Microscopic constantsd Tyrosine 7.2 Microscopic constantsd Compounds resembling E-NH: group of lysine side chain 10.4 10.4' ROOC-(CHz) s-NH: 10.4 Lysine 10.79 Electrostaticc Compounds resembling SH group of cysteine side chain 9.5 HO- (CHI)z-SH 9.5 9.1 Cysteine Microscopic constantsd Compounds resembling phenolic group of tyrosine side chain 9.5 9.5j Polyt yrosine 9.7 Tyrosine 10.05 Electrostatic 9.8 Tyrosine Microscopic constantsd Compound resembling guanidyl group of arginine side chain Arginine ca. 12.5 Electrostatic" ca. 12.0 Compounds resembling protein-linked phosphate 1.3 Glycerol-2-phosphate (pKI) 1.34 6.6 Glycerol-2-phosphate (pK2) 6.65 6.5 6.50 Glucose-1-phosphate (pKz) 6.02 Phosphoserine peptides (pKa)
-
-
74
CHARLES TANFORD
TABLE I-Continued Most of the data are taken from Edsall (1943) or Edsall and Wyman (1958). b Aversge value for four isomers. c A charged group is present on the model compound a t considerable distance from the acidic group. An estimate for its effect on pK has been made by examining effects of similar charged groups on other acids. d By measurement of the dissociation constants of the amino acids, and of suitable esters and other derivatives. The data have been analyzed by Edsall and co-workers (Edsall and Wyman, 1958; Martin et al., 1958) so as to yield, after suitable assumptions, the twelve microscopic dissociation constants. The ones applicable here are those which refer to dissociation of the group in question from an otherwise uncharged molecule. A molecule with two identical dissociable groups will have a pIi which is twice the value for either group alone. f Interpolated between ROOC-(CH&-COOH and ROOC-(CH&--COOH. c From titration of a polymer with degree of polymerization 15 (Patchornik el al. 1957). h The state of knowledge regarding the titration of imidazole groups is unsatisfactory. The p K of the imidazole group of histidine is 6.0, which is in qualitative agreement with the value listed in the table, since the effects of the two charged groups of histidine should roughly cancel. On the other hand, imidazole itself has a pK = 7.0 and 4-metfhylimidazolehas a pK = 7.5. It is not easy to see why a polar group substitution on the ?-carbon atom (relative t o the nearest nitrogen atom) should produce so large a difference in pK. It should be noted the,t a similar problem is 9.7, whereas that of exists for amino groups. The pK of ROOC-(CH?)a-NH: CHs-(CH&-NH: is 10.7. (Added in proof: Koltun et aE. (1959) obtained pK = 6.42 for carbobenzoxy-Lprolyl-L-histidylglycinamide.) i By extrapolation from data for ROOC-(CH&-NH: with n = 1 to 4. i Essentially t h e observed average pK, for a polymer of average degree of polymerization 30, extrapolated to zero net charge of the polymer (Katchalski and Sela, 1953). k Electrostatic correction computed by the Kirkwood-Westheimer theory. 2 Folsch and Osterberg (1959) determined the pK values of several peptides containing phosphoserine and obtained results in the range of 5.4 t o 6.0. Each peptide carried one positive and one negative charge, and no attempt t o correct for their presence was made.
.
The dissociation of a hydrogen ion from an acidic group changes the charge by one unit. Either the acidic form is charged (as in -NHt) and in that case the basic form is uncharged (-NH2); or the acidic form is uncharged (as in -COOH), leading to a charged basic form (-COO-). One of the groups considered in Fig. 1 and in Table I is slightly more complicated, this being the phosphate group which may lose two hydrogen ions to acquire a double negative charge. The model compounds which are considered in Table I contain only those charges which are an inherent part of the dissociating group. No other charges are present (or, if present, have been corrected for). The pK values listed are therefore those which
HYDROGEN ION TITRATION CURVES O F PROTEIN
75
would be expected if each of the titratable groups were attached alone, without other charged groups, to a protein molecule. An actual protein molecule will contain, as we have pointed out, a large number of acidic groups per molecule. At any pH many of these groups will be in their charged form. Since electrostatic effects are long-range effects, we must expect the course of titration of any one group to be influenced by these other charges which are present. To calculate this effect of electrostatic interactions on protein titration curves has been a major preoccupation of those who have been concerned with the theoretical aspects of protein titration curves. We shall discuss these aspects in Sections VI and VII of this review. For the practical aspects of protein titration which are considered in Sections IV and V, the following qualitative conclusions of the theoretical treatment suffice. (1) It is clear from the pK values of the various dissociating groups that the charges on the protein molecule will be mostly positive at low pH and mostly negative at high pH. At some intermediate pH, positive and negative charges will be present in equal numbers. Since positive charges repel protons, and negative charges attract them, we expect pK’s to be reduced below the values of Table I at relatively low pH, and we expect them to be raised above those values at relatively high pH. The expected magnitude of the effect decreases with increasing ionic strength. For proteins with molecular weight below 100,000, at an ionic strength of 0.1 or above, the normal change in pK due to electrostatic interactions will not exceed a value of 1.5 or so, except as one approaches the extreme ends of the titration curve. Furthermore, electrostatic forces affect all groups alike, so that the pK differences between one type of group and another, at any pH, will be expected to remain the same as the differences given by Table I. (2) When a monobasic acid is titrated, the titration curve is described completely by the pK. We know for instance that, when pH = pK 1.0, 91 % of the molecules will be in the acid form, and, when pH = pK 1.0, 91 % will be in the basic form. These same relations would apply to a particular titratable group on a polybasic acid or on a protein molecule, if all other titratable groups on the same molecule were to remain unaffected. In fact, as the pH is changed, many groups on the same molecule are titrated together. Thus when a particular group on a protein molecule is in its acidic form on most of t,he molecules (pH well below pK), other groups like it will also be in their acidic form, and the net molecular electrostatic charge will be more positive than at a higher pH where this particular group is in its basic form on most of the molecules, and other groups like it are also in their basic form. The effect of electrostatic interaction will thus lead this particular group to have a lower pK at luw degrce of dissociation than at high degree of dissociation. The difference in pH required to
-
+
76
CHARLES TANFORD
go from 9 % dissociation to 91 % dissociation will not be 2.0, as it would be if the pK were independent of pH, but will be larger: the difference in p H could be as large as 3.0 or even more. Conclusion. If protein molecules exhibit no interactions that are not also present on smaller molecules, then the pK values of their titratable groups would be expected to be roughly those of Table I. Electrostatic forces may move them up or down by as much as 1.5 pK units, but relative values will be unaffected thereby. The pK changes during the course of titration, so that the titration curve for any one group will be broader than it would be for a monobasic acid. The assumption that protein molecules do not have unique interactions absent in smaller molecules is of course naive. It is in fact untrue. Special interactions occur and upset the “expectationsJJwith which this section has been concerned. It is the occurrence of such deviations from the expected result which lend interest and importance to the study of protein titration curves.
111. EXPERIMENTAL TITRATION DATA
A . Electrometric Titration The foundation for any study of hydrogen ion dissociation in proteins is the electrometric titration curve. To obtain such a curve, one begins with a protein solution of known concentration, a t an arbitrary reference pH, adds to it varying amounts of a strong acid or a strong base, and then measures the new pH attained. I n a separate experiment, or by means of calculations based on similar experiments, one determines how much acid or base is needed to take a solution which does not contain protein, but other wise has the same initial pH, ionic strength, volume, etc., to the same final pH, ionic strength, volume, etc. The amount of acid or base required for the protein solution is always larger (under most circumstances very much larger) than the amount required for the corresponding solution without protein. The difference between the two amounts is the amount of acid or base which is bound to the protein in going from the reference pH to the final pH: a plot of this quantity versus the final pH is the desired titration curve. In plotting this curve, OH- ions bound are counted as H+ ions dissociated, a procedure which is always permissible in aqueous solutions. A sample plot is shown in Pig. 2. The procedure described in the preceding paragraph will of course measure the number of hydrogen ions bound to or dissociated from all substances which are present in the solution under study. The accuracy of an experimental electrome tric titration curve depends to a considerable degree on the absence of buffers, carbon dioxide, and any other substance, other than the protein of interest, which is capable of acting as an acid or base.
HYDROGEN ION TITRATION CURVES OF PROTEIN
77
Titration studies are nearly always carried out so as to maintain the same ionic strength and protein concentration throughout t,he curve, but this is not essential for all applications. Titration curves are always dependent on ionic strength, and a curve which is not obtained at constant ionic strength can be duplicated only if the ionic strength changes are duplicated. Titration curves are often independent of protein concentration, but will depend on the concentration whenever the possibility of association between protein molecules exists.
-
i scale ZHscole PH FIG.2. Titration curve of 8-lactoglobulin a t ionic strength 0.15 and 25°C. T h e alkaline branch is time-dependent (cf. Fig. 12), and the figure shows d a t a extrapolated t o the time of mixing ( t = 0) and t o infinite time. The figure also shows how t8hecurve is divided into acid, neutral, and alkaline regions. Three ordinate scales with different reference points are given. (Data of Y. Nozaki.)
Titration curves may sometimes depend on the time which has elapsed, between addition of acid or base and the measurement of pH. (This is true, for instance, of the alkaline part of the curve shown in Fig. 2.) By the same token, the titration curve obtained by addition of successive increments of acid or base to the same protein solution will sometimes differ from the curve obtained by addition of successively larger increments of acid or base, each to a fresh aliquot of the initial protein solution. Some titration curves or parts of titration cruves are independent of the initial pH, i.e., the number of H+ ions which are bound in going from, say, a reference point a t pH 5 to pH 4, is the same as the number bound in
78
CHARLES TANFORD
going from pH 5 to pH 4 after starting at a reference point at pH 7. Likewise, the number of protons dissociated in going from pH 4 to pH 5 may be the same as the number bound in going from pH 5 to pH 4. However, in many instances the situation is not so simple. The titration curve shown in Fig. 2, for example, would be obtained from any initial pH between pH 2 and pH 9.75. A different curve is obtained after exposure to any pH greater than 9.75. (See Section V for further discussion of reversibility.)
B . Reference Points We have described titration curves as records of the number of hydrogen ions attached to a protein molecule at m y pH, relative to the number attached at an arbitrary reference pH. It is advantageous however to choose as reference point a position on the titration curve which has physical significance. There are three such positions: (1) Point of zero net proton charge. If an aqueous protein solution is passed through a mixed-bed ion-exchange resin column (Dintzis, 1952), the solution is freed entirely of all small ions except Hf and OH-. The emerging solution is called isoionic.’ The protein molecules in it usually have a very low average charge, often negligibly different from zero. When a neutral salt is added to such a solution to adjust the ionic strength to whatever value is desired, the net molecular charge may alter because the ions of the salt may be bound. However, only minute numbers of protons are bound or dissociated: the net proton charge, which is defined as the average molecular charge due to bound hydrogen ions, or to the presence of groups from which hydrogen ions have been dissociated, usually remains close to zero. We shall discuss this topic in more detail in Section IX, B. What is important here is that the net proton charge of such a solution can always be calculated exactly from the pH of the solution, as Section IX, B will show. Since the titration curve itself tells us how big a change in pH is needed to bind or dissociate any given number of hydrogen ions, it becomes a simple matter to calculate the pH at which the net proton charge is truly zero. Like all aspects of the titration curve, this pH will usually be dependent on the ionic strength. We have pointed out that the net proton charge of a n isoionic solution 1 The distinction between the isoelectric and isoionic states of a protein was first made in a classic paper by SZrensen et al. (1926). Three definitions of the isoionic point were proposed, one of these being the stoichiometrically defined point which we have called the point of zero net proton charge. The other two were operational definitions (summarized by Linderstr6m-Lang and Nielsen, 1959). The term “isoionic point,” as used here, corresponds t o one of these two operational definitions, chosen because i t always permits calculation of the point of zero net proton charge, which is the only parameter of real interest in the analysis of titration curves. The same choice has been made by Scatchard and Black (1949).
HYDROGEN ION TITRATION CURVES OF PROTEIN
79
is nearly always close to zero. It can be shown from the equations given in Section IX, B that it is in fact negligibly different from zero if (a) the isoionic point falls between pH 5 and pH 9, and ( b ) the protein concentration is 1 gm/100 ml or larger. Under these conditions the isoionic point and the point of zero net proton charge are indistinguishable. (These conditions apply to the p-lactoglobulin curve of Fig. 2, for example.) For this reason the terms “isoionic point” and “point of zero net proton charge” are often used interchangeably. It is important to be aware of the difference when proteins such as lysozyme (isoionic point at pH 11) are being studied, and in general whenever protein solutions of low concentration are being used. It is to be noted that the point of zero net proton charge can be determined only for proteins which can be deionized without precipitation or other change. The point would also have little significance (and probably could not in any event be determined unequivocally) if the pH lies in a region where the titration curve is not behaving reversibly. (2) Point of maximum proton charge. At relatively high ionic strength (0.1 or above) a definite acid end point of the titration curve can nearly always be established, an example being provided by Fig. 2. The point represents a plateau of the titration curve, a range of pH within which the number of hydrogen ions attached to the protein molecule remains unchanged. The number of attached hydrogen ions is effectively a “maximum” number: more could be bound only at much greater acidity, where the integrity of the protein molecule would probably be destroyed. In terms of the “expected” pK values of Table I, the acid end point is the point where all titratable groups listed there, except the phosphate group, are expected to have been converted to their acidic forms. (Considering that most proteins have a large positive charge at the acid end point, the expected pK1 for the phosphate group would be zero or less.) (3) Point of minimum proton charge. The shape of the titration cruve at high pH suggests that a similar end point is being approached there, and we have indicated its probable location by the dashed line of Fig. 2. Unfortunately, the experimental curve usually can not be extended to the vicinity of this end point, because irreversible degradation of the protein molecule sets in when a pH of 12 is approached. Thus the alkaline end point, representing a position of minimum proton charge, can not be defined as precisely by experimental measurement as the acid end point. In terms of the pK values of Table I, the alkaline end point of the titration curve is the point where all titratable groups listed in the table, except guanidyl groups, are expected to have been converted to their basic forms. To change the reference point of a titration curve from an arbitrary reference pH to one of the reference points just defined simply involves a
80
CHARLES TANFORD
change in the zero point of the ordinate scale of the titration curve: the difference between the scale values a t any two pH’s must remain the same regardless of the reference point used. For illustration, Fig. 2 shows three ordinate scales based on three different reference points.
C. Spectrophotometric Titration for Phenolic Groups Electrometric titration is simply a measure of the total number of hydrogeri ions bound to or dissociated from a protein molecule, with no discrimination between the various kinds of acidic or basic groups with which these hydrogen ions may be associated. Thus there is a need for alternative methods which focus specifically on hydrogen ions associated with particlar groups of the protein molecule. One such method which has been used extensively is based on the change in the ultraviolet absorption spectrum which occurs when a tyrosine phenolic group dissociates to a phenolate ion. As was first suggested by Crammer and Neuberger (1943)) this spectral change can be used to follow the progress of the dissociation of hydrogen ions from tyrosine phenolic groups of proteins, by measurement of absorbance a t an appropriate wavelength, usually in the range 290 to 300 mp. The ultraviolet spectroscopy of proteins is reviewed elsewhere in this volume (Wetlaufer, 1962), so that no detailed discussion of this method is required. It should be noted however that indole groups of tryptophan side chains absorb in the same region of the spectrum as phenolic groups, and that there are spectral shifts, both for indole and phenolic groups, which are not related to hydrogen ion dissociation. Furthermore, a general increase in absorbance in the ultraviolet takes place whenever aggregation of protein molecules increases the amount of light which is lost by Rayleigh scattering. These effects, undesirable from the present point of view, can to some extent be separated from the changes ascribable to titration of phenolic groups by measurement of absorbance over a wider wavelength range, and by auxiliary studies of molecular weight and conformation. In general the spectrophotometric titration of phenolic groups will yield more accurate results for proteins which have a low tryptophan content and which undergo no conformational change in the pH region in which phenolic groups are titrated.
D. Other Methods Spectroscopic methods for following the titration of other common titratable groups of protein molecules do not exist. The reason is that the peptide group and the aromatic rings of phenylalanine, tryptophan, and tyrosine side chains absorb strongly in the ultraviolet below 250 mp, making it essentially impossible to observe the relatively small changes in absorb-
HYDROGEN ION TITRATION CURVES OF PROTEIN
81
ance which imidazole, sulfhydryl, and other groups undergo in the ultraviolet when their state of ionization is altered. The use of infrared spectra is prevented by similar reasons: not only the various parts of the protein molecule but also the solvent (at least if it is HzO) have overlapping absorption bands, which reduce titration-induced changes to a small fraction of the total absorbance. It has been suggested (Ehrlich and Sutherland, 1954; Susi et al., 1959) that infrared absorption can be used to follow the titration of carboxyl groups of DzO is used as solvent, but no critical examination of this possibility has been made. Spectroscopic methods can be used to follow the dissociation of hydrogen ions from certain special groups present on prosthetic groups of proteins. An example is the Fe(H20)+group of proteins containing ferriheme. The dissociation of this group to Fe(0H) is accompanied by a well-defined change in the visible spectrum, which has been used to follow the reaction by Austin and Drabkin (1935), and subsequently b y other workers. The reaction is also accompanied by a change in the magnetic susceptibility of the iron atom, and this effect, too, can be used as a measure of the extent of titration (Coryell et al., 1937). A method of an entirely different kind has been proposed for following the dissociation of hydrogen ions from the imidazole groups of histidine side chains (Koltun et al., 1958). It is based on the catalysis of the hydrolysis of p-nitrophenylacetate by uncharged (basic) imidazole groups. The rate constant for this reaction is independent of pH. Moreover, the presence of the p-nitrophenylacetate appears not to affect the equilibrium between the acidic and basic forms of the imidazole group. The observed rate of the catalyzed reaction is thus a direct measure of thc fraction of imidazole groups in the basic form a t any pH. There is so far only a single recorded application of this method to proteins (myoglobin, studied by Breslow and Gurd, 1962). A disadvantage of the method is that uncharged amino groups also catalyze the hydrolysis of p-nitrophenylacetate, so that the method can be used unequivocally a t present only in a region of pH where all amino groups are in the charged acidic form. Mention should be made, finally, of the existence of indirect methods. Changes in some property which is not directly related to the dissociation of an acidic group may be observed to have a pH-dependence which resembles the pH-dependence of hydrogen ion dissociation from a single group. It may then be postulated that the change in this property is an indirect reflection of the dissociation of a single group, and that the dissociation curve and pK of the group can be obtained from it. The commonest example of this procedure involves the use of ultraviolet difference spectra (Wetlaufer, 1962), but optical rotation and other properties can be used as well. An instance of an application of such methods to proteins is provided b y Hermans and Scheraga (1961b).
82
CHARLES TANFORD
E . Effect of Solvent The brief survey of experimental methods which we have given has in general assumed that water or an aqueous salt solution is being used as solvent for the protein. When other solvents are used, two problems arise: (1) The dissociation constants of all acids and bases depend on the solvent being used. The characteristic pK’s of Table I would not apply, for instance, in a dioxane/water mixture. (2) The usual techniques for measuring pH are applicable to aqueous solutions only. The electrolytic cell normally used to measure pH, when standardized by appropriate buffer solutions, yields a value of pH which is not exactly the same as, but is very close to -log aH+as defined by other methods. (Bates, 1954; Tanford 1955a). There is no assurance that this will be true in other solvents. There is a simple way to avoid these problems. One can define a pH scale in a completely arbitrary manner relative to the emf of a suitable cell. One can then relate the pH on this scale to an arbitrarily defined “activity” of hydrogen ions, simply be setting pH = -log aH+. The dissociation constants of model compounds can then be determined in terms of this arbitrary scale. This method has been used by Donovan et al. (1959) for protein titrations in concentrated aqueous solutions of guanidine hydrochloride and of urea, and by Sage and Singer (1962) for titrations in ethylene glycol.
IV. COUNTING OF GROUPS The simplest information gained from titration curves is a count of the number of groups titrated. This count can be useful regardless of whether the titration curve is reversible and regardless of whether the protein remains native throughout the pH range of titration. The most interesting applications of group counting are in fact to situations where the titration curve depends on time, the direction of titration, and similar factors, The differences observed, between one set of conditions and another, often tell directly how the protein differs under the two conditions.
A . Counting Procedure Most titration curves, such as that of Fig. 2, consist of three well-separated parts: a steep portion between the acid end point and about pH 5.5, another steep portion between about pH 9 and the alkaline end point, and a relatively flat portion (relatively few groups titrated per pH unit) in between. It is thus quite generally possible to divide the titration curve into three S-shaped portions, as has been done in Fig. 2, and to count separately the groups which titrate in the acid, neutral, and alkaline re-
HYDROGEN ION TITRATION CURVES OF PROTEIN
83
gions. The separation between the three parts is better a t higher ionic strength, and one can also approach closer to the end points a t higher ionic strength. Thus group counting is usually best conducted at ionic strengths of 0.1 or above. Table I shows that organic molecules which have carboxyl groups that resemble the carboxyl groups of proteins generally have pK values from 3.5 to 4.7,when electrostatic effects of charges elsewhere on the molecule are absent, or have been corrected for. The electrostatic effects expected to arise from other charges on a protein molecule may shift these pK values and will broaden the range of pH within which these groups are titrated. However, the major part of all carboxyl groups should be in their acidic form at pH 2 and in their basic form at pH 6 or slightly higher. Furthermore, no other titratable groups are expected from Table I to have pK’s within 2 pK units of the carboxyl groups. (It should be recalled that electrostatic effects are expected to influence all kinds of groups about equally.) It is logical, therefore, tentatively to identify the groups titrated in the acid part of the electrometric titration curve as “carboxyl” groups, the quotation marks signifying that these groups may not all in fact be carboxyl groups. Similary, the neutral region of the curve may be identified with “imidaeole” and “a-amino” groups, the a-amino groups being the N-terminal groups of the polypeptide chains. The alkaline region, finally, may be taken to represent primarily “side-chain amino” and “phenolic” groups, with a contribution from sulfhydryl groups where these are important. The number of polypeptide chains of a protein molecule is usually known. In most cases each polypeptide chain is terminated by a titratable a-amino group at one end and a titratable a-carboxyl group a t the other end. By subtracting the numbers of these groups from the titration regions in which they are tentatively assumed to occur, we obtain a count of “side-chain carboxyl” groups and of “imidazole” groups, the quotation marks again signifying that the identification rests on an assumption of normal titration characteristics which further investigation may prove to be false. If the phenolic groups have been titrated separately by the spectrophotometric method, then a count of these groups is available. The change in absorbance at 295 mp on ionization of a 1 M solution of tyrosine (1-cm light path) is 2300, and, at the present level of approximation, the same figure may be taken for the phenolic groups of proteins, with the understanding that processes other than phenolic ionization may effect the absorbance at 295 mp (see above). Subtracting the number of phenolic groups from the number of groups titrated in the alkaline region as a whole gives a count of “side-chain amino” groups. If the point of zero net proton charge is known, then a count is available
84
CHARLES TANFORD
of the number of hydrogen ions which must be added to go from that point to the acid elid point (point of maximum net proton charge). Unless the protein contains phosphate groups, or other groups with pK’s well below that of carboxyl groups, only amino, imidazolo, and guanidyl groups (from arginine or homoarginine side chains) will hear charges at the acid end point. Moreover, all of these groups will be positively charged. The number of hydrogen ions required to go from the point of zero net proton charge to the acid end point is then simply a measure of the sum of all these groups, which we shall call EN+ for short. If the number of “imidazole” groups and of both kinds of “amino” groups is known from the counting procedures already described, then this number may be subtracted from 2” to yield the number of “guanidyl” groups. It should be noted here that guanidyl groups are normally in their acidic (charged) form throughout the pH range covered by the titration curve of a protein. The count of these groups as obtained here is essentially a count of the number of carboxyl and other groups which must be titrated to neutralize the positive charge present on guanidyl groups. It should also be noted that any metal ions coordinated to a protein may contribute to the maximum charge. A ferric iron atom coordinated to a heme protein, for example, normally bears a charge of +1 a t low pH. Phosphate groups, as previously mentioned, would have a charge of -1 at the acid end point. These charges, where present, will all make a contribution to ZN+. The division of the titration curve into three parts is subject to some arbitrariness. The uncertainty in the count of groups titrated in the acid and alkaline regions is typically about 5 %. The uncertainty in the count of groups titrated in the neutral p H range is numerically the same, but, since there are generally fewer groups in the neutral region, it represents a larger percentage of the number of groups. One way of refining the division of the titration curve into the three regions is based on the fact that carboxyl groups have a heat of dissociation close to zero, whereas imidazole groups have a heat of dissociation of about 6 kcal/mole. An apparent heat of dissociation may be measured from titration curves a t two or more different temperatures
AH,,,
=
-2.303~R[apH/a(l/T)];
(1)
the pH being measured at the same state of titration (same value of ?, see Fig. 2) a t each temperature. This value of AH,,, will generally be quite close to the true heat of dissociation, and may thus be used to define more sharply the transition from titration of L‘carboxyl’7groups to titration of “imidazole” groups. An example is provided by Fig. 3. The same method cannot be used to define the transition from t)heneutral
HYDROGEN ION TITRATION CURVES O F PROTEIN
85
range to the alkaline range, because a-amino groups (which titrate a t the upper end of the neutral region) would be expected to have the same AH (about 11 kcal/mole) as the €-amino groups of the alkaline region. Also, the phenolic groups of the alkaline region are expected to have AH N 6 kcal/mole, which is similar to the value for imidazole groups. (Heats of dissociation will be considered again in Section VI, E.) Other methods of refinement of the count of groups exist. The most important one occurs as an adjunct to the semiempirical analysis of the shape of each part of the titration curve, to be discussed in Sections VI, B and VI, C . It is often impossible to achieve a self-consistent interpre-
, 0 80
100
No.of groups titrated
120
FIG.3. Apparent heat of dissociation i n serum albumin. The break between AH = 1 kcal/mole and AH = 7 kcal/mole occurs very close t o P = 100, indicating the presence of a total of 100 carboxyl groups per molecule. (Tanford et al., 1955b.)
tation with the numbers deduced from visual examination of the curve, as described here. A self-consistent interpretation may then be obtained by minor alterations in these numbers. It may be noted finally that more classes of titratable groups than have been discussed so far may be discernible. Figure 4 is a particularly striking example. It shows that the phenolic groups of chymotrypsinogen may, simply on inspection, be divided into three separate classes.
B. Difference Counting Counting of titratable groups is particularly interesting when the titration curve for a protein depends on the conditions under which it is determined, as for instance in the example of Fig. 2, where the titration curve above pH 9.7 depends on time. What is the physical meaning of the
86
CHARLES TANFORD
difference between the zero-time curve of Fig. 2, presumably representing a continuation of the titration curve of the native protein, and the lower curve, which represents the titration after some slow molecular change has gone to completion? Titration curves provide one of the simplest methods of detecting the occurrence of conformational change, and numerous examples will be cited. To avoid cumbersome terminology we shall call striking conformational
PH
FIG.4. Spectrophotometric titration of the phenolic groups of a-chymotrypsino gen (Wilcox, 1961). The native curve was obtained a t 25°C in 0.1 M KCI. The arrows indicate time-dependent data. The upper curve was obtained a t 25°C in 6.4 M urea, containing 0.1 M KCI. Under these conditions the protein R i denatured. The total number of groups titrated is 4, a change in absorbance of about 2000 at 295 m p corresponding to titration of a single phenolic group.
changes of this kind “denaturation,” and shall refer to the altered protein as “denatured.” In most instances a combination of physical and chemical measurements is required to specify precisely the nature of a conformation change, and a detailed description is therefore beyond the scope of this paper, which is limited to information derived from titration curves. The term denaturation is to be taken as covering a variety of possible changes in conformation. Figure 2 is one type of situation in which titration curve differences are observed: in this case it is the difference between a native and a denatured protein. Differences of this kind may also be observed by examining the
HYDROGEN ION TITRATION CURVES OF PROTEIN
87
protein in different solvents, or before and after it has combined with a metal ion, or before and after it has reacted with oxygen, or before and after it has been subjected to the action of an activating enzyme, etc. Titration curve differences may be of three types. (1)The total number of groups titrated has altered, i.e., new titratable groups have appeared. (2) The total count of groups remains the same, but their division into the classes enumerated above has altered, e.g., some “imidazole” groups may have become “carboxyl” groups. (3) The numbers counted in each of the
PH FIQ.5. Difference between titration curves for a given type of group in two differ ent states of a given protein. The difference lies in the number of groups available for titration. The top figure shows the actual titration curves, the lower figure a plot of the difference between them.
classes remains the same, but the shape of the curve is altered. With the first two types of difference, the S-shaped curves representing titration of any one of the classes of groups differ in the way shown in Fig. 5. If it is merely the shape of the curve which differs, then the difference will appear as in Fig. 6. Unfortunately, one cannot always tell which type of difference is involved. The alkaline region of the zero-time titration curve of Plactoglobulin (native protein) shown in Fig. 2 cannot be carried above pH 11, because the rate of denaturation becomes too rapid. The segment which is available shows the titration in the alkaline region of about half as many groups as are titrated altogether in this region after denaturation.
88
CHARLES TANFORD
The curve for the native protein is also less steep. It is not possible, however, to tell whether an extension of this curve would continue with its same moderate slope until the total number of groups titrated became the same as for the denatured protein (analogous to Fig. 5 ) or whether it would level off earlier (analogous to Fig. 6). Titration curve differences may be measured directly by means of a pH-stat (Jacobsen et al., 1957). We begin with the protein in its initial native state a t a particular pH, and then allow denaturation, metal binding,
'!2x f
l -
0 r
PH
FIG.6. Difference between titration curves for a given type of group in two difl'crent states of a given protein. The difference lies in the shape of the curve, the number of groups available being the same. The top figure shows the actual titration curves, the lower figure a plot of the difference between them.
or whatever other reaction we wish to study, to occur. The pH-stat is designed to add acid or base so as to maintain the pH unchanged, and the total amount required between the beginning and end of the reaction is then a measure of the difference between the corresponding titration curves a t that pH. Measurements made a t a series of pH values will then produce difference titration curves such as shown in the lower parts of Figs. 5 and 6. By themselves these difference curves are less informative than the complete titration curves. They may however be more precise, and thus are often useful in conjunction with the complete curves. They also allow measurement of the rate of reaction a t the same time as the total difference is observed (see Section V, C ) .
HYDROGEN ION TITRATION CURVES O F PROTEIN
89
An example of direct difference counting, in a situation where the overall titration curve is not known, is provided by Fig. 7. It shows the difference between the titration curves of active carboxypeptidase A, which contains Zn++,and the inactive zinc-free protein, as determined b y Coleman and Vallee (1961). The measurements were made by the pH-stat method, and confined t o a relatively narrow pH range. The figure shows a difference of two groups per bound Zn++ ion, with pK values of 7.7 m d 9.1. The two groups involved lose their protons when Zn++ is bound. I n the absence of a complete titration curve a completely unequivocal interpretation
-n 0
-
V
I
$ 2.0.-
--&, \
q'
0
I
z G 1.5,-
\,
40
z 0 ++ c
\
5
pKt7.7
t\
\
\
b\-*
1.0,-
N
n
\ \
---a=-,
*\ '\
0.5 -
'\,
pK;9.1
w
-I
k!
5-
O.O.,
.
I
I
(CPD) con" _ taining Zn++, and the zinc-free apoenzyme. The data were obtained with a pH-stat, measuring the amount of base required to maintain constant pH as zinc was added t o apoenxyme. The data are for 2 5 T ,ionic strength 1.0.
cannot be made, but it is reasonable to suppose that Zn++ is chelated to two basic groups which are prevented from combining wit.h hydrogen ions as long as the metal remains attached. When Zn++ is removed, these groups bind hydrogen ions with pK values of 7.7 and 9.1. The experiments were carried out in 1 M NaC1, so that electrostatic effects should be suppressed, and pK values observed should correspond closely to those of Table I. Independent information on the strength of Zn++ion binding, suggests that the ion is chelated to a site containing a basic nitrogen atom and a sulfide group. The observed pK's are compatible with such a binding site, the pK of 7.7 corresponding to an a-amino group (or nn imidazole group), while the pK of 9.1 is the expected value for a thiol group.
90
CHARLES TANFORD
Several other interesting examples will be cited in Section X, where the titration curves of individual proteins will be analyzed. It will be seen from difference counting that the binding site for Zn++ in insulin involves two uncharged imidazole groups, and that the site for iron binding in conalbumin involves three ionized phenolic groups. Difference counting will reveal the presence of anomalous carboxyl groups in native 8-lactoglobulin and lysozyme. It will show that deviations from expected pK values, where they occur, are generally characteristic of the native conformation of a protein and that they disappear on denaturation. (One example is provided by Fig. 4. The three distinguishable classes of phenolic groups of chymotrypsinogen disappear on denaturation.) Difference counting will be seen to provide evidence concerning the action of thrombin on fibrinogen, and concerning the action of acid and base in the liberation of gelatin from collagen. Group counting may be used of course to study any chemical modification of proteins. A recent example is provided by the use of difference counting in a study of the effect of photo-oxidation on proteins (Vodrazka et al., 1961). V. REVERSIBILITY ; THERMODYNAMIC AND KINETICANALYSIS
A . Reversibility and Time-Dependence The dissociation of hydrogen ions from the model compounds discussed in Section I1 is ordinarily a very rapid reversible reaction. Measurements by ordinary methods represent thermodynamic equilibrium. They are independent of time and independent of the direction in which the reaction is carried out. Large sections of protein titration curves are often equally time-independent and reversible, as, for instance, the acid part of the titration curve of P-lactoglobulin shown in Fig. 2. Any such section of the titration curve will again represent thermodynamic equilibrium and it may be subjected to thermodynamic analysis, as outlined in Sections VI and VII. On the other hand, a titration curve may depend on the time between addition of acid or base and the pH measurement (as in the alkaline branch of Fig. 2). When this happens, the curve will also in general be irreversible. A titration curve may appear to be time-independent but irreversible, especially if a continuous titration method is employed in which successive increments of acid or base are added to the same solution for each successive pH measurement. A hypothetical example is shown in Fig. 8. When this situation occurs, a careful rerun of the curve, in which each experimental point is obtained with an entirely fresh solution, will usually show time-dependence, as shown by curves 3,4, and 5 of Fig. 8.
HYDROGEN ION TITRATION CURVES OF PROTEIN
91
In many time-dependent situations a zero-time reversible curve (curve 6 of Fig. 8) may be obtained, i.e., reversed points obtained by keeping a solution a t an extreme pH for various lengths of time, and then extrapolating to zero time of exposure to the extreme pH, may coincide with a forward titration curve, each point of which also represents an extrapolation to zero time. In the same situation, the protein represented by curve 2 of Fig. 8 will usually be different from the native protein by criteria such as optical
End with
Denatyed Protein Start with Native Protein
PH
FIG.8. Hypothetical titration curves illustrating time-dependence and irreversibility. Curve 1 is an apparently time-independent curve, obtained by continuous titration, waiting several minutes for each successive pH reading. Curve 2 is the reverse titration curve, beginning a t t h e acid end point. Curve 3 is the forward titration curve obtained by flow methods, each pH being measured on a freshly mixed solution within seconds of mixing. Curves 4 and 5 are obtained from freshly mixed solutions with longer time intervals between mixing and measurement. Curve 6 is the titration curve which one might speculatively draw t o represent “instantaneous” titration of the native protein.
rotation or ultraviolet absorption spectrum, i.e., it will represent titration of a denatured state of the protein. In some instances, curve 2 itself may be reversible, i.e., the denatured protein may be a stable product in rapid equilibrium with its environment. On the other hand, time-dependent reversion to the native form may occur, or further slow denaturation reactions may take place. The zero-time reversible curve can usually be obtained over a limited range of pH only. I n the hypothetical example of Fig. 8, the molecular change which leads to curve 2 as the observed titration curve would become too rapid for extrapolation to zero time well before the acid end point of
92
CHARLES TANFORD
the curve is reached. It is often possible in such situations to extend the zero-time reversible curve by using a lower temperature, or a different ionic strength, to reduce the rate of denaturation. An example is provided by the titration of ferrihemoglobiii. Figure ‘3 shows the titration data obtained by Steinhardt and Zaiser (1953) a t ionic strength 0.02, a t 25°C. It is not possible to see enough of the native titration curve to decide whether it differs from the back titration curve
PH
FIG.9. The acid region of the titration curve of ferrihemoglobin a t ionic strength 0.02, a t 25°C. The lower curves in the inset represent the difference between the 3-sec curve and the 2- t o 22-hr curve, the dotted line incorporating a correction (discussed in the original paper). The upper curves in the inset are not of interest for the present discussion. From Steinhardt and Zaiser (1953).
chiefly in the number of titratable groups or chiefly in the shape. Figure 10 shows similar data for the cyanide complex of ferrihemoglobin, a t ionic strength 0.3, at 05°C (Steinhardt el al., 1962). A difference in the count of groups is now evident on inspection. Another example is provided by the titration of the phenolic groups of ribonuclease (Tanford et al., 1955a), in 0.15 M KC1. At 25°C the data strongly suggest that only three out of six phenolic groups are titrated in the native protein. At 6°C this conclusion becomes unequivocal (Fig. 11). A summary of information on the reversibility of protein titration curves,
HYDROGEN ION TITRATION CURVES OF PROTEIN
93
obtained from recent detailed studies, is given in Table 11. It is seen that some proteins, like chymotrypsinogen, may be titrated reversibly and instantaneously over a wide range of pH. At the other extreme is pepsin, which undergoes autolysis below pH 5 and becomes denatured above pH 6. The fact that a protein titration curve is reversible over a given range 1.61
,
I
I
I
I
I
I
I
I
Cyonoferri hemoglobin 0.3 m CI Q5.C o Notlve Protein (Flowig)
-
1
I
/ 3
I
4
I
I
a
I
, 6
t
7
PH
FIG.10. Data similar t o those of Fig. 9, but for the cyanide complex of ferrihemoglobin, a t lower temperature and higher ionic strength. The curves without experimental points represent similar data for uncomplexed ferrihernoglobiu, at the same temperature and ionic strength. The inset shows the two difference curves obtained from these data. From Steinhardt et al. (1962).
of pH does not necessarily mean that the protein conformation remains unchanged over the same pH range. Several of the proteins listed in Table I1 undergo conformational change within this pH range. In every example cited, except ovalbumin, the evidence for such change first came from an analysis of titration curves by the methods of Section VI.
B. Thermodynamic Analysis Thermodynamic analysis can be carried out only for reversible portions of a titration curve. The commonly used methods of approach will be
94
CHARLES TANFORD
considered in Sections VI and VII. As indicated above it may be possible, over a limited range of pH, to obtain two reversible curves for analysis, one representing the native conformation of the prot.ein, the other representing a denatured state.
C . Kinetic Analysis Whenever a titration curve depends on time, a kinetic analysis becomes possible. As indicated above, the time-dependence usually reflects a
16
7' 2
1 B
X
P 10
\
1' 1
aC 2
t
48
'E 0
0
6
8
10
12
14
PH Fro. 11. Dissociation of the phenolic groups of ribonuclense at ionic strength 0.15. The dashed lines show regions of time-dependence. Half-filled circles represent measurements after reversal from pH 11.5 (middle curve) and after reversal from pH 12.7 (upper curve). 0--T = 25°C; 0--T = 6°C. From Tanford et al. (1955a).
change in protein conformation, and a kinetic study is then a measure of the rate of change of conformation. As an example, Fig. 12 shows pH-stat records of the uptake of hydroxyl ions by 0-lactoglobulin in the alkaline region (Nozaki and Bunville, 1959). These are a part of the data from which the two alkaline branches of the titration curve of Fig. 2 were constructed. They also provide, however, a measure of the rate of denaturation of the protein. No systematic review of kinetic studies of this kind will be attempted in this paper, since they should logically be considered in conjunction with other methods of following the rate of denaturation.
95
HYDROGEN ION TITRATION CURVES O F PROTEIN
TABLEI1 Reversibilitu of Protein Titration Curves at 26'CU or LLlUIlal
Chymotrypsinogen Conalbumin a-Corticotropin Hemoglobin Insulin 8-Lactoglobulin Lysoa yme Myoglobin Ovalbumin Pepsin Ribonuclease Serum albumin
9.4 6.8 8.6 7.0 5.6 5.3 (1l.l)Q 7.5 4.9 <2 9.6 5.4
2.5-11.8 4.2-11.2 2-12 4.4-11.5 2-12 1.5-9.7 2-12 4.5-11.5 2-12 5-6 2-11.5 2-12
No Yes -e
No No/ Yes No No Yesh No No Yes
References to titration studies of individual proteins are given in Section X. The isoionic pH depends on a variety of factors. For example, i t will be higher for ferrihemoglobin than for ferrohemoglobin. The figures given in the table are intended simply as an indication of the p H were & N 0. The pH range depends on the exact conditions. In many instances there has been no serious effort topin down exactly how far a titration m2ty be carried without irreversible change. The range of pH 2 t o 12 given for several proteins does not mean that irreversible changes are known to occur at pH 2 or 12, but only that no such changes were observed within that range. Minor changes and simple reversible association reactions have not been included. a-Corticotropin does not have a globular compact structure at any pH. f The titration curve can give no information on the subject of reversible conformational change in insulin because there is extensive pH-dependent association between insulin molecules. The absence of other conformational change is indicated by hydrodynamic measurements of Fredericq (1956). 0 The isoionic pH of lysoayme is in doubt. See Section X. A No conformational change is detected by analysis of the titration curve. A distinct change in sedimentation coefficient occurs below pH 4, however. (Charlwood and Ens, 1957.)
VI. SEMIEMPIRICAL THERMODYNAMIC ANALYSIS A . The Equation of Linderstr$m-Lang If a portion of a titration curve represents thermodynamic equilibrium, it may be treated by standard methods of thermodynamic analysis. If, for example, a protein molecule has 100 dissociable hydrogen ions, and if
96
CHARLES TANFORD
its titration curve is reversible from one extreme to the other, one could determine the 100 successive equilibrium constants for the reactions PHloo
+ P H Q Q+ H+
*
+ Hf PHi-1 + H+ ...
P H ~ Q PHm
PHi
PH$P+H+
TIME, MINUTES
FIG.12. The time-dependent uptake of base by p-lactoglohulin a t ionic strerigt,h 0.15 at 25"C, as measilred with a pH-stat. The initial point of each curve (filled circles) represents a measurement on a separate solution, with a flow apparatus c a p ble of determining pH within 1 sec of mixing (Nozaki and Bunville, 1959).
where PHi represents a protein molecule in which i of the hydrogen ions are still bound. The dissociation constants which one obtains in this way are however of limited value. They provide an algebraic representation of the titration curve, but they do not reflect the properties of individual acidic groups because a species designated as PHi is not a single species, but represents a mixture of molecules in each of which the i protons are attached to a different set of basic groups. A much more revealing treatment is one which specifically takes into account the fact that many of the titratable groups of a protein molecule are likely to be intrinsically identical, or very nearly so. Furthermore, it takes into account the one interaction between titratable groups which is
HYDROGEN ION TITRATION CURVES OF PROTEIN
97
known to be of predominant importance, this being the Coulombic interaction between their charges, and introduces suitable theoretical expressions for this interaction into the equation for the titration curve. The first and simplest treatment of this kind was developed by Linderstrgm-Lang (1924). Its basic assumption is that the charge on a protein molecule is evenly smeared over the surface of a sphere. The sphere is assumed impenetrable to the solvent. The electrostatic interaction between charges enters into the theory by virtue of the fact that each hydrogen ion to be added has to increase the molecular charge by one unit, being opposed by the charge already present. As a result, the equilibrium constant K , for dissociation of any one titratable group of type i,
Ki
xiaH+ 1 - xi
= __
where xi is the average degree of dissociation and aH+the thermodynamic activity of hydrogen ions in the solution, becomes dependent on the average charge per protein molecule. This dependence takes an exponential form,
z
Ki
=
Kikl e2w'
(3)
where KM: is the intrinsic dissociation constant for the group in question, this being the value of Ki in the absence of electrostatic interaction, i.e., when 2 is zero. The constant w of Eq. (3) is the same for all titratable groups and is given by the equation 2
DRkT
(4)
where e is the unit of electron charge (4.80 X lo-'' esu), D is the dielectric constant of the solvent, k is Boltzmann's constant, T is the temperature, K is the Debye-Huckel parameter proportional to the square root of the ionic strength (at 25°C in water it is 3.87 X 1071"2, where I is the ionic strength), R is the radius of the sphere which represents the protein molecule, and a is the radial distance of closest approach to the center of this sphere of the center of the average ion of the salt which is being used to create the ionic strength. Generally the distance a will be of the order R 4- 2.5 A. A detailed derivation of Eqs. ( 3 ) and (4) has been given elsewhere (Tanford, 1961a). Combining Eqs. (2) and (3), we get
98
CHARLES TANFORD
or, in logarithmic form, X.
log A 1 - xi
=
pH
- pK:At
+ 0.868~2
(6)
In using these equations we should note that neither K , nor K/fl is a true thermodynamic equilibrium constant because the ratio xi/( 1 - x i ) is a concentration ratio instead of an activity ratio. Ionic strength is however the only factor which should influence its value measurably (see Section VI, D),and K:;: should remain essentially constant over the entire titration curve as long as ionic strength remains constant. It should be noted also that the “pH” which is ordinarily measured in the laboratory is not exactly -log uH+if uH+is to have the meaning assigned to it in Eq. (2). In dilute aqueous solutions, however, the difference must be quite small ( Tanford, 1955a). In using these equations we need to know the average net charge 2 at each pH. The titration curve gives us the major part of this charge, namely the part 2, which is due to hydrogen ions bound to basic groups or dissociated from neutral acidic groups. This quantity is given directly by the titration curve if the reference point for the ordinate is the point of zero net proton charge, i.e., if the isoionic pH has been determined. However, even if the pH of zero net proton charge has not been experimentally measured, it can be calculated after the group counting procedure is completed. If we know the maximum value of 2, at the acid end point of the curve, ( Z H ) m a x , then, at any other pH, ZH
and 2,
=
=
(2H)msx
-
f
(7)
0 at that pH where F =
In the absence of any other information we have no choice but to choose
2 as equal to 2, . However, if the salt ions, which are present in the solution to maintain a moderately high ionic strength, are known to be bound to the protein, then 2 must be corrected appropriately. If the electrolyte used is KC1, for example,
2 = 2,
+
- acl-
(8 1 where a is the average number of ions of a given type bound to the protein. The D values may be determined in separate experiments (Scatchard et al., 1950, 1957; Carr, 19,55), but these experiments are more difficult than the determination of titration curves and in many cases the a values for the ions are not known. We now introduce the second important feature of the LinderstrgmLang treatment, that many of the titratable groups of any protein molecule are intrinsically identical. In other words, we can divide the titratable rK+
HYDROGEN ION TITRATION CURVES OF PROTEIN
99
groups into a small number of classes: n1 groups of type 1, n2 groups of type 2, etc. All the groups of a given type have the same intrinsic pK, so that each group has the same degree of dissociation at any pH. The total number of groups of type i which are dissociated at any pH is then fj =
with
5, given
ngg
(9)
by Eq. ( 5 ) or (6). The total of all groups dissociated is
the summation extending over all types of dissociable groups. With Eqs. ( 5 ) and (9) the equation for the titration curve as a whole becomes eZwUB/a,+ P=C 1niKjf; + Kiffie2wa/aH+ i
(11)
The derivation of Eq. (11) is based on two assumptions which cannot be far from true for many globular proteins: ( a ) that the titratable groups are located on an approximately spherical particle which is impenetrable to the solvent and to small ions which it may contain, and ( b ) that the most important interaction between the titratable groups is the result of Coulombic forces between their charges. It will be seen in Section VLI however that the Linderstrgm-Lang method of calculating this interaction cannot be correct, although it will also be seen that the error introduced thereby will not be a serious one under favorable circumstances. The more sophisticated treatment of the electrostatic interaction which will be introduced in Section VII cannot be used as the basis of a practical analysis of protein titration curves because it requires precise knowledge of the location of titratable groups on the protein molecule, which is not available. One is therefore compelled to use Eq. (11) as the only available equation for the titration curve, with the understanding that it can be expected to apply only approximately. On the following pages we shall find many experimental deviations from the results predicted by the Linderstrgm-Lang treatment, and shall often use them as evidence that one of the basic assumptions of the treatment is inapplicable : the protein under examination may not be a compact, globular protein, or the interactions between titratable groups may not be purely electrostatic. However it will also be kept in mind that deviations may result from the approximate nature of the dculation, in situations where the lmic assumptions remain true.
R . Empirical Procedure In a practical procedure for applying the Linderstrgm-Lang treatment to experimental data, one modification is made. The electrostatic interac-
100
CHARLES TANFORD
tion factor w is treated as an empirical parameter, not necessarily equal to that calculated by Eq. (4). If necessary, w is considered to be a variable, its value depending on 2. It is generally assumed (tentatively) that all groups which are chemically identical, and fall into the same region of the titration curve, have the same pK i nt . The carboxyl groups of aspartic and glutamic acid are considered identical for this purpose because uncharged model compounds resembling these groups have essentially identical pK values, as shown in Table I. The a-carboxyl groups at the ends of polypeptide chains (if their number is known) are however assigned a different pKint, on the basis of the information of Table I. An exception is made of course if the group counting procedure shows that groups which are chemically alike clearly do not have the same P K ~ ,,, ~ as in the case of the phenolic groups of chymotrypsinogen (Fig. 4). The major terms of Eqs. (10) or (11) are now analyzed separately. The simplest is the term for the phenolic groups, the degree of dissociation of which is obtained independently of other groups by use of spectrophotometric titration. For other kinds of groups, where the electrometric titration curve must be used, we pick a region of pH within which only one of the iiof Eq. (10) is an unknown function of 2. Tn the acid region for example we may tentatively assume that the pKint of the usually small number of a-carboxyl groups is known. Side-chain carboxyl groups are the only other groups which are titrated in this range, so that Eq. (10) becomes F
=
nlxl
+ n2xz
(12)
where subscript 1 refers to a-carboxyl groups and subscript 2 to side-chain carboxyl groups. With x1 calculable, because pK/Ai has been assigned, we obtain x2 as a function of 2. I n general, in a region where groups of type m are the principal groups being titrated, so that variation in x, is the major factor in the change in i with 2, we wiIl have i-m-2
7 =
C
i=l
ni
+ nm--lxm-i+ nmxn+ nm+lxm+l -.
(13)
where groups designated by subscripts 1, 2, , ?n - 2, are known to be completely dissociated, so that xi for these groups is equal to unity, while groups with subscripts m 2, m 3, etc., are still completely in the acidic form, so that xi = 0. The values of xm--l and xpn+l,which make a relatively small contribution to the change in F, may either be known (they may be the phenolic or side-chain carboxyl groups), or they may be tcntatively calculated with assumed values for pKint and w by Eq. (6). The
+
+
HYDROGEN ION TITRATION CURVES OF PROTEIN
net result is that we can get 2% as a function of We can now rearrange Eq. (6)to give pH
- log[xm/(l - x,)]
=
101
zfor any kind of group.
pK!,mt) - 0.868~2
(14)
and plot the left-hand side of the equation versus 2. This will give a value for pK%) as the intercept at 2 = 0, and will show whether w can be considered a constant or whether it must be regarded as variable. The value of w is obtained from the slope if it is a constant, or by calculation using Eq. (14) and the value of pIC!,m,‘ if it is variable. The entire calculation must now be repeated whenever, in an equation of the type of Eq. (13), the calculation depended on tentative values of zm--l and x,+~, New values of these parameters are now used, based on PKint and w values which were obtained from the first round of calculations. By successive approximations of this type, there soon emerge values for each xi as a function of pH which no longer change with further reiteration of the mathematical procedure. Each of these will yield a value for pKi’hl and a single value or a set of values for w. Results obtained in this way will be discussed in the following section. It should be noted that the procedure outlined here often leads to refinement of the values of niwhich were originally obtained by the group counting procedure. If, for example, the successive evaluations of 2% consistently lead to a sharp rise in pH - log[s,/( 1 - z,)] at the high pH end, this suggests that the last one or two of the groups of type m which are being titrated have a higher pK than the rest. At least one possible explanation is that one or two groups which have been counted as being of type m really belong to type m 1. On the other hand, if pH - log [xm/(l z,)] decreases markedly at the high pH end, this suggests that we have counted too small a number of groups as being of type m.
+
+
C. The Electrostatic Interaction Factor The “expected” value of the electrostatic interaction factor w is given by Eq. (4). To estimate the radius R of the sphere representing the protein molecule, we can use an experimental value of the partial specific volume 8 and a reasonable estimate for the bound water, 61 grams per gram of protein (usually 61 0.2 is chosen). The volume per protein molecule becomes (Tanford, 196lb)
-
4 M - 7rR3 = - ( V 3 3t
+ 61~:)
where M is the molecular weight of the protein, 51 is Avogadro’s number, and vy is the specific volume of the solvent, which in the case of water is 1.00 at normal temperatures.
102
CHARLES TANFORD
Ail alt,eriiative procedure is to calculate the radius of a sphere equivalent to the protein molecule from hydrodynamic measurements. The value of R obtained in this way is always larger than that calculated by Eq. (15), even for the native protein conformation, the reason being that protein molecules are in fact never perfectly spherical. The resulting values of w are roughly 20 % less than those obtained with a radius calculated by Eq. (15). Some sample calculations are shown in Table 111. It is seen that w is larger for a smaller sphere and larger at low ionic strength. The data in the table are for 25°C. The effect of temperature, however, is quite small. Figures 13 to 17 show some experimental plots of titration data according to Eq. (14). For some of the examples chosen, ion binding data were available, so that the left-hand side of Eq. (14) could be plotted against 2.
TABLE111 Calculated Values for w at 36%' and Several Ionic Strengths Protein
a-Corticotropin Ribonuclease &Lactoglobulin Serum albumin
I
I
I
Mol. wt.
Calculated radiusa (A)
4541 13,683 35,500 65,000
12.0 17.1 23.7 28.9
Value of w
I
I = 0.01 I = 0.03 Z = 0.15 I = 1.00 0.218 0.137 0.088 0.066
0.186 0.113 0.069 0.051
0.138 0.079 0.046 0.033
0.094 0.051 0.029 0.020
Radius calculated by Eq. (15).
In most instances however only 2, is known, so that this has had to be used as abscissa. Figure 13 shows the data for the three phenolic groups of ribonuclease which ionize reversibly (Tanford et at., 19558), based on spectrophotometric titration curves such as Fig. 11. A straight-line plot is obtained, in agreement with Eq. (14). The values of 20 are 0.112, 0.093, and 0.061, respectively, at ionic strengths 0.01, 0.03, and 0.15. (The salt used to produce the ionic strength was KCI, and there is evidence that neither K+ nor C1is bound to an appreciable extent. The use of 2, as abscissa is therefore presumably acceptable.) Comparison with the calculated values of Table 111 shows that the experimental values are lower than predicted by about 20%. Such a deviation must be considered almost within the error of calculation. [If the radius of the hydrodynamically equivalent sphere (19 A) had been used as the basis of calculation, the calculated values of w would have become 0.119,0.096, and 0.066, respectively.] Another example showing good agreement with the theory is provided
HYDROGEN ION TITRATION CURVES O F PROTEIN
103
by the data for the side-chain carboxyl groups of p-lactoglobulin, shown in Fig. 14. The values of w are 0.072 and 0.039, respectively, at ionic strengths 0.01 and 0.15. Thus they are again somewhat smaller than the calculated values (0.088 and 0.046) given in Table 111. The electrolyte
10.0 CI
10.4 . I ( U
\
n
3 10.2 I
%
10.0
9.8
0
2
-4
-2 Charge
-0
-8
FIG.13. Titration data for the three reversibly titrated phenolic groups of riboiiuclease a t 25°C and a t three ionic strengths, plotted according to Eq. (14) (Tanford et al., 1955a).
n
U
4.6 r(
Y
1
s16
I 4.0
x,
0 0
P
3.6 -
1
30
X
I
20
10
ZH
I
0
- 10
FIG.14. Titration data for the normal aide-chain carboxyl groups of p-lactoglobulin a t 25°C and ionic strengths 0.01 (steeper line) and 0.15, plotted according to Eq. (14) (Nozaki et al., 1959).
104
CHARLES TANFORD
used was again KC1. Both ions of this salt are bound by ,@-lactoglobulin, and very approximate estimates for the extent of binding as a function of
$o. t
9.8
I
8
I
2
-
I
4
I
6
I
8
I
10
kZ)
FIG.15. Titration data for the phenolic groups of or-corticotropin, a t 25°C and ionic strength 0.1, plotted according to Eq. (14) (LBonis and Li,1959).
-
2,
FIG.16. Titration data for the carboxyl groups of lysozyme, a t 25"C, at three ionic strengths ( I ) . Plotted from data of Tanford and Wagner (1954).
pH are known. These indicate that the change in 2 over the acid pH range is about 0.8 of the change in 2, a t ionic strength 0.01 and about 0.67 of the change in 2, at ionic strength 0.15. The corresponding values of w become 0.090 and 0.058. These values are somewhat larger than the cal-
HYDROGEN ION TITRATION CURVES O F PROTEIN
105
culated values of Table 111. They should not be taken seriously because of the poor precision of the binding data, but they do serve to indicate that the difference between 2 and 2, is of such magnitude as to bring w values quite close to the values calculated by Eq. (4). An entirely different] situation exists in the titration of a-corticotropin (LBonis and Li, 1959). Data for the phenolic groups, obtained in 0.1 M KCl, are shown in Fig. 15. A straight line is obtained, but w is only 0.034 instead of the value of 0.16 expected a t that ionic strength from Table 111. This difference is far outside the limits of error of the calculation. The carboxyl groups of lysozyme (Tanford and Wagner, 1954) by con-
n
H
I 4.0 l-l
Y H
8
'
.-(
E
3.5
3.0 60
40
z
20
0
-20
FIG.17. Titration data for the carboxyl groups of bovine serum albumin, a t 25°C and ionic strengths 0.01 (lowest curve), 0.03, 0.08, and 0.15 (upper curve), plotted according t o Eq. (14) (Tanford et al., 1955b).
trast give a value of w much larger than expected. This protein has essentially the same size as ribonuclease, so that the expected values of w at ionic strengths 0.03, 0.15, and 1.00 are 0.11, 0.08, and 0.05, respectively. Experimental plots of pH - log[z/(l - z)] versus 2, , shown in Fig. 16, give the much higher values 0.23, 0.175, and 0.11. Moreover lysozyme is known to bind chloride ion strongly, the extent of binding increasing with increasing 2, , so that the values of w based on 2 would be even higher. Figure 17 shows an even more striking anomaly. It represents the titration of the carboxyl groups of bovine serum albumin (Tanford et aZ., 1955b), and it is seen that a constant value of w will not fit the data at all, the plots of pH - log[z/(l - z)] versus 2 being curved. It should be noted however that the steepest parts of the plots (near 2 = 0) are linear, and that the slopes give w values of 0.054, 0.036, 0.028, and 0.023, respec-
10G
CHARLES TANFORD
tively, at ionic strengths 0.01, 0.03, 0.08,and 0.15. These values are not too far below the calculated values of Table 111. The most interesting aspect of the foregoing results is that they indicate that the Linderstrfim-Lang equations are obeyed in some of the examples cited, whereas quite different results are obtained in other instances. We shall now examine some of the possible reasons for deviations from the Linderstrgim-Lang model. 1 . Trivial Factors It is seen from the discussion of the data for ribonuclease and p-lactoglobulin (Figs. 13 and 14) that two simple factors can lead to w values which are less than those given in Table 111, but only by a small amount. There is uncertainty in the choice of a radius for the sphere used to represent the molecule, and experimental uncertainty as to the relation between 2 and i?, . The uncertainty in the radius can account only for smaller values of w than those of Table 111, because the values in the table are based on what is essentially the minimum possible radius. The uncertainty in 2 can account only for smaller values of w (if 2, is used as abscissa in the logarithmic plots) because any change in i?, will favor the binding of oppositely charged ions, so that increments in i? are less than those in 2, . These two factors, plus the approximate nature of the theory as a whole, suggest that experimental values of w which are up to one-third less than those predicted by Eq. (4)should not be regarded as significant. On the other hand, values larger than those calculated by the equation [with Eq. (15) used for R] are more likely to carry significance. 2'. Unfolding or Swelling of the Protein Molecule
When unfolding or swelling of a protein molecule occurs, permitting the solvent to penetrate into the molecular domain, there is a drastic reduction in w,especially at higher ionic strength (Tanford, 195513, 1961a). As the sample calculation of Table IV shows, a swelling to twice the original radius (eight times the original volume) reduces w to an exceedingly small value at ionic strength 0.15. Unfolding of a globular structure to a random coil would lead to an increase in effective radius which would be of this order of magnitude. This is the most likely explanation for the low values for w which are obtained for a-corticotropin. The titration curve therefore suggests that this molecule exists in water in an unfolded rather than a compact globular conformation. This conclusion is supported by measurement of hydrodynamic properties of a-corticotropin. 3. Rod-Shaped Molecules Electrostatic interaction between charges on a rigid thin rod is also less than interaction between the same number of charges on an impenetrable
107
HYDROGEN ION TITRATION CURVES OF PROTEIN
sphere (Hill, 1955), but the effect of ionic strength is not nearly so great as it is for an unfolded molecule. It is likely that an unequivocal differentiation between a rod and a loose flexible coil could be made on this basis, though no actual examples have been reported.
4. Reversible Transition from Native to Denatured Conformations within the Titration Region We have already pointed out (Section V, A ) that titration curves can provide evidence for the existence of slow and irreversible changes in conformation. Where such changes occur, two separate curves can often be TABLEIV Theoretical Effect of Swelling, Dissociation, and Aggregation orL the Electrostatic Interaction Factor w at M ° C Value of w
I Impenetrable sphere, R = 25 A Same sphere, swollen by solvent penetration to R = 50 A Impenetrable spheres formed by dissociation to half molecules, R = 25/2'/* = 19.8 A Same, dissociated to quarter molecules, R = 25/4]/3 = 15.7 A Aggregation to impenetrable sphere with R = 25 X 4 '/3= 39.7 A
= 0.01
I = 0.15
0.081 0.027
0.042 0.004
0.056~
0. 031a
0.0380
0.023"
0.1630
0. 074a
a These are the apparent values of w which would be obtained with 2 calculated for the original molecular weight, assuming neither dissociation nor aggregation to take place.
obtained, at least over a limited range of pH, one representing the native and the other the altered conformation. If a change in conformation occurs rapidly and reversibly iL would not be detectable in that way, for a single smooth reversible titration curve would be observed. This curve might, for example, resemble curve 1 of Fig. 8. If the data from such a curve are plotted according to Eq. (14), the plot would initially (high pH end in this case) be that for the native protein, but, in the region of conformational change, the increased uptake or dissociation of hydrogen ions would be reflected by a break in this plot. Separate curves for native and denatured proteins being unavailable in this situation, it will not normally be possible to decide whether the conformational change which occurs is one which involves a change in the number of titratable groups or whether it involves a change in w or pKint without change in group number. If the process is one of unfolding, with-
108
CHARLES TANFORD
out change in the number of groups or in any pKint , then the reversible conformational change would correspond to a change from a curve like the flatter curve of Fig. 6 to the steeper one, the greater steepness being due to the large decrease in w which accompanies unfolding. The logarithmic plots which might correspond to native and unfolded proteins, each stable over a wide range of pH, are shown in Fig. 18, as is the experimental curve which would be obtained if SL transition from one conformation to the other occurs during the titration. This curve resembles the titration curve observed for the carboxyl groups of serum albumin (Fig. 17), and therefore indicates that one possible interpretation of the titration data for serum
+
-
-2-
-
FIG.18. Hypothetical plot of titration data for groups which all have the same pKi,t . The three curves show the data as they might appear for a protein which is native throughout the pH range of titration, for a similar protein which is unfolded throughout the same pH range, and for a protein which undergoes a rapid reversible transition from a compact, native to an unfolded conformation.
albumin is that this protein undergoes reversible unfolding in the region of titration of the carboxyl groups. That such unfolding occurs has in fact been shown be several other techniques (e.g., Yang and Foster, 1954; Tanford et al., 1955c) The curve of Fig. 18 which represents the unfolded protein has been drawn as a curve rather than a straight line because unfolded proteins have a flexible conformation and are expected to expand with increasing charge, with a resulting decrease in w. The titration curves of simple polyelectrolytes, when plotted according to Eq. (14), do not result in linear plots (Tanford, 196la). Although the titration data for the carboxyl groups of serum albumin are qualitatively compatible with the idea that serum albumin undergoes re-
HYDROGEN ION TITRATION CURVES O F PROTEIN
109
versible expansion during their titration, this does not exclude other possibilities. A conformational change which irivolves a change ill tlhe number of titratable carboxyl groups, or in the PI<,,^ of these groups, could lead to experimental data which resemble those shown in Fig. 18. A quantitative explanation of the data of Fig. 17 is in fact not yet available (see Section X). All that the limited discussion given here can really demonstrate is that a conformational change takes place. A detailed understanding of the titration curve requires a study of the conformational change by all available methods. 5. Dissociation and Aggregation Unfolding has been cited as one way to account for electrostatic interaction which is much smaller than expected. Another possibility is dissociation into smaller units without other change, i.e., the smaller units are considered spherical and impenetrable to solvent, and Eq. (4) is assumed applicable to the calculation of w. If Ml is the true molecular weight of the protein, w1the corresponding value of the electrostatic interaction factor, and Zl the corresponding charge at any pH, then, for any titratable group with given pKint, pH - log[z/(l
- x)] = pKint - 0.868wlZ1
(16)
If the observed titration curve is based on a false molecular weight Mz , then the values of 2 which one uses in Eq. (14) are equal to ( M z / M l ) Z l . Thus the term wZ of the equation is equal to w ( M ~ / M ~,)aid, ~ I by comparison with Ey. (16), the apparent value of w becomes
w
= Wl(MljM2)
(17)
The anticipated decrease in w resulting from this effect is shown in Table IV. It is seen that dissociation is less effective than unfolding in reducing w at moderate ionic strengths. Aggregation of proteins will have the opposite effect of dissociation: the apparent value of w, obtained from an analysis which assumes that no aggregation has occurred, will be larger than expected. Since aggregation can proceed to formation of very large aggregates, quite larger increases in w could occur in this way. One experimental titration curve which probably reflects aggregation is that of zinc-free insulin (Tanford and Epstein, 1954). Between pH 4 and pH 7 this protein becomes insoluble, but the precipitate is gelatinous, so that water and hydrogen and hydroxyl ions appear able to react with the acidic and basic groups rapidly and reversibly. The only abnormality is that the observed values of w, in the region of precipitation, become more than three times larger than at pH’s where the protein is soluble. The presence of large aggregates in the precipitate is the most likely explanation.
110
CHARLES TANFORD
6. Erroneous Calculation o j the Electrostatic Interaction
The titration of the carboxyl groups of lysozyme (Fig. 16) is an instance of the occurrence of unexpectedly large values of w. I n this example aggregation does not provide a possible explanation, for molecular weights of lysozyme have been determined at different pH’s without indicating any tendency for aggregation. An alternative explanation must be sought, and the simplest place to seek it is in the basic assumptions which underlie the Linderstrom-Lang treatment. A more sophisticated method for calculation of the electrostatic interaction between charged groups will be considered in Section VII. It will be seen that sizable deviations from the Linderstrom-Lang theory will occur whenever a titratable group is located in close proximity to another charged group. If the vicinal charged group is charged throughout the range of titration of the group being analyzed (e.g., an amino group near a carboxyl group), then the anomalously high interaction between the groups will affect the pKint term of Eq. (14) (see Section VI, 0).If the groups in close proximity are titrated within the same range of pH, on the other hand, the result will be an interaction between them which is stronger than the Linderstrom-Lang treatment would predict. The result will be an anomalously large value for w, and it is possible that the anomalously high w values found for lysozyme are ascribable to this cause.
7. Identical Groups Do Not Have the Same Intrinsic p K In the Linderstrflm-Lang treatment, groups which are chemically identical are assumed to have the same pKint. The fact that the dissociation constant of Eq. (2) (for titration of several chemically identical groups) depends on the extent of titration is ascribed entirely to electrostatic interaction between the groups. It is true of course that a spread in apparent pK over the titration region could be obtained without electrostatic interaction if the various groups of a given chemical kind have different intrinsic pK values (e.g., Karush and Sonenberg, 1949). This effect is not used as a hypothetical explanation for the normal variation of pK with extent of titration for two reasons: ( a ) because this effect would be independent of ionic strength, whereas the normal result is that w depends on ionic strength in the way expected for electrostatic interactions; and ( b ) because the very nature of a protein molecule requires that electrostatic interaction between charged groups exist, and no mechanism is conceivable whereby it could be eliminated. On the other hand, large differences between pKint values for chemically identical groups do exist. The four phenolic groups of chymotrypsinogen (Fig. 4) for example clearly fall into three distinctly different classes with very different dissociation constants. If smaller differences between pKint
HYDROGEN ION TITRATION CURVES OF PROTEIN
111
values of chemically similar groups were to exist, then the groups would be titrated in the same range of pH, but the curve would be more spread out than if the spread were due to electrostatic interaction alone. I n terms of the Linderstrgm-Lang treatment, this would reflect itself in abnormally large values of w. The abnormally large w values for lysozyme could be due to such an effect, then, as well as to abnormally strong vicinal electrostatic interactions. (As was pointed out earlier, however, changes in pKi,t can within the present treatment be themselves the result of strong vicinal electrostatic interactions.)
D. Intrinsic pK’s and Their Interpretation 1. Experimental Values
The preceding section has considered the electrostatic interaction factor w of the Linderstrgm-Lang equation. We consider now the value of the intrinsic pK, which is obtained formally as the value of pH - log[x/. (1 - z)], for any given kind of titratable group, a t the point 2 = 0. The value of pKint can be determined directly from a logarithmic plot of the data if, as in the example of Figs. 13 and 14, the point 2 = 0 falls within the range of titration of the type of group under consideration, If, on the other hand, the titration range is removed from the point 2 = 0, then an extrapolation is required to determine pKi,t. If the logarithmic plot has roughly the theoretical slope, and if similar plots, for other groups which do titrate in the region near 2 = 0, also show normal values of w, then such an extrapolation can be performed with some confidence that the result will be meaningful. Another criterion for validity of a pKint value determined by extrapolation is the dependence on ionic strength. Theoretically the effect of ionic strength should be small (see Section VI, D, 4). These criteria indicate that in a situation of the kind shown in Fig. 16, where the slope is abnormally large, and where a different intercept at 2 = 0 is obtained a t each ionic strength, the value of this intercept would have no meaning. It cannot be considered to represent the intrinsic pK of the groups being titrated. We have compiled in Table V the reliable values of pKi,t which have been reported in the literature. The values are to be regarded as having a significance of &O.l to 0.2, except that the values for imidazole groups are subject to somewhat greater error. These groups are usually present in smaller numbers than carboxyl and amino groups, so that the overlap between the different titration regions makes proportionately a larger contribution. The values of pKint for a-carboxyl and a-amino groups cannot usually be derived from experiment a t all because they represent such a small portion of the groups titrated.
112
CHARLES TANFORD
TABLEV Intrinsic p K Values at M°Ca Type of group
a-Carboxyl Expected value (Table I) Insulin Side-chain carboxyl Expected value (Table I) Lysozyme (3 of ca. 16 groups) Serum albumin Ovalbuinin Conalbumin Corticotropin Insulin @-Lactoglobulin(49 of 51 groups) p-Lactoglobulin (2 of 51 groups) Imidazole Expected value (Table I) Myoglobin (6 of 12 groups) Hemoglobin (ca. 22 of ca. 38 groups) Insulin R i bonuclease Myoglobin (6 of 12 groups) Chymotrypsinogen Ovalbumin Conalbumin Lysoz yme Serum albumin ,%Lactoglobulin a-tlmino Expected value (Table I) None of the experimental values can be considered reliable Phenolic Expected value (Tahle I) Conalbumin (11 of 18 groups) Insu 1in Chymotrypsinogen (1 of 4 groups) Corticotropin Papain (11 of 17 groups) Ribonuclease (3 of 6 groups) Serum albumin Chymotrypsinogen (1 of 4 groups) Lysoz yme Chymotrypsinogen (2 of 4 groups) Conalbumin (7 of 18 groups) Ovalbumin Papaiii (6 of 17 groups)
PK 3.8 3.6 4.6 - w
4.0 4.3 4.4 4.6 4.7 4.8 7.3 6.3b --m --m
6.4 6.5 6.6 6.7 6.7 6.8 6.8 6.9 7.4 7.5
9.6 9.4 9.6 9.7 9.8 9.8 9.9 10.4 10.6 10.8
113
HYDROGEN ION TITRATION CURVES OF PROTEIN
TABLE V-Continued Type of group
Ribonuclease (3 of 6 groups) Side chain amino Expected value (Table 1) Conalbumin Serum albumin &Lactoglobulin a-Corticotropin Ovalbumin Ribonuclease Lysoa yme Chyniotrypsinogen (3 of 13 groups) Guanidyl Expected value (Table I ) Insulin a-Corticotropin Ferriheme waterC Myoglobin Hemoglobin
PK 00
10.4 9.6 9.8 9.9 10.0 10.1 10.2 10.4 W
> 12 11.9 -12 8.9 8.W
a Only pK values observed for the native conformation of proteins are listed. A value of m is used to indicate that a group appears incapable of dissociation in the native molecule, a value of - co that i t appears incapable of binding a proton. References t o all data are given in Section X. * See text. There is considerable question as to the pKi,t value expected for an unperturbed imidazole group. c No suitable model compounds are available on which an expected value could be based. d Estimated from the data of George and Hanania (1953).
Table V shows that the vast majority of the titratable groups of the smaller protein molecules have pKint values which are quite close to the values predicted from the pK’s of model compounds. This feature of protein titration curves has been well known for a long time, and is accepted as normal. It is however really an astonishing result, for it implies that most of the titratable groups of the smaller protein molecules are in as intimate contact with the solvent as similar groups on smaller molecules, and that they are able to accept or release hydrogen ions in this location without requiring any modification of the protein conformation in the vicinity of the titratable group. Since most of the proteins examined have been globular proteins, tightly folded so as to exclude solvent from most of the interior portions, the titratable groups must be nearly always at the surface. When the molecular weight of a globular protein exceeds 100,000, the surface per unit mass begins to be too small to accommodate all the titrat-
114
CHARLES TANFORD
able groups. Titration anomalies should therefore become more frequent as the molecular weight is increased. No complete titration curves of globular proteins with molecular weights above 100,000 have been reported so far. 2. Major Anomalies
The most striking anomaly observed among the pK values of Table V is the existence of groups which cannot be titrated at all as long as the native conformation of a protein is retained. This observation is made most frequently for phenolic groups. All of the phenolic groups in ovalbumin, half of those in ribonuelease and ehymotrypsinogen, seven out of eighteen in conalbumin, and about one-third of those in myosin are not titrated even partly at a pH where normal phenolic groups have been converted almost 100% to their anionic form. The simplest explanation is that these phenolic groups are buried in the hydrophobic interior of the protein molecule and thus are inaccessible to the solvent. Only a major change in conformation will then permit the transfer of the dissociable hydrogen ion to a water molecule or OH- ion, and as long as it does not occur the phenolic group will remain intact. This explanation is especially attractive because phenolic groups are more frequently involved than other groups. The uncharged tyrosine side chain happens to be among the most hydrophobic of all the parts of a protein molecule, and, since the phenolic group is uncharged at neutral pH, where the choice of protein conformation is made in the native environment, it is in fact expected to be an “inside” group much of the time. It should be observed that uncharged lysine and histidine side chains are also quite strongly hydrophobic. When these side chains are charged, however, they require the normal hydration for the charged nitrogen group, and that group of the side chain becomes strongly hydrophilic. Since amino groups are charged at neutral pH, we do not expect them to enter the hydrophobic interior of the native protein, and indeed no titration anomaly suggesting buried uncharged amino groups has been reported. Lysine side chains can of course be partly buried, in such a way as to leave the charged group exposed at the protein/solvent interface, and this is undoubtedly the way they often exist. They could also be buried totally even a t neutral pH if they were first converted to their basic form. However, the free energy it would cost, to become dissociated at neutral pH, would be large [it would be equal to 2.303RT (pK - pH)], and would thus vitiate the free energy gained by elimination of the hydrophobic interaction with the solvent. Buried imidazole groups should be more frequent than buried side-chain amino groups, because they are at neutral pH close to their expected pK. Indeed, six imidazole groups in myoglobin and about twenty-two in hemo-
HYDROGEN ION TITRATION CURVES OF PROTEIN
115
globin are not titratable in the native state, and this presumably means that they are buried in the interior in the same way as nontitratable phenolic groups. Another way in which a normally titratable group can be made nontitratable is by formation of a very strong complex. I n the complex of conalbumin with iron, each of the two bound iron atoms is attached to three phenolate groups with an association constant of about lo3"(Warner and Weber, 1953). The six phenolic groups involved in this interaction are not titratable, being held in their anionic form as long as the iron complex retains its stability (Wishnia et al., 1961). These six groups are among the eleven which in conalbumin show normal behavior, and that they are still at the protein/solvent interface in the iron complex is shown by the fact that the iron atoms can be reversibly dissociated from the complex by reagents such as citrate which themselves form strong iron complexes. It is worth noting that the seven phenolic groups which are buried in the interior as uncharged groups in conalbumin remain in the same state in the iron complex. One of the abnormal imidazole groups in myoglobin, and four of those in hemoglobin (one for each iron atom) are also nontitratable by virtue of a covalent bond to an iron atom, these being the imidazole groups by which the hemes are attached to the protein portions of the two molecules. In addition, these groups are buried and inaccessible to the solvent. Two instances have been reported of nontitratable groups which are present on the native molecule in charged form, these being three carboxyl groups in lysozyme and three side-chain amino groups in chymotrypsinogen. No logical explanation exists. The removal of a single charged group from contact with solvent is accompanied by an unfavorable free energy of 10,000 to 100,OOO cal/mole (see footnote 14 in Tanford and Kirkwood, 1957). This unfavorable free energy would be greatly reduced if two oppositely charged groups were removed from contact with solvent as an ion pair, but in the two instances cited here there is no evidence which suggests that there are anomalous groups opposite in charge to the nontitratable groups. It is possible that titratable groups in their uncharged states can be buried in the hydrophobic interior of a protein molecule, but that a reversible change in conformation can bring them to the exterior where they are accessible to the solvent. If the change in conformation is such as to alter the titration characteristics of just a single group, and if no unfolding occurs (an example might be a refolding of a single loop of a polypeptide chain), then the only titration anomaly will be a shift in the pK of the group which is involved: the equation for titration of the group will still be Eq. (14), with the same value of w as applies to other groups. If the buried group is an uncharged acidic group, the anomalous pK (des-
116
CHARLES TANFORD
ignated by an asterisk) is given by (Tanford, 1961c)
*
pKint = PKint
+ log (1 + k)
(18)
where pKint is the normal pK of the group and k is the equilibrium constant which favors the native form a t a pH where the group would normally be uncharged. If the buried group is an uncharged basic group, the corresponding equation is PKrnt = pKint
- log (1 + k)
(19)
It is clear that large changes in pKint can arise in this way. The intrinsic pK of 7.3 found for two anomalous carboxyl groups of p-lactoglobulin (Tanford and Taggart, 1961) has been ascribed to this mechanism. A conformational change is observed to accompany the titration of the groups by optical rotation and other measurements. Although there are two anomalous groups, they are in separate polypeptide chains, and the titration and conformational change occur independently in each chain, so that the equations for a single group apply. If two or more groups are buried in the same hydrophobic region, and they can be brought to the surface only by a conformational change which involves all of the groups together, then a steepening of the titration curve is expected, as well as an anomalous pK. A steepening of the curve is also observed if a change in w accompanies the conformational change which brings buried groups to the surface. These situations were discussed in Section VI, C . 3. Minor Anomalies
Apart from the major anomalies which have already been discussed, all pKintvalues in Table V lie within about 1.0 pK unit of their expected value. Most of them are even closer. There is however a consistency in the direction of deviations from expectation which merits discussion. As the table shows, phenolic and imidazole groups tend to have pKint values larger than expected, and side-chain amino groups tend to have pKint values smaller than expected. In other words, the native structure tends to favor the uncharged form of the phenolic and amino groups; the special problem of the imidazole groups is discussed below. At least as far as the lysine amino groups and tyrosine phenolic groups are concerned, this cannot be considered an artifact of the way the data have been obtained, for the relative ease of dissociation of these groups can be determined unequivocally by comparing the course of titration of the phenolic groups (determined spectroscopically) with the course of titration of side-chain amino plus phenolic groups (determined electrometrically) . Even after discounting the phenolic groups which do not titrate at all, we find that amino groups dissociate at lower pH than phenolic groups in @-lac-
HYDROGEN ION TITRATION CURVES OF PROTEIN
117
toglobulin, serum albumin, and lysozyme. The data of Table I, however, predict that lysine amino groups should have a pK which is 0.8 higher than that of phenolic groups. Since the major part of tyrosine and lysine side chains is strongly hydrophobic, as was discussed above, in connection with the occurrence of nontitratability of phenolic groups, a simple explanation of these minor anomalies is to suppose that the hydrophobic parts of tyrosine and lysine side chains tend to be buried in the interior of the native structure. Although the titratable parts must project outward into the solvent to be titratable, it is possible that they may vary in the precise extent to which they can free themselves from the influence of the hydrophobic region. If they do not project sufficiently far, the uncharged form of the group will become stabilized relative to the charged form, and the pK will be altered accordingly. This same explanation clearly cannot be invoked to account for the observed pK values of imidazole groups, since these pK’s differ from expectation in the wrong direction. This result should perhaps not he taken too seriously since the intrinsic pK’s of protein imidazole groups are relatively imprecise. Moreover, there is some question about the expected pK of an imidazole group (Table I, h ) . On the other hand, the volume changes which accompany dissociation of imidazole groups, to be discussed in Section VIII, are also anomalous, and the over-all conclusion must be that some effect of protein structure on the titration of imidazole groups has so far been overlooked in theoretical treatments of the problem. Some other explanations which have been or could be offered for the smaller deviations of observed pKint values from expected values are the following. ( 1 ) Inadequacy of the Linderstr@-Lang treatment. The approximate way in which electrostatic interactions are treated in this section would have no effect on the estimation of pKint if the point 2 = 0 in fact represents a point where electrostatic interactions vanish. The value of pKint which one obtains by this treatment for a polymer containing only one kind of dissociable group (polyglutamic acid or polytyrosine, for example) must be the correct pKint reflecting only the chemical environment of the dissociable groups, with no contribution from long-range electrostatic interactions. In a protein, however, the distribution of charges may be quite irregular and, when 2 = 0, some groups may well be in an environment rich in positive charges and others may be in an environment rich in negative charges. For these groups the observed pK at 2 = 0 will not be the true intrinsic pK, a positive environment leading to a reduced value for pKint as defined by Eq. (14), and a negative environment to an increased value (Tanford, 1957b). A possible example of this kind of effect occurs in the three basic proteins
118
CHARLES TANFORD
which have been examined in detail. Figure 16 shows the anomalous titration behavior of the carboxyl groups of lysozyme, and, as was pointed out earlier, a possible explanation is the existence of apparently different pKintvalues among the side-chain carboxyl groups. Application of Eq. (14) to the carboxyl groups of chymotrypsinogen (Wilcox, 1961) and ribonuclease (Tanford and Hauenstein, 1956b) leads to a similar result, though the anomaly is not as pronounced as in lysozyme. The fact that all three of these proteins possess a large number of positively charged basic groups in the pH region where carboxyl groups are titrated makes an explanation based on irregular distribution of charged groups an attractive one. It is quite likely that some carboxyl groups of each of these proteins may lie on a region of the surface which is rich in basic groups, and these carboxyl groups would appear to have a lower pKin++than carboxyl groups elsewhere on the molecules. (2) Binding of salt ions. Ions of the neutral salt which is used to adjust the ionic strength of protein solutions may be bound to the protein. If the binding has been measured and corrected for, then no anomaly will result unless the binding sites are the groups whose pK values are being determined (see Section IX, A ) . But if the binding of salt ions occurs without the knowledge of the investigator, and if it is incorrectly assumed that 2, = 2, then an erroneous pKint value is obtained. The error is equal to ~i0.868qniziwhere pi is the number of ions of charge zi bound a t the point 2, = 0. (3) Conformational changes between conformations which d i f e r little in free energy. Equations (18) and (19) have been cited as providing a possible explanation for large anomalies in PKint . With small values of k they could clearly also account for small deviations from the expected pKint values. ( 4 ) Side-chain hydrogen bonds. It has often been suggested that sidechain hydrogen bonds may be responsible for small anomalies in pKint. The suggestion is supported by a calculation made by Laskowski and Scheraga (1954), but their calculation is based on an erroneous value for the heat of formation of a hydrogen bond in an aqueous medium. It is improbable that such hydrogen bonds can in fact have any stability unless the side chains which are involved are removed, at least to some extent, from contact with water. ( 5 ) Salt bridges. It has sometimes been suggested that attraction between positive and negative charges may lead to “salt bridges” (intramolecular ion pairs) which would favor the charged forms of titratable groups, and would account for anomalously low PKint values for carboxyl groups and for high values for amino or imidazole groups. That such attractions should exist in an aqueous environment is improbable. I n any
HYDROGEN ION TITRATION CURVES O F PROTEIN
119
event, anomalously high pK values for amino or imidazole groups have not been observed. All attempts to explain minor variations in pKint by any of the foregoing possibilities must be regarded as purely speculative. Of considerable interest in connection with speculations of this kind is an experiment by Klote and Ayers ( 1957). An S-(p-dimethylaminobenzeneazophenylmercuric) derivative of serum albumin was prepared. Only a single group of this kind can be attached to a serum albumin molecule because the protein contains only a single sulfhydryl group. The titration of the dimethylamino moiety of the group can be followed spectroscopically, because the attached group is a dye which has a different color in its acidic and basic forms. It was found that the group had a pK of about 1.8 in aqueous solution, whereas the same dye attached t o cysteine had a pK of about 3.3. In 8 M urea, on the other hand, the protein-bound dye had a pK of 3.3, and the cysteine-bound dye a pK of 3.4. This result, and the corresponding heats and entropies (KlOt5 and Mittleman, 1962) can be interpreted by several mechanisms, discussion of which is out of place here. (The interpretation of the data is hampered by the fact that native albumin, under the conditions of the experiment, is wholly or partly unfolded.) The really important conclusion to be drawn is that the work points out the possibility of new and imaginative experiments which may prove more instructive at the present time than further speculation concerning the data already in existence. 4. E$ect of Ionic Strength on Intrinsic pK’s The value of pKint in Eq. (14) should depend on ionic strength. The electrostatic term in this equation includes the effect of ionic strength on the interaction between charges, but not the effect on the chemical potential of the isolated dissociating group in its acidic and basic forms, and this effect therefore remains a factor in pKint. The effect of ionic strength is to stabilize charged groups, and pKint should therefore decrease with increasing ionic strength for dissociation of an uncharged acidic group, whereas it should increase for dissociation of positively charged groups. The magnitude of the effect at low ionic strength should be roughly the same as 6iT In y for a univalent ion (y being the activity coefficient), so that, a t 25°C in water, there should be a difference of about 0.05 between pKint values a t ionic strengths 0.01 and 0.15, and a difference of about 0.08 between values a t ionic strengths 0.15 and 1.00. The experimental error in determining pKjnt usually exceeds these differences. E. Heats and Entropies of Dissociation If values of pKint for a dissociable group are measured at several temperatures, heats and entropies of dissociation can be determined from the
120
CHARLES TANFORD
temperature-dependence. An alternative procedure is to obtain apparent heats of dissociation by Eq. ( I ) from regions of the electrometric titration can curve where a single kind of group predominates. The value of then be determined from a value of pKint at a single temperature. The latter procedure introduces an uncertainty because apparent heats of disTABLE VI Heats and Entropies of Dissociation i n Natiee Proteins Type of group Carboxyl Normal valuese Conalbuminb Serum albumin" Phenolic Normal values" Conalbuminb Lysozymed Ribonucleasee Serum albuminc Imidazole Normal values" Conalbumin6 Amino Normal values' Conalbumin* Iodinated insulin, Ferriheme water M yoglobing Hemoglobin9 ~
AH
ASiO,t
(kcal/mole)
(cal/deg/mole)
0 to 1 2 -2
-18 t o -21 - 13 - 12
6 8 6 to 7
-24 - 15
7 11.5 7 8 13 t o 14 I1 13.4 6 4
-26 t o -29 -22 -9 -8
-3 -3 to -8 -6
-s
-22
-27
~~~~~~
Edsall and Wyman (1958), Edsall (1943). Wishnia et aE. (1961). Tanford et al. (1955b). Donovan et al. (1961). * Tanford et al. (1955a). Gruen et al. (1959a). 0 George and Hanania (1952, 1953, 1957). No model compounds are available on which 60 base normal values. a
6
sociation may differ by 1 or 2 kcal from true AHint values obtained from the temperature coefficient of pKint . However, pKint values themselves are not very precise, as we have pointed out, so that a AH value obtained from their temperature-dependence is also quite imprecise. Some heats and entropies obtained in this way are listed in Table VI, and comparative values for model compounds are included. The uncertainties in the experimental determination of these values, coupled with
HYDROGEN ION TITRATION CURVES O F PROTEIN
121
the uncertain meaning of pKint when obtained by the empirical procedures of this section, suggest that no interpretation of the data is warranted. It should be noted in particular that the strikingly anomalous figures for ionization of the phenolic groups of serum albumin, which have been the subject of considerable speculation in the past, are now known to be without significance. They are based on an apparent AH, obtained in a pH range where serum albumin is now known to undergo unfolding, and must reflect in part the thermodynamic parameters for the unfolding process.
VII. MOREEXACT THERMODYNAMIC ANALYSIS The major deficiency of the Linderstrprm-Lang theory of titration curves lies in the calculation of the electrostatic interaction between charged groups. It is an oversimplificationto regard all charges as smeared evenly over the surface of the molecule. In reality these charges are discrete, and a given charged group is influenced much more by other charged groups which lie close to it than by more distant groups. The exact location of neighboring charged groups will thus necessarily have an important effect on the titration curve, as we have already noted qualitatively in Sections VI, C and D. An attempt has been made (Tanford and Kirkwood, 1957) to include this effect in the equation for the titration curve. We assume as before that many groups will have the same intrinsic pK, but no longer assume that all groups with the same intrinsic pK will be titrated simultaneously, allowing for variations which depend on the specific electrostatic interactions to which each group is subject. Since the theory has been described in three separate places (Linderstrgm-Lang and Nielsen, 1959; Rice and Nagasawa, 1961; Tanford, 1961a), in addition to the original publication, we shall not describe it here. It suffices to say that the theory involves calculation of the concentrations of individual species P, PH, . . -,PH, , . PHn-I , 1’15, , where v is the number of dissociable protons per molecule in a given species and n the maximum number of such protons which the molecule van contain. Each species is further subdivided into subspecies PHI”, PHf”’, PHI”, the superscripts indicating different arrangements of protons among groups which differ in intrinsic dissociation constants. There may he v1 protons bound to sites of class 1, vz to sites of class 2, and in general v I to sites of v j = v. Each different set of values for classj, with the proviso that Vl , VZ 1 . . , v j leads to a subspecies identified by a different superscript (i). It should be noted that each subspecies still has many possible distinguishable configurations, for the v j protons on sites of class j can be distributed in general in different ways over the total number ( n j ) of groups which belong to classj. We now let k:’) represent the equilibrium constant for the reaction
122
CHARLES TANFORD
P
+ vH+
PHP'
The value of this constant will be the product of an intrinsic term independent of configuration, and an electrostatic term which will depend on configuration, and is averaged by an appropriate statistical procedure over all configurations. The basis for the calculation of the electrostatic term in a given configuration is the generalized multicharge model of Kirkwood (1934). It depends on knowing the precise positions of all charges on the protein molecule, and also on knowing an effective distance ( d ) of closest approach of solvent to each charge. Since it seems improbable that two similar groups can have the same intrinsic pK without also being equally accessible to the solvent, it is likely that the parameter d has the same value for all groups of a given class. Moreover, if the intrinsic properties of titratable groups on protein molecules are the same as the properties of such groups on simpler molecules, then the parameter d should be the same on protein molecules as on simple molecules. Consideration of appropriate simple molecules (Tanford, 1957b) suggests that at 25°C in water d = 1.0 A for all pertinent groups. With this value of d one gets over-all electrostatic effects of about the same magnitude as would be calculated by the Linderstrplm-Lang treatment (Tanford, 1957b), but one also predicts variations whenever the distribution of charged groups over the protein molecule is an uneven one. The ways in which such variations might account for deviations from the Linderstrplm-Lang theory have ,already been brought out. There is a typographical error in three of the equations of one of the references cited above (Tanford, 1961a, Eqs. 30-10 to 30-12). The correct form of these equations should be obtained as follows. The concentration of species PH;" at any pH is
[PHI"']= ~L"[P]u;I+
(20)
where square brackets represent concentrations. The concentration of all species PH, is
cy
The total protein concentration is simply (PH,), the sum extending from v = 0 to v = n so that the average number of bound protons per molecule ( J ) is
Y
U.i
One can also calculate the average number (rj) of protons bound at any pH
HYDROGEN ION TITRATION CURVES OF PROTEIN
123
specifically to groups of class j,
where
vj
is the number of protons bound to groups of class j in the species
PHS". One can also compute the average of any desired function of
v.
For example,
The preceding equations have not yet been used as basis for the analysis of any actual titration curve of a protein. It should be feasible soon to apply the theory to the titration curve of myoglobin, the complete threedimensional structure of which should be available soon (Kendrew et al., 1961). This will permit exact localization of all titratable groups on the protein molecule and, hence, the theoretical evaluation of the kJi' from assumed intrinsic pK's. It is not to be expected that these kii' values will accurately reproduce the titration curve, but it is to be expected that the cause of observed deviations will be relatively easy to determine. Essentially three possibilities would be looked for in such a first application of the theory. (1) That the theory itself is incorrect. (The model on which it is based is still very crude even though exact locations of discrete charges are specified.) It is likely that an error in the theory can be corrected by a small change in the parameter d, from the value of 1.0 A which has been used for computations so far (Tanford, 1957b). (2) That some groups do not have expected pKint values. If the exact structure is known, it should be possible to identify such groups. (As was previously mentioned, six imidazole groups of myoglobin are already known to be highly anomalous. They will presumably be identifiable on immediate inspection of the final structure since they should be in positions that are inaccessible to solvent.) (3) That the structure determined by X-ray diffraction of protein crystals is not exactly the same as the structure in solution. To test this possibility one would have to make a guess as to the probable location of possible structural changes (e.g., at points of contact between neighboring molecules in the crystal), and would then determine whether they improve the agreement between calculated and experimental titration curves. It is likely that a computer program will have to be devised to perform actual calculations for a molecule as complicated as myoglobin.
124
CHARLES TANFORD
VIlI. VOLUMECHANGES ACCOMPANYING TITRATION The change in volume which accompanies the titration of acidic and basic groups of proteins was measured by Weber and Nachmansohn (1929; also Weber, 1930) more than 30 years ago. The results were approximately in accord with the volume changes measured on suitable model compounds, and they are of historical importance because they helped demonstrate that proteins are amphoteric ions, containing a t neutral pH charged carboxyl and amino groups which do not differ greatly from similar groups on small molecules. New and more precise measurements of volume changes have been made recently by Kauzmann and co-workers (Kauzmann, 1958; Rasper and Kauzmann, 1962; Kauzmann et al., 1962). These measurements show TABLEVII Volume Change for the Binding of Hydrogen Ions by Cmboxylate Groups at 30°C and Ionic Strength 0 . 1 ~ 5 ~
a
Protein
A V (ml/H+ ion bound)
Lysozyme Ovalbumin Ribonuclease Serum albumin (above pH 4) Serum albumin (below pH 4)
10
12 11 11 -5
Data from Kauzmann (1958), Rasper and Kauzmann (1962).
that there are in fact significant differences between the behavior of proteins and model compounds. Kauzmann and co-workers measured AV for the addition of Hf ions to proteins below pH 5 . Normally only carboxyl groups are titrated in this range of pH, so that the results represent AV values for the reaction -COO-
+ H+ + -COOH
In simple molecules, the AV for this reaction is sensitive to the environment of the carboxyl group. Normal values lie between 10 and 14 ml per mole of hydrogen ions bound. If there is a positively charged group near the carboxyl group, AV is decreased to 6 or 7 ml, and near a negatively charged group AV rises to 15-20 ml and even larger values. If anomalous values were obtained for proteins they could thus be readily rationalized. In fact, as the results of Table VII show, anomalous results are not obtained, except for serum albumin, below pH 4.3. As serum albumin undergoes configurational changes below pH 4.3, the A V value must reflect the volume change which accompanies the conformational change, in addition to that which
HYDROGEN ION TITRATION CURVES OF PROTEIN
125
accompanies hydrogen ion binding. An anomalous value is therefore not unexpected. Kauzmann and co-workers also measured AV for the addition of OH- ions to proteins from pH 5 to pH 10 or above. These values mostly represent AV for the reactions -COOH
+ OH+ OH-
--t
-COO-
+ HzO
+ H20 -+ -NH2 + HzO -NH: + OH-CJIaOH + OH- -+ --Ce,H50- + HzO -1mHf
+ -1m
where I m represents the imidazole group. The carboxyl groups make a significant contribution only at the lowest part of the pH range studied. The phenolic groups make a contribution only at the highest pH values, and so do lysine amino groups. Terminal a-amino groups and imidazole groups are titrated entirely within the region of pH which was studied. The number of a-amino groups, however, is always small, so that the measured AV values represent primarily the volume change accompanying titration of imidazole groups. In small molecules containing basic nitrogen atoms, the value of AV for the reaction -NH+
+ OH- -+ -N + H20
is 23 to 25 ml per mole of OH- ions added. In contrast to what one finds in the case of carboxyl groups, this value is insensitive to the nature of other groups which may be present on the small molecule. Even closely located charges have little effect, and there is no significant difference between imidazole groups, primary amines, secondary amines, and tertiary amines. Therefore it is of considerable significance that the A V values found in proteins are always substantially below the expected value of 23 to 25 ml, as the results in Table VIII show. No explanation for this phenomenon exists as yet. The more nearly normal value of AV found for pepsin, which is denatured in the pH range of the measurements, suggests that the anomaly is associated only with the native state of the protein. It is possible then that the anomaly is caused by the large hydrophobic groups to which the basic nitrogen atoms, both of imidazole groups and of lysine amino groups, are attached. These hydrophobic groups might tend to pull the entire group towards the hydrophobic interior of t,he protein molecule, diminishing the amount of tightly bound water about the group in its acidic form, and thereby decreasing the value of AV, which must
126
CHARLES TANFORD
reflect in part the bound water which is released when the group is converted to its uncharged basic form. If this were the correct explanation, we should also expect a decrease in pKint , since the acidic form of imidazole and amino groups would become less stable if it could not be hydrated to its normal extent. As Table V shows, there is in fact a tendency for the pKintof amino groups to be below expected values. The pKlntvalues for imidazole groups however are above rather than below expected values. Moreover, the amino groups of lysozyme do not have an anomalous pKint , so that AV for lysozyme should approach a normal value at higher pH, where these groups begin to make an important contribution to the titration curve. In fact the opposite result TABLEVIII Volume Change for the Binding of Hydroxyl Ions by Proteins (Primarily by Imidazole GTOOUPS) at 30°C and Ionic Strength 0.16"
a
b
Protein
Range of pH
Chymotrypsin Hemoglobin Lysosyme Lysoz yme Ovalbumin Oval bumin Pepsin Ribonuclease Serum albumin
7.5-11 5.5-7.5 7.5-10.5 5.2-6.8 6.8-10 7-10 5.5-10 6-10
8-8
AV (ml/OH- ion
bound)
17 18.6 17 15 6.7 16 20b 16 17
Data from Rasper and Kauamann (1962). Pepsin is denatured in the pH range covered.
is obtained, and AV falls to even lower values than are observed for other proteins. Table VIII shows that the volume changes for ovalbumin between pH 5.2 and 6.8 are especially low. This cannot be explained in a simple way, e.g., by the fact that there are phosphate groups in ovalbumin which titrate in this range of pH (because only four of the twenty groups which are titrated are phosphate groups). There is also no evidence that ovalbumin undergoes a significant conformational change between pH 5 and pH 7. It is clear from this discussion that the measurement of volume changes which accompany titration of proteins and of model compounds represents a potentially fruitful area of research. It would be of special interest to have available AV values for synthetic polypeptides (or for naturally occurring substances such as a-corticotropin) , which exist in aqueous solutions in a randomly coiled conformation.
HYDROGEN ION TITRATION CURVES OF PROTEIN
127
IX. MISCELLANEOUS TOPICS
A . Binding of Ions Other Than Hydrogen Ions If a titratable group on a protein molecule can combine with substances, other than hydrogen ions, which may be present in a protein solution, then its titration properties will be altered. If the combining substance is present a t a thermodynamic activity a M, and if it combines with the basic form of the titratable group only, the equilibrium constant for association being k M , then the apparent hydrogen ion dissociation constant Kappof the group becomes
Kapp= K(1
+ kaM)
(25)
where K is the true dissociation constant. If, on the other hand, the combining substance reacts only with the acidic group, Kapp
= K/(1
+
~ U M )
(26)
In these equations both K and k will depend on pH through interactions with other charges, the effect on K being given in the LinderstrGm-Lang approximation by Eq. (3). The effect on k depends on the charge of the combining substance. If combination requires two titratable sites on the protein molecule, more complex equations are of course obtained, as discussed elsewhere (Tanford, 1961a). Most easily recognized are those examples in which kax is very large. In that case, the group in question will be combined with the combining substances throughout the normal range of titration, and the group will be titrated with a highly anomalous pK, i.e., Kapp = KkaM or K a p p = K / k a M , the titration being in fact a substitution of H+ or OH- for the combining substance, It is an association of this type which is responsible for the large displacements observed, for example, when zinc combines with carboxypeptidase (Fig. 7 ) . Weak binding (small haM)could produce smaller effects, leading to small changes in observed pK values. However, no example ascribable to this cause has been found, and none of the variations in pKint shown in Table V can be reasonably ascribed to such ion binding. If ions are bound to any part of a protein molecule other than the groups which are being titrated in a particular pH range, they will not directly influence the dissociation of hydrogen ions. They will influence it indirectly however by contributing to the electrostatic interactions. In terms of the treatment of Section VI this influence enters solely into the value of the net molecular charge If binding occurs, but is not recognized, the values used in the analysis of the titration curve will be false, leading to false values of w and pKi,t, as discussed in Sections VI, C and D.
z
z.
128
CHARLES TANFORD
B. The Isoionic Point An isoionic solution is defined as a solution which contains only protein ions of average charge H+ ions, and OH- ions. No other ions may be present. An isoionic solution can be prepared by means of a mixed-bed ion-exchange column (Dintzis, 1952), or by electrodialysis, and only for proteins which are soluble in salt-free water. (Refer to Section 111, B ) . The condition that any solution must be electrically neutral requires that in an isoionic protein solution, containing protein a t a concentration Cp moles/liter,
z,
CpZ
+
CH+
=
(27)
COH-
where C,+ and CoH- also represent molar concentrations. be noted that Z = ZH, and that
It should also
KL (28) where KL is the apparent ionization constant of water, which will be equal t o the thermodynamic ionization constant K,, under most, condit,ions, since activity coefficients are essentially unity unless the total ion concentration becomes large. A third equation applicable to the isoionic solution is the titration curve of the protein, which may be written for the present purpose as C,+Co,-
Z,
=
= unique function of
C,+
=
cp(C,+)
(29)
This equation will usually be independent of Cp as long as Cp is reasonably small. Only if a protein is subject to aggregation will there be a dependence of Eq. (29) on Cp . Equations (27-29) can be solved to yield values for CH+and for 2, (or 2 ) a t the isoionic point. We get
c@(c,+)= K ~ / c ~-+c,+
(30)
zH
from which it is clear that CH+and hence do not have unique values, but depend on protein concentration. It is for this reason that we earlier (Section 111, B ) chose the point a t which 2, = 0 as a reference point for titration curves, rather than the isoionic point. It may be noted that the value of 2, a t the isoionic point can be cslculated by Eq. (27) if the pH of the isoionic solution is measured, even if t,he equation for the titration curve is not known. Setting pH = -log C,+ we obtain the close approximation
Z
2,
(KL/c,+
- cH+>/c~
(31 1 It is evident from this equation that ZH will not bt: significantly different from zero if the measured p H lies between pH 5 and p H 9, and if the protein =
=
HYDROGEN ION TITRATION CURVES OF PROTEIN
129
concentration is of the order 1 gm/100 ml, corresponding to C, of order For salt-free isoionic p-lactoglobulin, for example, the pH at 25°C is 5.39. At a concentration of 1 gm/lOO ml, Cp is 2.8 X lo-*, so that 2, = 0.015. We consider now the addition of a neutral salt to an isoionic protein solution. Equation (27) still applies if 2 is replaced by 2, , since the charges provided by the neutral salt balance each other, regardless of whether salt ions are bound to the protein. Thus 2, can still be calculated by Eq. (31). It is generally found that the pH of an isoionic solution is changed when a neutral salt is added. The theory of this effect has been discussed by Scatchard and Black (1949), with the assumption that 2, is negligibly different from zero. The analysis is within the framework of the Linderstrcim-Lang treatment, and thus only approximate. Scatchard and Black give the following equation for the effect of ionic strength on the isoionic pH if no binding of ions occurs:
In this equation, I is the ionic strength, and w is given by ICq. (4). The derivatives of 2, are obtained directly from the titration curve, and must be evaluated at 2, = 0. The effect of ionic strength on the isoionic pH, which is predicted by Eq. (32), is usually extremely small, and, in particular, is often much smaller than the experimentally observed variation. Large effects of ionic strength on the pH have been generally considered as evidence for binding of the ions of the added salt. If the salt is composed of cations of charge x+ and anions of charge x- , respectively, and if a+ and T- are the average numbers of each kind bound at a given concentration of the salt,
-
z
= B+Z+
+
(33)
a-2-
(where x- is of course a negative number). Since 2, = 0 at each ionic strength, the degree of dissociation of all groups remains unchanged, so that Eq. (14) gives for the pH, at any ionic strength, pH
=
- 0 . 8 6 8 ~ (a+z+
+ a d - ) + constant
(34)
At zero ionic strength, a+ and F- must both be equal to zero, so that pH
-
(pH)I=o = - 0 . 8 6 8 ~ (a++
+ PA-)
(35)
where w is the value appropriate to the ionic strength at which the pH is measured. Equation (35) shows that the pH will fall with increasing I if cations are bound, but will rise if anions are bound.
130
CHARLES TANFORD
Some experimental data on the effect of KC1 on the pH of isoionic protein solutions are shown in Fig. 19. (All the isoionic pH values lie close enough to pH 7 so that 2, = 0 is within experimental error.) The data indicate that C1- ions are bound to serum albumin, that K+ ions are bound to p-lactoglobulin, and that little or no binding to hemoglobin occurs, the small changes in pH observed for this protein being of the order of magnitude predicted by Eq. (32). These results agree with results obtained by direct binding studies in the case of serum albumin (Scatchard et al., 1950, 1957) and of hemoglobin (Nozaki, 1959; Scatchard et al., 1962).
I
I
0.2 (Concentration) "e
-0.b
I
I
0.4
FIG.19. The effect of KC1 on the isoionic pH of three proteins. The serum albumin data are from Scatchard and Black (1949), the hemoglobin data from Noeaki (1959), the &lactoglobulin data from Noeaki et al. (1959).
Direct binding studies for p-lactoglobulin, however, show a contrary result. Saroff (1961) found that K+ is bound a t p H 7, but not at the isoionic pH. The discrepancy is unresolved at present.
C. Charge Fluctuations Throughout this paper we have so far discussed only the average number of hydrogen ions bound to or dissociated from a protein molecule a t any pH. It should be pointed out that individual protein molecules may a t any given instant have ZH values which can be larger or smaller than the average value 2,. Edsall (1943) has shown for example that in hemoglobin at pH 6.4, where 'v 0, 4 % of all molecules have Z , = +3, 9 % have 2, = +2,17% have ZH = $1, 22% have ZH = 0, 21 % have
zH
131
HYDROGEN ION TITRATION CURVES O F PROTEIN
-
1, 14 % have 2, = -2, 7 % have 2, = -3, with smaller fractions having 2, > +3 or < -3. A given molecule will have a fluctuating charge, varying about the mean charge ZH = gH. Titration curves cannot measure these variations in charge. Only is determinable. However, theoretical equations relate charge fluctuations to the titration curve, so that they can be calculated. One obvious result , and this is in of charge fluctuations is that (ZH)2is not the same as fact the parameter by which the spread of molecules among different values of 2, is usually characterized. The difference between and (ZH)2is identical with the difference between 3 and (5)' given by Eqs. (22) and (24). Differentiating Eq. (22), we get 2, =
z,
As is shown elsewhere (Tanford, 1961a), the left-hand side of Eq. (36) may be evaluated by use of the LinderstrGm-Lang equation, giving
-
(ZH)2
- .j>
= Cnj~j(1 i
(37)
where nj is the number of groups of class j, and xj the degree of dissociation of groups of that class, as given by Eq. (6). It should be noted that Eq. (36) is given incorrectly in the reference just cited (Tanford, l96la).
X. RESULTSFOR INDIVIDUAL PROTEINS A . Chymolrypsinogen Titration curves for bovine a-chymotrypsinogen have been determined under a variety of conditions by Wilcox (1961). The results are summarized in Table IX. The spectrophotometric titration of the phenolic groups is shown in Fig. 4. It is seen that all four phenolic groups are titrated together in 6.4 M urea, whereas only two are titrated in the native protein. Even these two have sufficiently different pK's so that the titration of one is essentially complete before that of the second has begun. The count of side-chain amino groups corresponds to the analytical figure for lysine side chains in a denaturing solvent (8 M urea). I n the native protein, however, three of the thirteen lysine groups cannot be observed to titrate. The maximum positive proton charge ( ZN') is however the same in the native and denatured states, within the uncertainty of about
132
CHARLES TANFORD
f l in the determination of the quantity in the native state. This means presumably that the native structure stabilizes the charged form of three lysine side chains, preventing their dissociation to an uncharged state. The count of “carboxyl” groups is larger than expected from analysis. No explanation has been offered. The usual explanation for this observation is that the analytical figure for free carboxyl groups is too small because of too high an estimate for amide nitrogen. This explanation is unlikely here because asparagine and glutamine were determined by direct analysis. TABLEIX Titration Data for a-Chymotrypsinogenil
Type of group
a-Carboxyl Side-chain carboxyl Imidaeole a-Amino Phenolic Side-chain amino Guanidyl ZNi ~
Titration Analysis
Native protein
2 1
4 13 4 20
(native protein)
14
-
3
3
G.7c
2d
4 -13 10.5
9 . 7 , 10.6” -
-14
l j
Denatured proteinb
[>Kin&, 25°C
10d -20
_.
-
~~~
a Molecular weight is 25,000. Titration data are from Wilcox (1961), analytical d a t a from Wilcox et al. (1957). * Data were obtained in 4 M guanidine hydrochloride and in 6.4 M or 8 M urea. c The same P K , , ~was assigned to all three groups which titrate in the neutral region. d More phenolic and amino groups are titrated slowly above pH 12 as the protein becomes denatured. See Fig. 4. 6 Each phenolic group has a different pK.
Wilcox (1961) has also obtained a partial titration curve for a guanidinated derivative of chymotrypsinogcn (lysine side chains converted to homoarginine). The only major difference was the disappearance of the ten side-chain amino groups. No spectrophotometric titration was carried out, but the electrometric titration curve in the alkaline region suggests that the two phenolic groups which are inaccessible to titration in the native protein are still inaccessible in the guanidinated derivative. The titration curve of native chymotrypsinogen is reversible between pH 2.5 and pH 11. Analysis hy the methods of Section VI shows that the neutral pH region can be described by a single intrinsic dissociation constant for all three groups which titrate in the region, together with a w
HYDROGEN ION TITRATION CURVES O F PROTEIX
133
value of 0.065 (at ionic strength 0.1). As Table 111shows, this is a reasonable value for a compact globular protein of the size of chymotrypsinogen. The carboxyl region of the titration curve cannot be described by a single pK and a reasonable value of w. It is likely that the explanation lies in the fact that chymotrypsinogen is rich in basic nitrogen groups (isoionic point is at pH 9.4),so that the carboxyl groups are titrated in an environment rich in positive charges. If these charges are unevenly distributed with respect to the carboxyl groups, then the latter will appear not to have identical pKint values, as was discussed in Section VI, D. The major problems which this titration curve poses are identification of the two unexplained groups in the carboxyl region, and an explanation for the failure to titrate three of the thirteen side-chain amino groups in the native protein.
B. Chymotrypsin Havsteen and Hess ( 1 9 6 2 ) have studied the titration of the phenolic groups of a-chymotrypsin and of its diisopropylphosphoryl (DIP) derivative. The result is similar to that observed with chymotrypsinogen (Fig. 4) in that oiily two of the four groups are available for titration in the native protein, as well as in the DIP derivative. The data do not have sufficient precision to determine whether the two titratable groups have different pK’s, as they do in chymotrypsinogen, but the wide spread of the titration curve suggests that they do. All four tyrosyl groups are titrated normally in solvents which denature chymotrypsin. C. Collagen Fibrils Martin et al. (1961) have shown that the titration curve of suspensions of freshly precipitated collagen fibrils depends markedly on the pH at which aggregation to fibrils takes place. As this pH increases the count of “imidazole” groups decreases, and there is a corresponding increase in the number of groups titrated in the carboxyl region (in which the fibrils go into solution). There is a correlation between the titration characteristics and the occurrence of periodic banding in the fibrils, the incidence of thc latter increasing with pH. It is suggested therefore that the banded regions contain uncharged imidazole groups which cannot be titrated as long as the fibrils remain intact.
D. Conalbumin The titration curve of conalbumin (Wishnia et al., 1 9 6 1 ) is complicated by time-dependent reactions in the acid range. These reactions, however, appear to influence only the shape of the titration curve, and the count of
134
CHARLES TANFORD
various kinds of groups presented in Table X is taken to apply to the native and modified protein alike. The agreement between the titration count and the result expected from analysis is remarkably good, the only discrepancy being in the figure for the maximum proton charge ( E N + ) , where the titration value is seven less than the analytical value for the total of all basic nitrogen groups. No explanation for the discrepancy exists. The small difference between the number of observed “carboxyl” groups and the analytical value is well within the experimental error of the latter. A spectrophotometric titration of phenolic groups was carried out. The TABLEX Titration Data for Cona16umina ~
Type of group
a-Carboxyl Side-chain carboxyl Imidazole a-Amino Phenolic Side-chain amino Guanidyl ZN+
Number of groups
Analysis
8t
Titration 86
131
14
19 52 33 99
11
52
-
92
PKnt
25°C
:1
-
5°C
4.54 7.20
9.41
9.64
-
-
9.85 10.20 -
-
aMolecular weight is 76,600. Titration data are from Wishnia et al. (1961). Analytical data are as given by Wishnia et al. (196l), derived from studies by Lewis et al. (1960). b Eleven groups are titrated in the native protein, the remaining seven are titrated upon denaturation.
results are qualitatively similar to those for ribonuclease shown in Fig. 11, except that the total number of groups per molecule is larger. Eleven of the eighteen phenolic groups are titrated reversibly in the native protein, another seven appear with accompanying denaturation, and their appearance can be delayed by reducing the temperature. An interesting example of difference counting is provided in the conalbumin study. Conalbumin can bind two iron atoms very tightly and it had been concluded earlier (Warner and Weber, 1953) that each iron atom might be bound to three phenolic groups, which would remain ionized at all pH’s where the iron complex is stable. This earlier conclusion was firmly established by spectrophotometric titration of the iron complex. Only five phenolic groups were titrated between pH 8 and 12, compared to eleven in native iron-free conalbumin. The result shows, incidentally,
HYDROGEN ION TITRATION CURVES O F PROTEIN
135
that the seven phenolic groups inaccessible to titration in the native protein (presumably buried as un-ionized groups) are still inaccessible in the iron complex. At 25°C the titration curve was reversible and independent of time between pH 4.2 and pH 11.2. By use of a flow method the reversible portion could be extended to somewhat lower pH. At 5°C it was possible to extend it on the alkaline side to pH 12. The treatment of Section VI was applied to the reversible region of the curve. It was possible to account for the major part of this region by using a single value of w, which was somewhat below that calculated by Eq. (4)) but the difference is only of the order of 25% and does not suggest that conalbumin is not a globular protein in its native state. Below pH 4 and above pH 11.2 (at 25OC) there is a marked decrease in electrostatic interaction (resembling that shown for serum albumin in Fig. 17)) indicative of a reversible transition, presumably to an expanded conformation. It is interesting that the over-all change in conformation which takes place in acid solution is by these measurements found to occur in two parts: a rapid and reversible part when 2, N 20 and then a slower irreversible reaction when 2, ‘v 32. The intrinsic pK values deduced from the part of the curve which could be fitted with a constant w are shown in Table X. The values are comparable to those found for other proteins (Table V) . The heats and entropies for dissociation are given in Table VI and are also found to be unremarkable. The over-all conclusion is that the titratable groups of conalbumin are largely accessible to the solvent, except for the phenolic groups discussed earlier.
E. a-corticotropin a-Corticotropin ( ACTH) has a molecular weight of only 4541 and should contain less than twenty titratable groups. The titration curve (of sheep corticotropin) has been studied by LBonis and Li ( 1959), the results being shown in Table XI. The corticotropin is usually isolated as thc trichloroacetate, and the titration was first carried out on this salt. The count of groups was in accord with expectation, except for the presence of one anomalous group in the neutral region. The corticotropin was then deionized by ion exchange, but the anomalous group remained. Moreover, two additional anomalous groups appeared as a result of deionization, one being a “carboxyl,” the other a “side-chain amino” group. LBonis and Li present evidence that the neutral anomalous group is due to an impurity. An unusual feature of the titration curve of corticotropin is the relatively low pH for titration of the guanidine groups. The titration of these groups
136
CHARLES TANFORD
is not visually distinct from the titration of the side-chain amino groups, though the distinction becomes apparent on mathematical analysis of the alkaline region of the curve. The titration curve of beef corticotropin (LBonis and Li, 1959) and a partial curve for pork corticotropin (Danckwerts, 1952) are essentially identical to the curve for the sheep product. The titration curve is reversible and has been analyzed by the methods of Section VI. The values of w which are obtained from titration of the phenolic and amino groups at ionic strength 0.1 are near 0.03. A somewhat TABLEXI Titration Data f o r a-Corticotropin" Number of groups
Titration
Type of group
pKint
Trichloro- Deionized acetate salt sample a-Carboxyl Side-chain carboxyl Imidaaole a-Amino Phenolic Side-chain amino Guanidyl ZNi
7
8
1 1 l i 2
2
2
3 4 9 }
7"
8"
-
5 or 6*
3
10
a These dat>aare for sheep corticotropin, with molecular weight 4541. Titration data from LBonis and Li (1959);analytical d a t a from Li et al. (1955). * There are 5 residues of glutamic acid and 2 of aspartic acid. Either one or two of them may be present as arnides. c The titration of guanidyl groups overlaps t h a t of amino groups, so t h a t they cannot be differerhiated on inspection.
higher value (0.0'3) is obtained from the titration of carboxyl groups, but it is probable that this is in part due to the fact that some of the carboxyl groups are bunched together on the corticotropin chain: the sequence -Gln.Asp.hsp.Clu.occurs at one point and accounts for more than half the carboxyl groups. As explained in Section VI, C , such close juxtaposition of titratable groups is expected to lead to a high value of w. In any event, the value of 0.03 is far below the value of w expected on the basis of the compact sphere model (Table III), and thus the titration curve clearly indicates that a-corticotropin does not have a globular structure. This conclusion agrees with the results of other measurements which all indicate that this molecule has a flexibly coiled conformation.
HYDROGEN ION TITRATION CURVES O F PROTEIN
137
As might be expected of a molecule with an essentially random structure, the pKint values which are given in Table X I are essentially normal. I t is especially noteworthy that the phenolic groups have a lower pKint than the side-chain amino groups, as is expected from the data of Table I. In most proteins with a typical globular structure this order is reversed (see Table V). The pKint values given in Table X I for the imidazole and aamino groups must be regarded as quite uncertain, because of the anomalous group which is titrated in the same range of pH.
F. Cytochrome c Titration curves of oxidized and reduced cytochrome c (from beef heart or horse heart) have been determined by Theorell and ikeson (1941) and by PalBus (1954). A major problem concerns the titration of the imidazole groups, of which there are three in the protein (Margoliash et al., 1962). According to Theorell and Akeson, there are two groups titrated with a pK approximately that of imidazole groups. Since cytochrome c has no free a-amino group, a reasonable interpretation is that both of these are in fact imidazole groups, and that the one imidazole group which is not titrated is linked to the heme iron atom of the protein. This result is compatible with the proposal of Margoliash et al. (1959)) that the two basic groups coordinated to the heme iron atom are one imidazole group and one lysine amino group. Theorell and ikeson, on rather weak evidence, suggested that one of the groups titrated in the neutral region is actually not an imidazole group, and PalBus, with more accurate data, comes to the same conclusion, demonstrating quite convincingly that just a single group with pK near 7 (actually pK = 6.8) is being titrated. This would seem to favor the hypothesis that two imidazole groups are coordinated to the heme iron atom. An undecapeptide which can be obtained from peptic hydrolysis has also been subjected to titration (PalBus et al., 1955). This peptide contains the heme group, attached to the polypeptide chain through two half-cystine residues, and possesses one histidine residue, which is thought to be covalently linked to the iron atom. There is not a unique way of interpreting the titration curve. It is possible to arrive at an interpretation which assigns a pK of 3.5 to the imidazole group, but it must be regarded as specultLtive. Seventeen groups arc titrated in the carboxyl region on the basis of a molecular weight of 12,500. This figure agrees with expectation. There are twelve side-chain carboxyl groups and one terminal a-carboxyl group (Margoliash, 1962). The other four titratable groups represent the two free propionic acid groups of the heme, and the two iron-linked basic groups, which should be freed from their bonds to iron, and titrated, when
138
CHARLES TANFORD
the pH becomes sufficiently low. (This process does not lead to separation of the heme from the protein in the case of cytochrome c, because the heme is linked to the protein through its side chains, which form thio-ether bonds to the two half-cystine residues mentioned earlier.) The absorption spectrum of ferricytochrome c changes with pH, and indicates that several hydrogen ions are dissociated with accompanyin effect on the electronic structure of the heme iron atom (Theorell and keson, 1941;Boeri et al., 1953). The results are complicated by an influence of chloride ion, and a simple interpretation of all the changes is not possible. However, a change involving two H+ ions, titrated simultaneously at pH 2.1, appears to reflect the dissociation of the basic nitrogen groups from the heme iron atom. In the undecapeptide studied by Paleus et al. (1955), the corresponding change occurs when Hf ions are added or removed at pH 3.4 and 5.8.
f
G . Fetuin Fetuin is a glycoprotein which has titratable groups associated with its carbohydrate moiety (sialic acid) in addition to those present on the protein. Spiro (1960) has determined the titration curve of the native protein, as well as that of a preparation from which sialic acid had been removed. The group count differed only in the number of groups assignable to sialic acid. In particular, the value of 2 N + was the same for both proteins. It is clear from these results that the combination of sialic acid with the protein does not involve any of the basic groups of the protein.
H . F i b h o g e n and Fibrin The difference between the titration curves of fibrinogen and fibrin, in 3.3 M urea solution, has been determined by Mihalyi (1954a). It was found that the reaction fibrinogen -+ fibrin leads to the production of 3.5 new groups per molecule, with a pK near 8.0. These groups are presumably a-amino groups which arise from the proteolytic nature of the activation process. It is known that two peptide bonds per molecule of fibrinogen are broken in this process, two peptides being split off. At pH 5.5 (the reference point of Mihalyi's data) no net difference in dissociated hydrogen ions is involved, since one end of each split bond will be in the form -COOand the other end in the form -NHt. The resulting amino groups will however be titratable, losing their hydrogen ions near pH 8. The fact that 3.5 new groups, rather than two were observed, is perhaps the result of some nonspecific splitting of peptide bonds by thrombin. Mihalyi (1954b) studied in a similar way the difference in the titration curves of fibrinogen and of polymerized (clotted) fibrin. By subtracting from this difference the difference described above between fibrinogen and
HYDROGEN ION TITRATION CURVES O F PROTEIN
139
fibrin, he was able to compute the difference due to the polymerization of fibrin alone. (The clotting experiments were carried out in 0.3 M KCI, whereas the fibrinogen -+ fibrin experiment was carried out in the presence of urea. Urea was found to have only a small effect on the titration curve of fibrinogen in the pH range of interest. It was assumed that the effect on unpolymerized fibrin would be the same, and the small correction required for the change in solvent was applied on this basis.) The observed change on clotting consisted of the appearance of three or four new titratable groups with a pK of 7.0 and the disappearance of three or four groups which originally had a pK of 8.2. Several different explanations have been proposed by Mihalyi and others, none of which carries conviction. Scheraga and Laskowski (1957) consider that the altered titration curve arises not from the disappearance of groups with pK of 8.2 and the appearance of new groups with pK of 7.0, but from a small pK shift of a larger number of groups. This may well be correct, as the original interpretation of Mihalyi depends on ignoring some of the experimental points. However, the elaborate mechanism used by Scheraga and Laskowski to account for such a shift in pK is an improbable one. It may be that nothing more complicated is involved than the necessity to maintain charge neutrality of areas of contact between fibrin molecules in the polymer.
I . Gelatin Kenchington and Ward (1954) have used titration studies to resolve the molecular difference between gelatin extracted by processes employing acid and alkaline media. Acid-processed gelatin was found to have an isoionic point a t pH 9.1 and to possess 85 titratable carboxyl groups per 100,000 grams. The alkali-processed material was isoionic at pH 4.92 and contained 123 titratable carboxyl groups, essentially equivalent to the total amount of glutamic acid and aspartic acid plus the corresponding amides, which gelatin is known to contain. It is clear that processing in alkaline solution hydrolyzes side-chain amide groups to the corresponding carboxyl groups.
J . Hemoglobin Titration studies of hemoglobin have made important contributions to our knowledge of this protein. The three outstanding features are : (1) Four groups are titrated (pK near 8.0) in ferrihemoglobin,which are not titrated in hemoglobin itself, nor in the complexes of hemoglobin with 0 2 or CO (German and Wyman, 1937; Wyman and Ingalls, 1941). The same groups, with similar pK, are observed by spectrophotometric titration (Austin and Drakbin, 1935; George and Hananis, 1953), and by observation of the effect of pH on magnetic properties (Coryell et al., 1937).
140
CHARLES TANFORD
+
These groups represent the dissociation Fe(HzO)+s Fe(0H) H’, from the heme iron atom. (2) Four groups, which have a pK near 7.9 in hemoglobin, are titrated at much lower pH (pK near 6.7) in the hemoglobin-oxygen complex. These are the four “heme-linked” imidazole groups, which are responsible for the effect of pH and of COzon the hemoglobin-oxygenequilibrium (BohrHasselbach-Krogh effect). Pour other groups have an altered pK when oxygen is combined with hemoglobin, the pK being near 5.25 in hemoglobin and near 5.75 in the oxygen complex. These are also believed to be imidazole groups, though the identification is less secure. It is of interest that all eight of these groups have the same pK in ferrihemoglobin as in the hemoglobin-oxygen complex (Wyman and Ingalls, 1941), so that the pK differences observed are presumably associated with a conformational difference in the region of the molecule which contains the heme, rather than with a specific effect of oxygen (Wyman and Allen, 1951). The subject of the heme-linked groups has been previously reviewed (Wyman, 1948), and the reader is referred to that review for a detailed discussion of the subject. (3) The titration curve in the acid region is time-dependent and irreversible, as was first clearly demonstrated by Steinhardt and Zaiser (1951). This aspect of the titration of hemoglobin has also been reviewed previously (Steinhardt and Zaiser, 1955), but there was some ambiguity about the meaning of this phenomenon at the time of the review, which has been removed by more recent work, as will be briefly described here. The original observation (Fig. 9) was that a difference of up to thirtyeight groups could be obtained in the number of groups required to titrate an initially neutral protein to pH 3.5, depending on whether data were obtained by a rapid-flow method or by slower procedures. A parallel study of spectral changes indicated that protein examined immediately on attainment of pH 3.5 would be largely in its native state, but that conversion to a denatured state takes place rapidly. It was not possible however to determine whether the extra thirty-eight groups which were titrated represent an effect of conformation on the number of groups titrated in the acid range, or whether they reflect a change only in the shape of the acid part of the titration curve, brought about by unfolding and hence decreased electrostatic interaction. It appeared that at least a part of the effect, must be due to the latter phenomenon (Tanford, 1957). The question has been essentially decided by more recent experiments (Beychok and Steinhardt, 1959; Steinhardt et al., 19G2). One procedure was to reduce the temperature and increase the ionic strength, the increase in ionic strength being for the purpose of diminishing the importance of electrostatic interactions. A second procedure was to use hemoglobin derivatives which are especially stable in the native state, these being the
HYDROQEN ION TITRATION CURVES OF PROTEIN
141
CO-hemoglobin complex and the cyanide complex of ferrihemoglobin. Both procedures resolved the ambiguity in the interpretation of the earlier results. Figure 10 shows the titration curves of the stable complexes and the rapid back titration of the corresponding denatured proteins over a wide range of pH. What these curves show is that the number of groups in the carboxyl region is essentially the same in the native and denatured states, but that the number of groups titrated in the neutral (imidazole) region is about twenty-two greater for the denatured protein. These twenty-two groups (all assumed to be imidazole groups) are in their uncharged form in the native state and cannot be titrated with acid. Upon denaturation they become free from the restraint to which they were subject, so that, in going from a neutral reference point towards lower pH, under conditions where denaturation occurs, these groups are titrated as an accompaniment of denaturation. Reverse titration of the denatured protein then shows these groups as titrating in the normal region for imidazole groups. (The maximum difference of thirty-eight hydrogen ions per mole observed in Fig. 9 must represent these twenty-two imidazole groups plus an additional difference of sixteen hydrogen ions per mole which reflects the difference in electrostatic interactions between native and denatured protein.) It should be noted that four of the anomalous imidazole groups are the four groups by which the heme iron atoms are attached to the protein. These cannot be titrated as long as the hemes remain attached. The other groups must be simply “buried” in the interior. Such groups occur in myoglobin (see below), as well as in hemoglobin. Nozaki (1959) has carried out careful titration curves of bovine ferrihemoglobin over the entire accessible pH range. A preliminary group count was made. Starting with native isoionic protein, titration to the acid end point (where the protein is of course denatured) required 102 hydrogen ions/mole, so that the maximum positive charge is 102. The expected figure (based on 14 arginine, 48 lysine, 36 histidine, and 4 iron atoms, each contributing one charge) is 106. Starting with native protein and titrating towards the alkaline side gave a count of 28 groups in the neutral pH region. The expected figure is 44 [36 histidine, 4 N-terminal amino groups, and 4 Fe(Hz0)+ groups], so that there is a discrepancy of 16. This discrepancy confirms the conclusion of Steinhardt et al., given above, that many of the imidazole groups of the native protein are inaccessible to titration, the difference between the actual group numbers (22 versus 161 being probably within the experimental error of both determinations. Nozaki’s figure for the alkaline range is 60 groups per mole, in agreement with expectation (48 lysine, 12 tyrosine). The total number of groups titrated in the carboxyl region, including those released on denaturation, was 88,
142
CHARLES TANFORD
which is approximately the expected figure when the eight carhoxyl groups on the side chains of the four heme groups are included. Another titration curve, this time for human CO-hemoglobin, has been determined by Vodrazka and Cejka (1961). Unfortunately, their group counting procedure is not valid: all groups titrating below pH 4.7 were arbitrarily assigned to the “carboxyl” region and those titrating between pH 4.7 and pH 8.5 were assigned to the “imidazole plus a-amino” region. This procedure assigns too large a number of groups to the latter region and led the authors to the conclusion that the anomalous groups which titrate in the acid pH range are basic groups other than imidazole groups. The data in fact do not support this conclusion. Vodrazka and Cejka (1961) titrated the phenolic groups spectrophotometrically, and found no evidence for buried groups. Hermans ( 1962), has reported from a similar study that four groups per molecule (one per polypeptide chain) are not titratable in the native protein. Hermans’ study employed absorption at 245 mp instead of the customary wavelength of 295 mp.
K . Insulin Titration curves of insulin have been determined by Tanford and Epstein (1954) and by Fredericq (1954, 1956). As Table XI1 shows, the count of groups obtained for zinc-free insulin is in agreement with analytical data. This is true in spite of the fact that insulin is insoluble between pH and pH 7. The precipitate is evidently highly hydrated, so that titration of the acidic groups occurs as if they were in solution. Tanford and Epstein also determined the titration curve of crystalline zinc insulin, containing one atom of Zn++ per two insulin molecules. The titration curve of this material differs from that of zinc-free insulin in two ways. (1) Two new groups are titrated for each zinc ion, one near pH 8, the other near pH 12. These presumably represent acidic water molecules attached to Zn“, Zn(H20)2++ -+ ZnOH(H20)+
-+
Zn(OH)z
(2) The count of imidazole groups is reduced from four (per two insulin molecules) to two, and, at the same time, two new “carboxyl” groups appear. The titration of these new groups (in the direction of hydrogen ion addition) parallels the dissociation of Zn++ from the zinc insulin. Each Zn++ ion is evidently associated with two imidazole groups in their basic uncharged form. The value of the binding constant for zinc confirms this conclusion. Gurd and Wilcox (1956) have pointed out that the binding groups could
143
HYDROGEN ION TITRATION CURVES OF PROTEIN
be terminal amino groups rather than imidazole groups, since these groups have essentially identical pK's in the free state. Marcker (1960) has presented evidence that the amino groups are in fact the more likely binding sites. The curve for zinc-free insulin was analyzed by the method of Section TABLExrr Titration Data for Insulin at $ 6 ' 0 Number of groups Type of group
a-Carboxyl Side-chain carboxyl Imidazole a-Amino Side-chain amino Phenolic Guanidyl Zn(H,O)++
Titration
pKint
Analysis
Zinc-free insulin
Zinc insulin
8.5* 4 4
12.5
14.56
2
-
4 4
2 4
6.4 7.4
10
10
9.6'
2
2 2
11.9
-
-
a Data of Tanford and Epstein (1954; see also Fredericq 1954, 1956), calculated for an insulin dimer of molecular weight 11,466. The zinc insulin preparation contained one zinc atom per dimer molecule. The pKint values are for the zinc-free protein. * The fractional number arises from the probable presence of two forms of insulin which differ in the number of free carboxyl groups. c Two of the groups titrating as carboxyl groups are the imidazole groups t o which the Znf+ ion is bound. I, This pK was determinable because the titration curve of the carboxyl groups was clearly not compatible with the presence of 12.5 identical groups. Assuming 4 groups with a lower pK, this was the value required. 6 No attempt was made t o distinguish between amino and phenolic groups in the analysis.
VI. The pKint values are shown in Table XI1 and reveal no important anomalies. The apparent value of w rises to very high values in the region where the protein is precipitated. (See Section VI, C . ) The dissociation of the single lysine amino group of iodinated insulin has been studied by Gruen et al. (1959a). (The iodination separated the titration region of phenolic residues from the titration region of the amine group. ) No abnormalities were observed. The over-all difference between the titration curves of iodinated and nor-
144
CHARLES TANFORD
ma1 insulin corresponded to that which is expected as due to the lowered pK characteristic of iodjnated phenolic groups (Gruen et al., 1959b).
L. @-Lactoglobulin Complete titration curves of 0-lactoglobulin have been determined by Cannan et al. (1942) and by Nozaki et al. (1959). Between the acid end point and the onset of alkaline denaturation near pH 9.7, the data of the two studies are indistinguishable. Above pH 9.7, Nozaki et al., by use of the pH-stat were able to obtain two curves, one representing an extrapolation to zero time (corresponding to the titration of the native protein), the other representing infinite time (corresponding to titration of denatured protein). Both curves are shown in Fig. 2. The curve for the native protein could be obtained over a limited range of pH only, so that it is not possible to decide whether the difference between the two curves represents a difference in the number of groups accessible to titration, or whether it lies in the shape of the curve alone. It was possible to fit the data for the native protein by assuming that all amino and phenolic groups are accessible, and that the value of 20 is the same as that which is applicable to the reversible part of the titration curve. The side-chain amino and phenolic groups were assumed to have the same pKintand a reasonable value of 9.95 was obtained from this analysis. This suggests that the difference between the curves lies primarily in the steepness, and not in the count of groups. It was assumed, however, that the four thiol groups of the protein are not titrated in the native state because they are found to be quite unreactive by other methods. Since thiol groups are expected to have a somewhat lower pK than amino or phenolic groups (Table I ) , it would probably have been difficult in any case to fit the native curve with reasonable pKint values if the thiol groups had been included. The titration curve of 0-lactoglobulin denatured a t pH 12.5 has also been determined (Tanford el al., 1959). It was found that the curve is reversible. The denatured protein is insoluble near its isoelectric point and this region was not studied in detail. The group counting results obtained from these studies are reported in the last two columns of Table XITI. ( I n comparing these results with the analytical data of the first two columns of this table it should be noted that the p-lactoglobulin studied was a mixture of the two genetic isomers, 0-lactoglobulins A and B.) The most significant feature of the analysis is that the native protein appears to contain six imidazole groups, compared to the analytical figure of four. At the same time, the number of carboxyl groups titrated is less than the analytical figure by two groups. After denaturation, however, the group count agrees with the amino acid analysis. It is evident that two carboxyl groups of the native protein are titrated with
HYDROGEN ION TITRATION CURVES OF PROTEIN
145
a pK which is characteristic of imidazole groups. The probable reason has been discussed in Section VI, D. Titration curves of pure /3-lactoglobulins A and B have also been determined (Tanford and Nozaki, 1959). The two genetic variants differ in isoionic point, but they possess the same maximum positive charge (Fig. TABLE XI11 Group Counting for @-Lactoglobulin Amino acid analysisType of group
a-COOH Side-chain COOH Imidazole a-NHz Thiol Phenolic Side-chain NH2 Guanidyl
ZN+
@-LactA
8-Lact R
2 52 4 2 2 8 28 6 40
4 2 2 8 28 6 40
Titration curveb
Native B-Lact A
1
Native @-IdactB
1
52
I
50
I
Native lenatured mixturec mixtureC 51
a Gordon et al. (1961), Pie2 et al. (1961). The figures have been adjusted t o the nearest even integer for a two-chain molecule of molecular weight 35,500. * Titration data of Nozaki et al. (1959), Tanford et al. (1959), Tanford and Noeaki (1959). The mixture contained essentially equimolar amounts of the two genetic isomers, &lactoglobulins A and B. d Figures in parentheses are subject t o considerable uncertainty. The number of phenolic groups was determined from the total change i n extinction a t 295 mfi in going from native protein with undissociated phenolic groups t o denatured protein with all phenolic groups dissociated. No correction was made for the change i n extinction a t this wavelength which results from unfolding of the protein as a result of the emergence of the tryptophan residues from the inside of the native structure. There are four tryptophan residues per molecule, so that this change can be expected t o be quite large, certainly large enough t o account for an error of two groups in the count of phenolic groups.
20) and the same two anomalous carboxyl groups with pKintof 7.5. Thus the difference between them lies in the number of normally exposed carboxyI groups, two more of these being in form A than in form B. This result has since been confirmed by amino acid analysis (Gordon et al., 1961; I’iez et al., 1961). Another interesting feature of the titration studies (Nozaki et al., 1959) is the fact that addition of KC1 and CaClz depresses the pH of isoionic protein solutions. This means that I(+ and Ca++ ions are bound by the iso-
140
CHARLES TANFORD
ionic protein, a result which is apparently in agreement with published studies of ion binding by direct means (Carr, 1953, 1956). These studies show that K+ and Ca++ ions are bound at pH 7.4, and the quantitative difference between the number found bound at that pH, and the number calculated as bound from the pH change at the isoionic point (Section IX, B ) , is of the order of magnitude expected on the basis of the difference in protein charge a t the two pH's. More recently, however, Saroff (1961) has measured ion binding as a function of pH and has observed that there is
I
2.0
PH
2.5
I 3.0
FIG.20. Approach t o the acid end point of the titration curves of p-lactoglobulins A and B, and for the normal equimolar mixture of the two, a t 25°C and ionic strength 0.15. The value of 2 , is calculated relative t o the point of zero net proton charge, which occurs a t a different pH for each of the three samples (Tanford and Nozaki, 1959).
essentially no binding of K+ at the isoionic pH. He observes binding a t higher pH, the appearance of binding sites occurring in parallel with the conformational change during which the two anomalous carboxyl groups are titrated. The discrepancy between his results and those derived from the titration studies is at present unresolved. The values of w which one obtains from the carboxyl region of the titration curve by application of Eq. (4), assuming 2, = 2, are 0.072 and 0.039, respectively, at ionic strength 0.01 and 0.15, i.e., they are somewhat below the calculated values of Table 111. (As was mentioned earlier, the same values are compatible with the entire titration curve of the native protein.) If one attempts to evaluate the difference between 2 and 2, at all pH's
147
HYDROGEN ION TITRATION CIJRVES OF PROTEIN
from the few values of ion binding at different pH's which are available, larger values are observed (w = 0.090 and 0.058 at ionic strengths 0.01 and 0.15). Both sets of values are within the range expected for a compact globular protein. The slopes of logarithmic plots for the alkaline titration curve at t = (Fig. 2) are however very much less, showing that the alkali-denatured protein is randomly coiled. The intrinsic pK values obtained from these studies at 25OC and ionic strength 0.15, without correcting for K+ ion binding, are 4.7 for the sidechain carboxyl groups, 7.3 for the imidazole groups, and 9.9 for phenolic Q,
TABLEXIV Carboxyl Groups of LysozymeR Carboxyl groups titrated Lot number
in 0.15 M KCl
in 8 M GHCl or 5 M GHCl 1.2 M urea*
+
003L1 381-187 D638040 381-187Methylated
10.5 9 14 1 (or 2)
13.5 12
381-187Acetylated
12
12
9
12
381-187Guanidinated
-
1 (or 2)
Remarks
-
12.2Methoxyl groupsper molecule Acetylation occurs principally at amino groups Guanidination converts amino groups to hornoarginine groups
= Data from Tanford and Wagner (1954);Donovan et al. (1960,1961). b GHCl = guanidine hydrochloride.
and lysine amino groups. Correction for K+ ion binding increases the pKint values by 0.15.
M . Lysozyme Titration studies of lysozyme have revealed two unique features, both occurring in the carboxyl region of the titration curve. The pertinent data are shown in Table XIV. It is seen ( a ) that the count of carboxyl groups varies widely from one preparation of lysoByme to another, and ( b ) that three extra carboxyl groups appear in denaturing solvents such as 8 M guanidine hydrochloride. The three extra carboxyl groups which appear in denaturing solvents were apparently in their carboxylate ion form in the native protein. Since these groups are not detectable at all in the titration of the native protein
148
CHARLES TANFORD
down to pH 2, a true plateau being approached at pH 2, they must in effect be inaccessible to the solvent. It is possible for a charged group to be so located only if it is in close contact with a similarly inaccessible group of opposite charge. There is no evidence that any of the titratable cationic groups of lysozyme are so located. However, the twelve guanidyl groups are never titrated, so that three of these could be located away from the protein/solvent interface. A number of chemical derivatives of lysozyme were studied by Donovan et al. (1960), with the results shown in Table XIV. The guanidinated derivative, in which charged lysine groups are simply replaced by similarly charged homoarginine groups, showed behavior similar to that of the native protein. The acetylated derivative, in which lysine side chains are rcplaced by uncharged acetyllysine groups, titrated like denatured untreated lysozyme. The most interesting result was that obtained with the methylated derivative, in which most of the carboxyl groups are esterified and only a single titratable group in the carboxyl region is observed. The number of methoxyl groups introduced was found by analysis to be equal to the number of carboxyl groups titrated in the denatured protein, essentially confirming that the three extra groups titrated upon denaturation are in fact carboxyl groups. The only aspect of the titration data which raises a question about the existence of buried carboxylate ions in the native protein is the maximum positive charge estimated by Tanford and Wagner (1954). The figure is based on an assumed location of the point of zero net proton charge at pH 11.1. (Lysozyme precipitates on deionization, so that the normal procedure for determining this reference point is not possible.) This assumed pH, however, is supported by the fact that titration curves at three different ionic strengths, which should intersect a t Z = 0, do intersect at pH 11.1, and by the fact that the isoelectric point has been determined electrophoretically to be at pH 11.1. With this assumed pH of zero net proton charge, the maximum positive charge ( Z N + , Section IV, A ) becomes 19, which is just the analytical figure for the total number of cationic groups. If three carboxyl groups are still in their anionic form a t the acid end point of the titration curve, the experimental maximum positive charge should have been 3 less than the anlaytical figure. It would appear that the problem of the carboxyl groups of lysozyme merits further investigation. The count of other titratable groups of lysozyme agrees with analytical data. Moreover, no variation between different preparations has been reported. The titration curve of the native protein is reversible and has been analyzed by the methods of Section VI. As we have already noted (Fig. 16),
HYDROGEN ION TITRATION CURVES OF PROTEIN
149
the carboxyl region does not obey Eq. (14) if all carboxyl groups are assumed to have the same pKint. This is not an anomaly unique to lysozyme, but is shared by other proteins (chymotrypsinogen, ribonuclease) which are rich in basic nitrogen groups. The anomaly presumably reflects uneven spatial distribution of these groups, relative to the carboxyl groups. The neutral and alkaline region of the titration curve is compatible with values of w of magnitude similar to those calculated by Eq. (4). The intrinsic pK values are not remarkable (see Table V ) , except that pKint for the phenolic groups is 1.2 higher than the expected value. The likely reason has already been discussed in Section VI, D . It may be noted that Tanford and Wagner (1954) found the spectral change corresponding to dissociation of the phenolic groups to be quite abnormal, though an approximate pK for the dissociation could be determined. Their difficulties have been elegantly explained by Donovan el al. (1961) as arising from changes in the spectrum of tryptophan side chains, which occur in the same region of pH as the ionization of phenolic groups. When these changes are corrected for, the residual spectral change becomes that which is normally expected for phenolic ionization.
N . Myoglobin Special interest attaches to the titration of sperm whale myoglobin because the three-dimensional structure of this protein is well on the way to being completely elucidated (Kendrew el al., 1961). The speculative structural features, which have been invoked to explain titration data that do not conform to expectation, will in this protein soon be subject to actual test. A titration curve for sperm whale myoglobin has been reported by Breslow and Curd (1962). The most striking feature is that it exhibits a timedependent acid denaturation, which resembles that observed for the similar protein hemoglobin. To elucidate the physical nature of this reaction, emphasis was placed on the back titration to neutral pH of denatured protein. As in the case of hemoglobin (mentioned earlier) ,there are two major differences between the titration curves of native and denatured myoglobin, as shown by the data of Table XV. The first difference is in the count of imidazole groups. Only six of the twelve groups known to be present are titrated in the normal pH range in the native protein. When the native protein is titrated towards acid pH, these six groups are titrated in the carboxyl region as the protein becomes denatured. In the back titration of the denatured protein, all twelve of the groups are titrated with approximately the expected pK. To confirm the identification of the groups concerned, the kinetics of hydrolysis of p-nitrophenylacetate (Section 111, D) was studied. This method gives
150
CHARLES TANFORD
direct information as to the number of accessible uncharged imidazole groups, and in the present study confirmed exactly the conculsions reached from the titration curve as a whole. It is concluded therefore that six of the twelve imidazole groups of native myoglobin are buried in the interior in their uncharged form. One of these is of course the imidazole group by which the heme iron atom is attached to the protein. The other five have not been identified. The expectation from the present study is that the complete three-dimensional structure of the protein, when available, will show these groups in positions where they are not in contact with the solvent. The second difference between native and denatured myoglobin lies in TABLEXV Titration of Myoglobin in the Neutral pH Region" ~
Native protein Number of imidaaole groups: By analysis By titration By reaction with NPAh pKi.t for imidazole groups w At ionic strength 0.16 w At ionic strength 0.06
After acid denaturation 12
6 6 6.62 0.050
0.085
12 12 6.48 0.034" 0.044O
a Titration data of Breslow and Gurd (1962). Analytical data by Edmundsori and Hirs (1961). The protein studied was ferrimyoglobin from the sperm whale. Its molecular weight is 17,816. NPA p-nitrophenyIacetate. See Section 111, D. Determined from the carboxyl region rather than the imidazole region, assuming that all 23 carboxyl groups found by analysis are titratable with the same pKint. =i
0
the value of the electrostatic interaction factor w. In the native molecule this factor is somewhat, but not much smaller than calculated by Eq. (4). Using a radius derived from the volume of the molecule as it appears in myoglobin crystals, Breslow and Gurd (1962) calculated w = 0.106 and 0.065, respectively, at ionic strengths 0.06 and 0.16. The experimental values at the same ionic strengths are 0.085 and 0.050. (It was assumed that = ? .& , which is likely to be essentially correct for myoglobin.) The values of w for the denatured protein are considerably smaller, indicating that unfolding of the protein occurs. The values given in Table XV are obtained by assuming that all of the carboxyl groups of myoglobin have identical pKint values. If this assumption is in error, the actual values would be smaller than those given in the table. (The intrinsic pK of the carboxyl groups, obtained by a rather long extrapolation to = 0, is 4.4.) Apart from the imidazole groups of myoglobin, only one other group has
z
z
HYDROGEN ION TITRATION CURVES O F PROTEIN
151
been studied in any detail, this being the 17e(HzO)+group, which dissociates to Fc(OH) near pK 9. The pK value for this group has been determined by Theorell and Ehrenberg (1951), George and Hanania (1952), and Rreslow and Gurd (1962) from the spectral change which accompanies the dissociation. The values are given in Table V, together with comparable data from other proteins. In a recent study, Hermans (1962) has indicated that only two of the three tyrosyl phenolic groups of myoglobin titrate below pH 13, this result being obtained for both whale and horse myoglobins.
0. Myosin The titration of myosin has been studied by Mihalyi ( 1950). The count of groups in the various regions of the curve agrees with analysis to better than lo%, except for the carboxyl region, where titration indicates 165 groups per 100,OOO gm, as compared with the analytical figure of 132. It is likely that the analytical assay for amide groups is the source of the error. Mihalyi’s titration curves were obtained a t a series of ionic strengths, ranging from quite a low value to I = 1.2 M . The curves were reversible over a wide range of pH, and were considered to represent equilibrium. They were not analyzed by the methods of Section VI, but even a cursory examination shows that the carboxyl region at least would not obey the behavior predicted by the Linderstrfim-Lang equations. The carboxyl regions of the titration curves are quite flat, indicating fairly strong interaction between the groups, but if the data were plotted according to Eq. (14), with 2, as abscissa, the w values would be essentially independent of ionic strength. On the other hand, the pKint values would depend strongly on the ionic strength. Mihalyi suggests that strong anion binding in the acid region would be responsible for this kind of behavior. A spectrophotometric titration of the phenolic groups of myosin and its subunits has been reported by Stracher ( 1960). The data resemble those shown for ribonuclease in Fig. 11. About two-thirds of the tyrosine residues are titrated normally, and about one-third appear inaccessible in native myosin. An interesting feature is that 6 M urea has no effect a t all on the titration curve. Similar studies were performed on the subunits L-meromyosin and Hmeromyosin. In the former 90 % of the phenolic groups appear abnormal, but they are titrated normally in 5 M urea. In H-meromyosin all of the groups are normal, even in aqueous solution.
P. Ovalbumin The very first electrometric titration curve of a protein to be reported in the literature is a study of ovalbumin (Bugarszky and Liebermann, 1898).
152
CHARLES TANFORD
The first detailed analysis of a protein titration curve, according to the semiempirical treatment used for most of the titration curves reviewed in this paper, also involves ovalbumin (Cannan el al., 1941). The first discovery of phenolic groups inaccessible to titration was again made with this protein (Crammer and Neuberger, 1943). Within the limits of error of amino acid analyses available a t the time, the count of groups obtained by Cannan et al. agreed with expectation, except in so far as the alkaline part of the curve was concerned. The number of groups titrated here is essentially the same as the number of amino groups, rather than the sum of amino and phenolic groups. This result is in accord with the later spectrophotometric titration of phenolic groups: essentially all of these groups are inaccessible to titration in the native protein. Cannan et al. (1941) were able to describe the entire titration curve, at eight different ionic strengths, by Linderstrgim-Lang’s equation, using a single value of w a t each ionic strength. The variation of w with ionic strength was essentially that of Eq. (4),experimental values being about 0.8 of those calculated by that equation. [Itwas found that the isoionic pH depends on the concentration of KCl which was used to vary the ionic strength, the direction of the effect indicating that chloride ion is bound. Correction for the effect was made by an adjustment factor introduced into Eq. (14), this factor being a measure of the number of chloride ions bound by the protein at its isoionic point. The possibility of variation in chloride ion binding with pH was not allowed for, and this is one reason why experimental values of w fall below calculated ones. The intrinsic pK values required to fit the data were not far from expected values: 4.3 for the carboxyl groups, 6.7 for imidazole groups, and 10.0 for lysine amino groups.] Harrington (1955) has used the pH-stat method to show that there is a large difference between the titration curves of native ovalbumin in 2.6 M guanidine hydrochloride, and the denatured protein which is formed by a measurably slow reaction in this solvent. The result has been interpreted as indicating the release of eight carboxyl and eight phenolic groups which are not accessible to titration in the native protein. Since the two branches of the difference titration curve reach plateaus a t extreme acid and alkaline pH (i.e., they resemble the curve shown in the lower part of Fig. 5 ) , the interpretation of the difference as newly liberated groups appears indisputable. That the phenolic groups should be so liberated on denaturation is not surprising, since these groups are known to be buried in the interior in the native protein. The liberation of carboxyl groups is surprising, however, since the expected number is titrated in the native protein. Furthermore, the carboxyl groups which are unavailable in the native protein appear to be in their anionic form. In this form they would reduce the maximum possible positive charge of the native protein by eight groups, whereas
HYDROGEN ION TITRATION CURVES OF PROTEIN
153
Cannan et al. (1941) find complete agreement between the experimental maximum positive charge and the analytical figure for Z N'.
Q . Papain Glazer and Smith (1961) have carried out a spectrophotometric titration of the phenolic groups of papain. Of the seventeen phenolic groups known to be present, eleven to twelve ionize normally (pKint = 9.8). The remainder ionize only upon denaturation, which takes place only slowly in the range of pH 12 to 13.
R. Paramyosin A detailed study of the hydrogen ion titration of clam paramyosin has recently been reported by Riddiford and Scheraga ( 1962). Essentially all the groups expected to be titratable from analytical data were found to be titrated reversibly both in 0.3 M KC1 and in a guanidine-urea mixture in which extensive denaturation had occurred. In the native state the protein is precipitated between pH 3.5 and 6.5, but this apparently did not interfere with titration, indicating that the precipitated protein is gellike in nature, permitting free passage of water and of ions to the individual molecules. Earlier Johnson and Kahn (1959) had reported that the titration curve of paramyosin has a plateau between pH 3 and pH 5. They concluded that the carboxyl groups were titrated in two distinct steps, one near pH 6 and one below pH 3. This finding would have suggested highly unusual interactions within the molecule. Riddiford and Scheraga (1962) did not find such a plateau in their studies. However, the fact that the protein is in an insoluble state between pH 3.5 and 6.5 suggests that the differences between the two sets of results may depend upon the particular conditions of preparation and handling of the protein, in a manner not yet adequately defined. The electrostatic interaction factor w for the native protein was found by Riddiford and Scheraga to be much smaller than would be expected for a compact spherical particle by Eq. ( 4 ) . In order of magnitude, the w value agreed with the value predicted for long cylindrical rods by Hill (1955). This is in agreement with the known dimensions of the paramyosin molecule. However, a considerably larger value of w was required to fit the alkaline part of the titration curve than was required for the acid branch. In the denaturing solvent, w values even smaller than those for the native state were observed. On the alkaline side, electrostatic interaction disappeared almost entirely, presumably because extensive unfolding with penetration of salt ions into the domain of the protein had occurred. The intrinsic pK values in the guanidine-urea mixture were found to be close to the normally expected values. The pK values for the native pro-
154
CHARLES TANFORD
tein were also remarkably close to expected values. Only the pK for the lysine amino groups deviated significantly from expectation, a low value of 9.65 being obtained. Spectrophotometric titration of the phenolic groups led to the conclusion that all of the fifty-eight phenolic groups present on each paramyosin molecule were titrated in the guanidine-urea mixture, but that only forty-nine were titrated in the native state in 0.3 M KC1. (All of these had an essentially normal pKint of 9.6.) This conclusion however was based entirely on the fact that the total change in absorbance at 295 mp, between neutral pH and pH 14, is about 15 % less in the native protein than in the denatured state. If the change in absorbance per group titrated were to differ in the two solvents, then the conclusion reached would have to be revised.
S. Pep& It is not possible to determine the titration curve of pepsin over a wide range of pH because autolysis, with liberation of free carboxyl and amino groups, occurs at acid pH. Titration studies have been performed (Edelhoch, 1958) in the range of pH 5 to 8, however. This is the pH range within which pepsin undergoes denaturation, and the data clearly show that more hydrogen ions are dissociated in this pH range from the denatured protein than from the native protein. The difference is greater at low ionic strength than at high ionic strength, but a difference of six H+ ions per molecule persists at ionic strengths from 0.4 to 1.0. These six H+ ions are thought to represent a difference in the number of titratable groups, the additional difference at lower ionic strength being a measure of the greater steepness of the titration curve of the denatured protein which is the result of diminished electrostatic interaction after unfolding has taken place. The simplest explanation of the difference in group count would be that native pepsin has six uncharged carboxyl groups inaccessible to titration, and that these are exposed during denaturation.
T . Peroxidase A titration study of a peroxidase from Japanese radish has been rcported by Morita and Kameda (1958). The titration curves of native protein, acid-denatured protein, and alkali-denatured protein are dramatically different. Unfortunately only continuous titration curves were obtained, so that an interpretation of the data is not possible at this time,
U. Ribonuclease The best-known feature of the titration of ribonuclease is the fact that only three of the six phenolic groups of this protein can be titrated while the protein is in its native state, as was first reported by Shugar (1952).
HYDROGEN ION TITRATION CURVES OF PROTEIN
155
These three groups have an essentially normal intrinsic pK and their titration curve yields essentially normal values for the interaction parameter w (Tanford et al., 1955a). The three phenolic groups which are not available for titration in the native protein are titrated a t 25°C near pH 13, where the protein becomes denatured. At 6"C, where the rate of denaturation is slower, a pH of 14 can be reached with only partial titration of these groups. The titration curve of ribonuclease (Tanford and Hauenstein, 1956b) is reversible between its acid end point and the onset of alkaline denaturation. All titratable groups, which are expected to be present on the basis of amino acid analysis, are found to be titrated in the expected parts of the titration curve, with the exception of the abnormal phenolic groups mentioned above. The amino and imidasole groups appear to have normal pK's, and the neutral and alkaline regions in which they occur are compatible with the same values of w as are required to fit the titration curves of the three normal phenolic groups. A second (minor) anomaly is found in the acid part of the titration curve. Although the expected number of groups is titrated, the observed values of w are anomalously large. It is possible that this is simply a manifestation of the inadequacy of the Linderstrgm-Lang treatment, as was pointed out in Section VI, C . Two alternate explanations have been proposed, neither of which deserves to be taken very seriously. (1) Tanford and Hauenstein showed that the acid part of the titration curve could be compatible with the same values of w as were used to fit the rest of the titration curve, if one were to assume that five of the ten sidechain carboxyl groups have pKi,t = 4.0, while five others have pKint = 4.7. (2) Hermans and Scheraga (1961b) showed that a fair fit of the titration data could be obtained with these same values of w, if one assumed that one of the side-chain carboxyl groups has pKint = 2.5, another has pKint = 3.65, and the remaining eight have pKint = 4.6. The existence of groups with pKint 2.5 and 3.65 was inferred from low pH conformational changes (Hermans and Scheraga, 1961a). It is to be expected that these special features of the titration curve of ribonuclease will disappear when the protein is unfolded. In agreement with this expectation, it has been found that all six phenolic groups are titrated together in ethylene glycol (Sage and Singer, 1958, 1962), in 8 M aqueous urea (Blumenfeld and Levy, 1958), and in aqueous solutions containing 5 M guanidine hydrochloride and 1.2 M urea (Cha and Scheraga, 1960). I n the guanidine-urea medium the electrometric titration curve has also been determined (Cha and Scheraga, 1960), and it was found possible to fit the entire curve with a single value of w,including all carboxyl groups as a single set with pKint = 4.6.
156
CHARLES TANFORD
The only unexplained feature in these experiments concerns the value of w. In the guanidine-urea medium the ionic strength is exceedingly large, so that a very small value of w would be expected even if the protein were not unfolded. For an unfolded protein, w should become essentially zero. The value observed by Cha and Scheraga, both for the titration curve as a whole and for the spectrophotometric titration of the phenolic groups alone, is 0.056, which is almost as large as the value found for the native protein at an ionic strength of 0.15. In 8 M urea, by contrast, the expected result is obtained. Blumenfeld and Levy (1958) obtained w = 0.018 at an ionic strength of 0.1, which is far below the value expected for the native protein a t this ionic strength, as predicted for an unfolded protein molecule. Bigelow (1960) has found that all six of the phenolic groups of performic acid-oxidized ribonuclease behave normally, which is to be expected since the oxidized protein is believed to be highly unfolded. More interesting is his finding that a pepsin-inactivated preparation of ribonuclease can be prepared which contains five normal phenolic groups and one buried one. Tanford and Hauenstein ( 1956a) observed that the chromatographic minor component of one commercial preparation of ribonuclease has an isoionic point of 9.23, whereas the major component (ribonuclease A) has an isoionic point of 9.65. This difference corresponds to a difference of one tit,ratable group, so that the minor component either has one fewer amino or guanidyl group than ribonuclease A, or else it has an extra carboxyl group. Titration curves of the two components indicated that the latter explanation is correct, for the two components gave the same maximum positive charge, but a difference of one in the number of carboxyl groups. T t appears now that these experimental data were incorrect, as Eaker (1961) has found by amino acid analysis that this minor component in fact consists of two sub-components, both of which lack the N-terminal lysine residue of ribonuclease A, so that the glutamic acid residue normally in the second position becomes the terminal residue. In one of the sub-components this glutamic acid residue has itself been converted to a pyroglutamyl residue. These findings are incompatible with the unaltered maximum positive charge found by titration. (The titration curve error presumably resulted from the fact that only a small amount of the second component was available for study, so that all the data depend on a single determination.)
V . Serum Albumin The titration curve of serum albumin (Tanford et aE., 1955b) is independent of time and essentially completely reversible. However, the protein undergoes at least three changes in conformation during the course of titration. These changes are themselves rapid and reversible, so that sepa-
157
HYDROGEN ION TITRATION CURVES OF PROTEIN
rate titration curves for individual conformations cannot be obtained. The interpretation of the titration curve is thus dependent on an interpretation of conformational changes which are observed by other means. The over-all count of titratable groups is shown in Table XVI. The agreement with analytical data is excellent, except that about nine more carboxyl groups are titrated than the analysis predicts. This discrepancy has been ascribed to an erroneously high analytical estimate of amide groups, and no recent data have appeared to support or gainsay this. When the data are analyzed according to the methods of Section VI, one obtains essentially normal values of w between pH 4.3 and 10.5. The TABLEXVI Titration Data for Serum Albumin" Number of groups Type of group
Analysis
Titration
pK
int,
25°C
curve
a-Carboxyl Side-chain carboxyl Imidazole a-Amino Thiol Side-chain amino Phenolic Guanidino EN+
90ll
'i
100
18
<57 18 I 22 97
57 19 96
1;:
-
4.0
10.35 -
a The data are for bovine albumin, with an assumed molecular weight of 65,OOO. Analytical data from Stein and Moore (1949); titration data from Tanford et al. (1955b). Titration data for human serum albumin (Tanford, 1950) are not significantly different.
actual values reported by Tanford et al. are somewhat below those calculated in Table 111, but Scatchard et al. (1957) have suggested that the calculated values themselves would fit the data about as well. Below pH 4.3 and above pH 10.5 there is a sharp decrease in the slopes of plots according to Eq. (14), as shown by Figs. 17 and 21. As we have pointed out in Section VI, C , such a break is an indication that a drastic conformational change occurs. The simplest possibility is that the protein becomes unfolded at these pH's. That such unfolding actually occurs is confirmed by numerous other studies by a variety of methods, including viscosity, optical rotation, etc. The experimental values of the degree of dissociation of carboxyl, amino, and phenolic groups, which were used to obtain the data of Figs. 17 and 21 were of course based on the notion that all titratable groups are accessible
158
CHARLES TANFORD
to titration throughout the entire pH range of the titration curve, because the group counting procedure had produced no evidence for inaccessible groups. Furthermore, it is an inherent part of the treatment of Section VI that any given group has the same pKint value throughout the course of titration. As was noted in Section VI, 6 ,however, these assumptions need no longer hold true if conformational changes occur which indicate (as they do here) that most of the groups are in fact titrated with the protein no longer in the native state. It now becomes possible that the count of
11.5 h
w
I rl \
v
w
2? 11.0 I %i 10.6 ~-
-40
-60 2
-80
-100
FIG.21. Titration data for the phenolic groups (upper curve) and the side-chain amino groups (lower curve) of bovine serum albumin, a t 25°C and ionic strength 0.15, plotted according t o Eq. (14) (Tanford rt al., 1955b).
titratable groups in Table XVI does not apply to the native protein or that, pKintvaluesin the native protein differ from those which apply to the conditions under which most of the groups are actually titrated. This possibility was not considered by Tanford et al. It was assumed t h a t the conformational change involved only unfolding, without ot,her change. The p I L values which then result from the customary analysis are shown in Table XVI. It is seen that they are all somewhat abnormal. The value for the phenolic groups is a little higher than expected, and the pK's for imidazole and side-chain amino groups are somewhat lower. As was pointed out in Section VI, D, deviations in the same directions occur for these groups in many proteins, and they may have their origin in the
159
HYDROGEN ION TITRATION CURVES OF PROTEIN
hydrophobic nature of the side chains of which these dissociable acidic groups are a part. A more unusual anomaly is the pKint of 4.0 for sidechain carboxyl groups. The anomaly is within the limits of variation allowed by the explanations for small differences in pKint which were given in Section VI, D, 3. It is however unexpected that any of the possible causes listed there should apply equally to almost one hundred side-chain carboxyl groups. (The data of Figs. 17 and 21 are based on true values of so that the anomaly cannot be due to a mistaken location of the point = 0.) The anomalous pKint for the carboxyl groups just cited is one reason which suggests the desirability of a deeper probe into the titration curve of serum albumin and its relation to the conformational changes which this protein undergoes. An even more compelling reason lies in the detailed study of the conformational changes themselves. (Only those on the acid side have been studied in detail.) Much of the work in this area has been done by Foster and co-workers (the earliest paper being by Yang and Foster, 1954), and it has been recently reviewed by Foster (1960). These studies show that the conformational change near pH 4 occurs in at least two stages, each of which may itself consist of several successive steps. The first stage is the so-called N + F transition, which does not involve an appreciable expansion of the molecule, but which may involve an uptake a t constant pH of about twelve hydrogen ions per molecule. In other words, in the region near pH 4 there appear to be present two kinds of serum albumin molecules, which do not differ in size but do differ in charge by 12, the form F having the greater number of bound hydrogen ions. The second stage of the reaction is the expansion of the F form, brought about by electrostatic repulsion between positive charges as the pH is reduced from pH 4 to 2. From the point of view of the analysis of the titration curve, these oonformational studies create a problem because the major part of the apparent decrease in w (Fig. 17) , at least at the higher ionic strengths, occurs during the first stage of the reaction, where little expansion occurs. I t is evident then that a large part of the anomalous behavior seen in Fig. 17 cannot in fact represent an actual decrease in w,due to expansion, without other change. It becomes necessary to explain the conformational change either by supposing that the number of titratable carboxyl groups is not the same in the two conformations, or that the number is the same, but pKint is different. The first alternative leads t o the conclusion that the native eonformation contains about thirty-six buried carboxyl groups, in the carboxylate ion form. It is not possible to bury charged groups in the interior of a protein molecule unless they are associated with an equal number of positively charged groups. There is however no evidence that thirty-six positively charged buried groups (i.e., groups with abnormally high pK)
z
z,
160
CHARLES TANFORD
exist. On the contrary, the side-chain amino groups, which do have an abnormal pK, deviate from expectation in the opposite direction. The second alternative is that the N and F forms contain the same number of groups, but with different pKint. Aoki and Foster (1957) have found that this possibility is indeed compatible with all known facts, the pKint values which are required being 3.7 for the native conformation, and an essentially normal value of 4.4 for the F conformation. The Aoki-Foster hypothesis, while satisfactorily correlating the data of Fig. 17 with data on the conformational change at low pH, thus increases the anomaly in the intrinsic pK of the carboxyl groups of the native protein. We now have pKint = 3.7 instead of the former value of 4.0. It should be noted in fact that the problem of the value of pKint is really independent of the mechanism proposed to account for the shape of the titration curve. The pH at which 2, of serum albumin is zero falls about 0.5 pH units lower than one would estimate on the basis of the expected pK’s of the groups known to be present, and to account for this is difficult regardless of the conformational changes which serum albumin undergoes. Of the explanations which were offered in Section VI, D for small pK anomalies, one (mistaken identification of 2, with 2 ) is not applicable here, and one (hydrophobic interactions) would lead to an anomalously high pKint. The other explanations can lead to a low pKint, but only if all one hundred carboxylate anions are involved in some stabilizing interaction which becomes weakened when the groups are converted to their acidic form. Only positively charged cationic groups would seem capable of interacting with carboxylate ions in this way, but if they did they would themselves acquire anomalously high pK values. Experimentally, it is found that the pKint of the fifty-seven side-chain amino groups is itself very low, and that of the seventeen imidazole groups is certainly not higher than is normal. These groups together comprise three-quarters of the available positively charged groups. The final solution to this problem must await future research.
W . Thyroglobulin Preliminary titration data have been reported (Edelhoch, 1960; Edelhoch and Metzger, 1961). They show that denaturation by various means facilitates titration of both phenolic and carboxyl groups. The data are not sufficiently detailed to permit a decision as to whether a difference in the count of titratable groups is involved.
X . Trypsinogen The titration of the phenolic groups of trypsinogen has been studied by Smillie and Kay ( 1961). Four groups are titrated reversibly with a normal
HYDROGEN ION TITRATION CURVES OF PROTEIN
161
pK. Four more are titrated at much higher pH, with a steep titration curve suggesting that their titration is accompanied by unfolding. That such unfolding occurs is confirmed by studies of sedimentation and viscosity. Both the titration and the conformational change are reversible at 10°C, but not at higher temperatures.
Y . Trypsin Titration curves of trypsin were obtained under a variety of conditions by Duke et al. (1952). The most noteworthy feature is a specific effect of calcium, which displaces the acid part of the titration curve to lower pH, and decreases the total number of groups which are titrated between pH 6 to 9. It is likely that the groups titrated between pH 6 and 9 in the absence of Ca++ are a-amino groups, produced by self-digestion of the enzyme. The effect of Ca” thus appears to result from a complex with the carboxyl groups of the protein, which stabilizes the anionic form of these groups so as to produce the displacement of the acid part of the titration curve. This complex is more resistant to self-digestion than the enzyme alone. ACKNOWLEDGMENTS The preparation of this review was supported by research grants from the National Science Foundation and from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Services. The author wishes to express his indebtedness to Drs. F. R. N. Gurd, G. P. Hess, W. Kauzmann, H. A. Saroff, J. Steinhardt, B. L. Vallee, and P. E. Wilcox, for providing him with results of investigations in advance of publication.
REFERENCES Aoki, K., and Foster, J. F. (1957). J . A m . Chem. SOC.79, 3393. Austin, J. H., and Drabkin, D. L. (1935). J . Biol. Chem. 112, 67. Bates, R. G . (1954). “Electrometric pH Determinations.” Wiley, New York. Beychok, S., and Steinhardt, J. (1959). J . Am. Chem. SOC.81, 5679. Bigelow, C. C. (1960). Compt. rend. trav. lab. Carlsberg 31,305. Blumenfeld, 0 . O., and Levy, M. (1958). Arch. Biochem. Biophys. 7 6 , 97. Boeri, E., Ehrenberg, A . , Paul, K . G., and Theorell, H. (1953). Biochim el Biophys. Acta 12, 273. Breslow, E., and Gurd, F. R. N. (1962). J . Biol. Chem. 297, 371. Bugarszky, S., and Liebermann, L. (1898). Arch. ges. Physiol. PjZuger’s 72, 51. Cannan, R. K., Kibrick, A., and Palmer, A. H. (1941). Ann. N . Y . Acad. Sci. 41, 247.
Cannan, R. K., PaImer, A. H., and Kibrick, A. C. (1942). J . Biol. Chem. 142,803. Carr, C. W. (1953). Arch. Biochem. Biophys. 46, 424. Carr, C. W. (1955). In “Electrochemistry in Biology and Medicine” (T. Shedlovsky, ed.), Chapter 14. Wiley, New York. Carr, C. W. (1956). Arhc. Biochem. Riophys. 62, 476. Cha, C.-Y., and Scheraga, H. A. (1960). J . Am. Chem. SOC.82, 54.
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REGULARITIES I N THE PRIMARY STRUCTURE OF PROTEINS By F. SORM and B. KElL Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Science, Progue, Czechorlovokia
I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 A. Homogeneity of Proteins ..................... 168 B. Development of Theories tructures of Proteins. 169 ..................... 171 11. Types of Structural Regularities in Proteins. A. Lower Peptide Sequences.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 B. Linear Analogies between Higher Peptide Structures. . . . . . . . . . . . . . . . . . 174 C. Repetition of Peptide Sequences.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 D. Symmetrical Arrangement of Peptide Chains. . . . . . . . . . . . . . . . . . . . . . . . . 185 E. Regularities in the Spacing of Amino Acid Residues. . . . . . . F. Simplified Structures Obtained When Applying Acid Interchangeability, . . . . . . . . . . . . . . . . . . . . . . . . . 111. Comparison of Proteins and the Search for Common Types of Structural Regularities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 IV. Mathematical Approach to the Evaluation of Structural Similarities between Proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Relevance of Structural Regularities t o Protein Sy References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. INTRODUCTION The investigation of each type of naturally occurring substance has always been carried out in three stages involving: (a) the isolation of the compound in the pure state, (b) the establishment of the internal structure, and (c) the classification of this individual structure with respect to its conformity to more general laws governing the constitution of matter. This approach offers possibilities of predictions which advance the deveiopment of our knowledge; it also represents the basic prerequisite for obtaining the compound in a different manner independently of the natural sources. The investigation of proteins, which together with nucleic acids represent the most complicated compounds found in living matter, has also developed through these three stages. The isolation of pure proteins opened the way toward the investigation of proteins, first from the physicochemical aspect and later with respect to their internal structure. As could be assumed, the structure of proteins has been found to be far more complicated than the structure of lowmolecular weight organic compounds. Therefore different laws of a more 167
168
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SORMAND
B. KEIL
complicated character may undoubtedly be expected to underlie the structure of proteins. An effort will be made in the forthcoming sections to summarize the present state of theories concerning the structure of proteins.
A . Homogeneity of Proteins As with all other organic compounds, a fundamental requirement for structural investigation of proteins is the homogeneity of the starting material. Seven years ago the problem as to whether proteins are uniform substances or rather a mixture of mutually related molecules was still unsolved. Sanger (1952) was the first to conclude from his study on the primary structure of insulin, that pure preparations of this protein consist entirely of a population of identical molecules. In contrast to this, Colvin et al. (1954) voiced the opinion that the primary or secondary structures of proteins may be regarded as statistical problems and that they cannot be determined in full. These authors supported the view that, within the definition of organic chemistry, the existence of a primary homogeneity is improbable. Problems pertaining to the primary homogeneity of proteins were considered in detail by Vaughan and Steinberg (1959). In the opinion of these authors, which seems to acquire further support in the light of recent studies, the primary structure of the protein can be expressed in one single formula. At the same time these authors emphasized the necessity of distinguishing between the homogeneity of the primary and of the secondary structure, since the failure to make such a distinction has resulted several times in the past in considerable controversy. The homogeneity of structure established by chemical methods does not necessarily guarantee secondary structural homogeneity of the given protein; similarly, a protein of homogeneous secondary structure, as indicated by physicochemical investigation, may possess a microheterogeneous primary structure. The term “sequential purity” was coined by Steinberg and Mihalyi (1957) for protein preparations comprised of molecules which all have an identical primary structure. The results of experimental work done so far on the analysis of the primary structures of proteins furnkh evidence in favor of the microhomogeneity of proteins. The elucidated structures of proteins ranging in molecular weight from 10,000 to 20,000 are in good agreement even though involved research was conducted in different laboratories and the starting protein was obtained from different individuals of the same species. Assuming that the initial protein samples were microheterogeneous within a certain area of the chain, the partial hydrolysis of two molecules would produce fragments differing in composition. However, when analyzing
REGULARITIES IN THE PRIMARY STRUCTURE OF PROTEINS
1G9
the peptide fragments derived from a homogeneous protein preparation, no peptide sequences have as yet been found which could not be reconciled with a unique structure. An exception which should be noted in this connection is the observation of Keil et al. (1962a, b) of two peptides isolated from a partial hydrolyzate of chymotrypsinogen. These peptides differed in one amino acid residue and cannot have existed in the same sequential order. In this case, the anomalous peptide was obtained in a poor yield. A possibility which cannot as yet be excluded is that chymotrypsinogen, a protein regarded as homogeneous according to the results obtained by present methods, also contains minor components slightly different from the major one, in analogy to the genetically different hemoglobins. However, even a small degree of microheterogeneity in the proteins investigated should not interfere with the unambiguous determination of the dominant structure,
B. Development of Theories Concerning the Primary Structures of Proteins Among all earlier basic theories of protein structure Fischer’s theory (1902, 1906) of the peptide bond is the only one which has not yet been shaken. His concept of proteins as open chains of a-amino acids linked by peptide bonds paved the way toward further knowledge of proteins. Since the first solution of the complete structures of proteins (1951-1953), the number of amino acid residues assigned unambiguously in either complete or partial structures of proteins has increased to 2200. The full structures of seven types of proteins of molecular weights below 20,000 have been elucidated. A complete list of known structures available in the first half of 1959 was published in two reviews by 8orm et aE. (1957, 1961). Proteins in which complete structures are known, or those in which at least the sequence of 15 amino acid residues has been determined, are given in Table I.’ The elucidation of the first complete structures of proteins in 1951-1953 supported the opinion that each protein has an individual specific composition and owes its difference from other proteins to its unique amino acid sequence. It has been suggested that peptide chains are arranged in random sequence (YEas, 1958). I n his paper introducing a series of theoretical studies gorm (1954) pointed out that “this opinion is not in agreement with all the known data concerning living matter. All our experience has shown the existence of only a small number of structurally well-defined groups of compounds in organisms; the formation of these compounds is governed by unique laws at each stage of the 1 Table I completes the data reported by Vaughan and Steinberg (1959). The following abbreviations are used throughout the tables and text : corticotropin, ACTH; melanocyte-stimulating hormone, MSH; tobacco mosaic virus, TMV.
170
P. ~ O R MAND B. KEIL
TABLE I Proteins with Determined Sequence o j at Least I6 A m i m Acids Protein
Origin
ChymotrypRinogen A Beef Clupeine Z Corticotropin
Cytochrome c Fibrinopeptides Glucagon Hemoglobin A Hemoglobin Fa Heme peptide Insulin Lysozyme Melanocyte-stimulating hormone Myoglobin Papain Pepsinogen Ribonuclease TMV protein Trypsinogen Turnip yellow mosaic virus 4
Largest fragment (number of residues) 34
+ 28, etc.
References
Keil (1961) Keil et al. (1962a) Hartley (1960, 1961) Clupea 28 Ando et al. (1901) Beef 39 Li et al. (1958) 39 White and Landmarin (1955) Hog (A) 39 Shepherd et al. (1956) Hog (0) 39 Li et al. (1956) Sheep 104 Horse Margoliash and Smith (1961) Beef fibrinoger 16 18 Sjoquist et al. (1961) 29 Bromer et al. (1956) Hog Human adult 141 146 Braunitzer et al. (1961a) Hill and Konigsberg (1961) Human fetal 37 30, etc. Schroeder et al. (1961) Chromatium 27 Dus et al. (1961) 51 Beef Ryle et al. (1955a, b) 51 Hog, sheep Brown et al. (1955) 51 Horse, whale Harris et al. (1956) 23 20, etc. J o l l h et al. (1960) Egg white Geschwind et al. (1957) 18 Hog-@), beef, horse Harris and Roo8 (1959) Monkey 18 Lee et al. (1961) Human 22 Harris (1959) Sperm whale 151 Kendrew et al. (1961) Edmundeon and Hirs (1961) Carica papaya 51 34, etc. Smith et al. (1961) Lokiina and OrechoviE 18 Hog (1960) Beef Hirs et al. (1960) 124 Tobacco leave! 157 Anderer et al. (1960) Tsugita et al. (1960) 15, etc. Beef Walsh et al. (1961) Mike6 et al. (1961) Keil (1961) Turnip 5 13 Harris and Hindley (1961)
+ +
+
+
+
+
See Table IV for other genetically different hemoglobins.
REGULARITIES IN THE PRIMARY STRUCTURE OF PROTEINS
171
phylogenetic evolution.” He concluded that the composition of protein molecules may also be assumed to be governed by definite and not unmeasurable regularities and that a random arrangement of amino acids is hardly to be expected. The necessary data for the interpretation of internal regularities in protein structures could be obtained from three different types of experimental approach involving the investigation : (a) of analogies in proteins produced by the same types of cells and the same organs of identical organisms, (b) of different proteins produced by the same organism, and lastly, (c) of proteins of corresponding function, which originated at different stages of ontogenetic and phylogenetic evolution. The third approach has been followed predominantly in many laboratories. A comparison of differences in the primary structures of insulins, corticotropins, and cytochromes c from different species (see Table I) may serve as typical examples of these efforts. The data from these studies provide evidence of the extensive structural relationship between functionally identical proteins from different species; this relationship decreases with the increased difference in species. The search for structural similarities between homologous proteins has been paralleled by efforts aimed at the determination of general regularities in protein structure. Whereas the similarities between homologous proteins are obvious at first sight, this approach represents a complex of observations, suggestions, and unanswered problems and is at present far from leading to a general theory. This subject will be dealt with in the following sections.
11. TYPES OF STRUCTURAL REGULARITIES IN PROTEINS The complete and partial protein structures known today obviously account for only an infinitesimal fraction of the macromolecular species contained in living organisms However, even the available data can become a valuable source of information provided they are examined from a biological point of view, bearing in mind that the chemical structures of proteins (and, of course, nucleic acids) reflect the evolution of species in the phylogenetic history of life on our planet. It may be assumed that the differentiation of living matter proceeded not only at the level of biological structures but also at the level of molecules and their interactions. If it is conceded that a comparison of metabolic processes supplies information about the evolution of living matter complementary to the purely biological evidence then it seems only fair to assume that the structures of those molecules which determine these processes should themselves bear the imprint of the evolutionary process; of the individually “random” genetic changes only those will survive which are in harmony with evolutionary
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AND B. KEIL
trends. Excellent evidence for the existence of such regularities in the primary structure of proteins has been provided by the discovery of the analogies in structure of the active centers in a number of enzymes. There is no reason t o suppose that these regularities, corresponding to specific structural requirements, are confined to such centers; the active centers play only a limited, though critical role in the function of the protein as a whole. From these considerations it appears legitimate to approach the analysis of protein primary structures on the assumption that the structures of individual proteins will not be unique in the sense to be expected if they were determined purely by random mathematical combination but that they will be governed by certain definite principles and laws, even though these may be highly complex and not as yet accessible to our understanding in their entirety; and though the factual knowledge a t present available may be too fragmentary to permit successful prediction even on a limited scale. The most valuable confirmation of this view to date is, without doubt, to be found in the known structures of homologous proteins and peptide hormones, that is compounds of identical biological function isolated from different species. As is well known, the primary stsuctures of the homologous insulins, corticotropins, hypertensins, posterior pituitary hormones, and heme peptide sequences from cytochromes c are closely similar and differ only a t certain definite sites in the peptide chains.2 These can, in particular, serve as a useful point of departure in a search for more general principles governing protein structure, and in the comparison of different proteins.
A . Lower Peptide Sequences The fundamental consideration in any discussion of general principles governing protein primary structure is, of course, the degree of regularity in the distribution of amino acid residues along the peptide chains. The first problem to arise concerns the frequency of short peptide sequences in proteins: are certain such sequences preferred and others discriminated against, or even “forbidden”? Certain conclusions about this problem can be reached by a straightforward analysis of the available experimental material (YEas, 1968; gorm, et aE., 1957, 196l), and by comparison of this material with a mathematical treatment (Williams et al., 1961, Borm and
* The specific differences in homologous proteins have been reviewed and discussed in several papers; see e.g., Anfinsen and Redfield (1956), V a u g h n and Steinberg (1959), Cibian (1961), Sorm et al. (1957), YEas (1961), Bonn (1961b).
173
REGULARITIES IN THE PRIMARY STRUCTURE OF PROTEINS
Knichal, 1962).3 Both approaches indicate that there is no general preference for any particular dipeptide sequence or sequences. It also seems likely that there will be no forbidden tripeptide sequences since of the 8000 possible combinations of three amino acids as many as 1032 had already TABLEI1 Recurring Lower Peptide Sequences's ~~
~~~
Dipeptides
Occurrences
R
Tripeptides
5
srl
z
sd
2 X 3 x 4 x
17.7 3.8 0.6
3.7 1.9 0.8
2.0 0.0
1.7 0.1
(0)
X x x x
24.0 6.9 2.2 0.4
3.1 2.5 1.4 0.7
(24) (8) (2) (0)
2 x 3 x 4 x 5 x
19.2 4.4 0.9 0.2
2 x 3 x 4 x 5 x
Tetrapeptides Z
sd
0.2
0.7
_
(2) (0)
-
3.9 0.0
1.9 0.2
(4) (0)
0.3
0.4
-
-
-
4.1 (15) 1.8 (2) 1.0 (2) (0) 0.4
3.1 0.1
1.8 0.3
(0) (0)
0.1
0.3
(0)
12.6 3.4 1.0 0.2
3.5 1.7 0.9 0.4
(7) (2) (0) (1)
2.2 0.1
1.9 0.2
(0)
H b A, a-chain 2 X 3 x
6.3 1.3
1.7 1.0
(7) (0)
0.8
HbA,b-chain 2 X 3 x
15.6 4.5
3.2 1.9
(13) (3)
TMV
2 3 4 5
C
T
~
-
_ _
- _ _
_ -
_
- _ _ _ -
_ _ _
_ _ _
(0)
_ -
(0)
0.2
-
0.4
(0)
(0)
1.0
(0)
0.1
0.3
(0)
2.7 1.5 0.02 0 . 1
(1) (0)
0.3
0.7
(0)
_
_
_
-
-
-
-
-
-
_
-
-
_
~~
Repetitions ofdi-, tri-, and tetrapeptides in random sequences corresponding t o ribonuclease (R), TMV rotein, chymotrypsinogen (C), trypsinogen (T), 01 and chains of human hem0 h i n . Means ( 2 ) and standard deviations (sd) for 50 sequences of each type. h g u r e s in parentheses are the values for the naturally occurring sequences. b Williams et al., 1961. 0
been encountered in protein structures by the end of 1959. On the other hand, considering the large number of possible combinations of four amino acids (160,000) it is not possible to exclude the existence of certain forbidden tetrapeptide sequences. 3 The mathematical treatment of structural resemblances in proteins is the subject of Section I V of this review; the numerical values quoted in this section are based on the procedures and results given there.
174
F.
BORM
AND R . KEIL
The mathematical probability that certain peptide sequences will recur in one protein molecule, or that they will occur in two or more proteins, can be determined by statistical methods; one such approach has recently been described by Williams et al. (1961) and will be discussed later. Table I1 (Williams et al., 1961) illustrates the recurrence of di-, tri-, and tetrapeptide units in randomly drawn sequences corresponding to ribonuclease and TMV protein and the partial structures of chymotrypsinogen, trypsinogen, and the a-and @-chainsof hemoglobin. As is shown in this table, the frequencies of occurrence of lower peptide units as determined experimentally by random drawing of sequences closely approach those found in the actual protein structures. More recently, additional complete structures have become available which could profitably be subjected to the same type of analysis, using the data given by Williams et al. for a protein of average amino acid composition. Certain deviations as against the random recurrence of tripeptide and tetrapeptide sequences are found in cytochrome c where one tripeptide unit, in the same sequence sense, occurs three times, and a tetrapeptide unit twice; in addition each of these units recurs once more with reversed sequence sense. Certain indications for a selectivity of bonding are obtained by a comparison of the occurrence of peptide bonds of the types AB and BA in some individual proteins (Sorm et al., 1961). In a sufficiently long peptide chain the statistical probability of both these combinations should, of course, be equal; in the majority of cases, indeed, the experimental data are in agreement with statistical expectation, but in some instances there are deviations, as in the structure of ribonuclease, or in the a- and P-chains of hemoglobin where there are differences in the incidence of several dipeptide combinations of the two sequences senses (Table 111).
B . Linear Analogies between Higher Peptide Structures The observed small deviations from statistical values in the incidence of lower peptide sequences (cytochrome c) are not in themselves useful for defining structural regularities in proteins but they can assist in the search for more complex relations between higher peptide structures within the same molecule of different proteins. As has been shown in several studies by the authors of this review, and later also by Gibian (1961) the best approach in such a search is based on the analysis of those peptide sequences in which the specific differences between homologous proteins are located. At these sites we frequently meet with the interchange of certain structurally or biogenetically related amino acids in corresponding positions of the chain (e.g., in the insulins), or with characteristic short-range rearrangements of identical amino acid residues (as in the corticotropins). The occurrence of such regularities in interspecific differences suggests the possibility that
REQULARITIES IN THE PRIMARY STRUCTURE OF PROTEINS
175
similar structural units, differing only by the interchange of certain definite amino acids, might also occur in nonhomologous proteins, or at different sites in the same protein molecule. The application of these principles of amino acid interchange and rearrangement in the analysis of known protein structures has, in fact, revealed interesting analogies in the structures of higher peptide sequences. In the course of this work, particularly the analysis of the known hemoprotein structures, it has proved possible to define a number of additional amino acid pairs as interchangeable, so that recently one of the authors (gorm, 1962a) has been able to present a relatively integrated, though still preliminary, picture of this phenomenon. TABLEI11 Occurrence of Dipeptide Sequences i n Hemoglobin A of the A B and BA T y p e AB
Number of occurrences
AB
a-Chain Leu.Ser Pro.Ala Val.Asp Val.Glu &Chain Ala.His Lys.Val Thr.Pro Pro.Glu Leu.G1y Gly.Lys Gly.Asp
6 3 3 3
4 4 3
3 3 3 3
BA
1 0
1 1 0
1 0 0
1 1 1
As will be shown in Section V, this picture is also in good accord with current views on the mechanism of genetic changes during phylogeny (Ingram, 1961). Since amino acid interchanges will figure largely in the considerations which follow, a scheme showing the interrelation of the amino acids as envisaged a t present is given in Fig. 1. Full lines directly connecting the symbols of two amino acids indicate standard amino acid interchanges, i.e., interchanges which have been encountered quite regularly; dottedline interchanges are probably also standard but have not been reliably established. As the figure indicates, glycine-the simplest of the amino acids-forms a sort of hub in the scheme.The amino acids interrelated by standard interchanges form four groups, each consisting of chemically and biogenetically
F.
176
BORM
AND B. KEIL
related amino acids. Aspartic acid and asparagine, and glutamic acid and glutamine, are considered as single entities since the conversion of the acids to the amides and vice versa probably occurs readily not only in ontogeny (protein metabolism) but also in phylogeny (differentiation). Eighteen amino acids in all are considered in the scheme. This number of species can, quite generally, be paired in (18 X 17)/2 different ways, i.e., in 153 ways; and since the number of standard interchanges actually assumed is sixteen (cf. Fig. 1) they represent only 10% of the theoretically possible combinations. Figure 2 lists some examples of recurrent linear sequences with standard amino acid interchanges and short-range rearrangements taken from the structures of two proteins! The occurrence of such regularities in all protein structures known so far has been pointed out by one of the authors Glu(N&) -Afp(NH,)
I
LYS
I
A%
I
- Cys Ala I Ser - --
-Gly
I
I I
I I
I I
Ty
Phe
I I
Val
-1
I I I
I
-
Leu -Met
-
I
I Try FIG. 1. Chart of amino acid interchanges.
(sorm, 1959, 1961a); linear analogies in the structure of ribonuclease have also been detected by Lanni (1960, 1961) using a different approach (“sequence vectors”). The same regularities were mentioned by Schwartz (1959) in the case of the corticotropins. It should be noted that analogous sequences may often occur with opposite sequence sense, i.e., with one sequence reversed with respect to the other. An example of this type of analogy is found in the Fig. 2c (a pair of sequences from ribonuclease). These undecapeptide units are remarkable in showing a completely regular interchange of aspartic and glutamic acid and in being terminated by identical (Met) or standard interchangeable (Ala, Thr) amino acid residues in “crossed-over” position. However, one pair of corresponding positions of the two structures contains residues completely unrelated in the sense of standard interchanges (Tyr and Ser, respectively) within the otherwise fully analogous sequences. In the figures, these unrelated residues are marked by vertical arrows. 4
Such sequences will be referred to as analogous.
63 Val. Ala.
[email protected](NH,)
a.
108 Val. Ala. Cys.%.
b.
~
69
c.
Gly. Asp(NH,)
x
Ribonuclease
(9 = 2.819X 10
68
Asp(NHJ Val. Ala. C y s . Lys. Asp(N€&). Gly
I
x
Thr. Asp(NHI,). Glu(NH,)Cys, Tyr.*(N&). 29
Ribonuclease (fi = 2.038X10-10)
1113’
55 61 Glu(N&), Ala. Val. Cys. Se r. Glu(NH,). Lys 62
~
68 Gly
79
Ser. T y r . Ser, Thr. Met
19 Met.Glu(N~).Asp(N~~}Cys.Tyr.A,Yp(NH,).Ser.Ser.Ser.Ala.Ala _I
71 77 Gly. Ala.Ileu. Asp. Arg. - Lys. - Lys
’
I
Myoglobin
143 Lys.*=
Ribonuclease * ( p = 0.703 xlO-l6,
Ala. Reu. Asp.
FIG.2 , Example of linear resemblances in ribonuclease and m>-oglobirl. * The probability of the simult,aneous occurrence of these two pairs of peptide structures, which, of course, do not account for the m-hole number of internal structural regularities in ribonuclease, is 1.968 X 10-27.
a.
50 Asp. Ala. Asp(N&). Lys. &p(N&) 2 Asp. Val, Glu--Lys.%68 Leu. - Glu-
Asp. Pro-
24
b.
Gly. LJ.
- Lys.--Gly. Lys Lys-I
57 Ileu
Cytochrome c
.
10
lleu n e
($NO.3-10
and NO. 68-76=0.8851X
37
Cytochrome c
42 I
Thr-Gly-
W
m
.
81
94
Ileu. P h e . 2 . @. (Lys),. Thr. Glu. Arg. Glu. Asp. 5 11
Val. Phe-
Iku.(Lys),. G l y -
x 4 2
L=.Glu.VJl.Asp
P
2:
Glu(NH,)
96 104 Ala.Tyr. + . ( L ~ S ) , . ~ ~ . & - - G ~ U
C.
)
76 Lys. Lys.Tyr.Ileu.PZ
31 H i s . Lys. Thr.Gly. Pro. Asp(N&)
Gly. Arg -Lys.
-10
10
I
Cytochrome c
d.
e.
20 25 Ala-3. His. Gly. - Lys. Leu 88 94 Ala. His. Ser. H i s Ala. Lys. Leu -
Myoglobin
38 43 Asp.& Leu. Glu. Lys. E. 147 143 Leu. Glu. L y s . m r Lys
Myoglobin
35
f.
I
41
_ Pro. _ -Glu. - Thr. Leu. Glu. Lys. Phe
130 136 G~J. Lys. Ala. Leu. Glu. Leu. Phe
t
FIG.3. Higher analogous peptiderfragments with “gaps.”
Myoglobin 0
q
‘d
5 0
Y
E 2
m
180
F. BORM AND B . KETL
On the basis of current ideas on the mechanism of protein synthesis it is difficult to rationalize the occurrence of analogous structures of inverted sequence sense, and their significance in terms of structural regularity is in itself highly debatable. Their discussion in this context should he regarded as purely phenomenological. It has been suggested that the “perturbation’) of analogous peptide sequences by interpolation of unrelated amino acids in corresponding positions, which has repeatedly been observed, might conceivably have the function of a certain L‘codesignal” in the cybernetic sense. Another structural feature frequently encountered in the analysis of protein structure is the interpolation of one or more amino acid residues in only one of the sequences, with gaps in the corresponding positions of another sequence. This type of perturbation appears to be very common (Borm and Keil, 1958; gorm, 1961a). Recently, it has been pointed out again by Braunitzer (1961b) in his analysis of hemoglobin. Figure 3 gives examples of extensive structural analogies with perturbations due to gaps or the interpolation of unrelated amino acid pairs in the molecules of cytochrome c and myoglobin. Some of these analogous structures show a repetition of residues of one amino acid or short runs of interchangeable residues. If this fact is allowed for, a further simplification and a more pronounced resemblance of the analogous sequences may be achieved. The pair of sequences from cytochrome c listed in Yig. 3c, arranged in this simplified form, may serve as an example. In the structural analogies discussed so far the identical or interchangeable amino acids occupied corresponding positions in the analogous sequences (allowing for gaps). However, in some cases identical or analogous sequences, commonly tripeptides, are extended a t opposite ends by the same amino acid, or identical or analogous sequences, often placed symmetrically. A good example of this kind is provided by insulin (Fig. 4a) where the dipeptide Leu.Tyr (13, 14) follows the sequence Val.Cys.Ser in chain A and (reversed) precedes the analogous sequence Val.Cys.Gly in chain B; both sequences are terminated at the carboxyl end by glutamic acid. This type of analogy is quite frequently encountered in protein structures and appears to have more general significance; it might conceivably arise during evolutionary differentiation of protein structures by a structural unit (in this case, Tyr.Leu) “skipping” one turn of a n a-helix. Further evidence for this type of regularity can be found by comparing the structures of compounds as distantly related as insulin and oxytocin (Fig. 4b). Analogous peptide sequences may occur not only twice, but even three or more times within the peptide chain of a given protein, as in both the
a.
10 15 Val. Cys. Ser. Leu. Tyr. Gh(N&) 16' 21' T z .1 ; Val. s . Cys. Gly Glu
16 Leu. Glu-
b.
'
20 A s ~ N H J Tyr. . Cys
1
(Insulin, A Chain)
5
Cys.Tyr. Ileu.Glu(N&) .Asp(NHJ
C.
(Insulin, beef)
122 126 His. Ala. Ser. Leu-Asp
x
(oxytocin)
8M
(Hemoglobin A, @-chain)
78 85 Asp. (Ala. Leu.*r),.Asp 112 108 His Ala, Leu T &
.
d.
23 31 Val. Gly. G>.%. Ala. Leu. Cly. Arg. Leu 1 3 6 X 143 Val. Ala. Asp.&. Leu. Ma. H i s
ws 70
78 Leu.Ala,His. Leu
4 (Hemoglobin A, 0-chain)
&.Phe.@*Asp.%. Fro. 4. Symmetrical esteusions of nnalogolls fragments.
( p = 1.056 X
-16
10
)
182
F. &OR,
AND B. KEIL
a- and @-chainsof hemoglobin (Fig. 4c, d).
A notable example is found in the a-chain of hemoglobin (Fig. 4c) where the tripeptide sequence Ala.Leu. Ser, doubled in positions 79-84, recurs (with certain modification) in positions 123-127 and (reversed) 108-111. Moreover, two of these sequences are terminated at both ends by aspartic acid or histidine residues, the remaining sequence at one end by histidine. It should be recalled that histidine and aspartic acid (or asparagine) are very probably interchangeable amino acids. As has been pointed out by Gibian (1961) and also by one of the authors (gorm, 1961b) structural analogies may sometimes extend for considerable distances along the peptide chain. In these cases isolated pairs of unrelated (by standard interchange) amino acids may again be encountered at corresponding positions in the two analogous sequences. However, there is a degree of regularity even in these interpolations since the same pair of unrelated amino acids often recurs at such sites, either with the same mem-
AcetyI.
165
FIG.5 . Structural resemblances between peptide fragments of the TMV protein.
ber of the pair in one sequence, or with the members of the pair reversed as between the two sequences at different sites. Examples of such sequences are given in Fig. 5 (TMV protein) and Fig. 6 (insulin).
C . Repetition of Peptide Sequences The repetition of dipeptide and tripeptide sequences in protein primary structures is not, as has already been stated, statistically significant in the case of proteins of high molecular weight. However, such repetitions may become worthy of note if the identical or analogous lower sequences immediately follow one another in the peptide chain (contiguous sequences) or if their recurrence is connected with other, additional regularities (gorm, 1961b, 1962a). Such special instances have been encountered especially in the structures of both hemoglobin chains, and examples are listed in Fig. 7. The remarkable fragment in positions 78-85 of the a-chain (Fig. 7b) not only shows contiguous repetition of a tripeptide sequence but the hexapeptide unit so constituted is bracketed between identical amino acid residues; and, moreover, a tetrapeptide fragment of this sequences has an analogy in positions 123-126 of the same molecule. Again, the sequence
I
REGULARITIES IN THE PRIMARY BTRIJCTURE OF PROTEINS
I
183
F. ~ O R MAND B. KEIL
184
in positions 91-100 of the same chain shows a similar regularity in that two contiguous analogous sequences are symmetrically terminated by two analogous dipeptide sequences (with the standard interchange Arg/Lys) . A further interesting sequence is found in positions 62-71 of the a-chain where two alternate, overlapping pairs of contiguous analogous tetrapeptide sequences can be discerned. Sometimes the regularity of such repetitions is disturbed by interpolations, as in the undecapeptide sequence from Dipeptides 18 23 Val. Asp. Val. Asp. Glu. Val
a.
0-chin
Tripeptides 78 85 Asp. (Ala. Leu. Ser). (Ala. Leu.Ser). Asp 123 126 Ala Ser. Leu. Asp
x .
b.
a- chain
( p = 1.074
100 91 Leu. Ar2. (Val. Asp.Ero). (Val. Asp. Phe) .Lys. Leu
c. d.
134 139 (Val. Ala.%).(Val.Ala.fia)
e.
( S s . Ala. Val). (Thr. Ala. Leg)
a-cliaiii (/I
105 ( x u . Leu.-).
= 2.65 X 10 -')
0-chain /;-chain
100 108 (Leu. Leu. Ser). His. Cys. Leu. (L4eu.Val.Thr)
g-
10 -6)
14
9
f.
X
(Y-
chain
115
Asp. ( V J . Leu.Val). Cys.
(s. Leu.&)
@-chain
Tetrapeptides h.
62 71 (Val. Ala. Asp.Ala).(Leu.Thr. Asp.Ala). (Val.Ala)
a- chain
FIO.7. Special types of recurrence of lower peptide sequences in heinoglohin A .
corticotropin where the second identical tetrapeptide sequence is broken into by a run of three basic (and interchangeable) amino acids (f3orm et al., 1957) :
.Gly.Lys.Pro.Val-Gly.Lys. (Lys.Arg.Arg) .Pro.Val. A particularly fine example of multiple recurrences of short as well as longer sequences, with standard interchanges and short-range rearrangements, is provided by the heme peptide isolated as a fragment from the heme protein of the photoanaerobe Chromatium (strain D) (Dus et al., 1961)
REGULARITIES IN THE PRIMARY STRUCTURE OF PROTEINS
185
probably related to the cytochromes c to which it shows extensive structural resemblances (gorm, 1962a, c) . The great regularity with which the peptide chain of this fragment is constructed is shown in Fig. 8. The probability of such an arrangement of the peptide chain resulting from the operation of chance is very low (4.27 X lo-”) compared with the theoretical number of permutations (4.36 X loz2);in other words, there are only some 100,000 structures of the same composition which would show the same degree of regularity. Yet another regularity in the arrangement of certain parts of protein molecules becomes apparent if the alternate residues in certain sequences are matched against other, full sequences (of the same or opposite sequence sense) (gorm, 1962a, b, c). In some proteins, e.g., the a- and @-chainsof hemoglobin, such a comparison reveals the frequent occurrence of sequence pairs of this type, with one member of the pair an “abbreviated” (by omission of alternate residues) version of the other; with standard interchanges 1
LSel?Ala
.Lys.Cys.His.Thr.Ee__
27 Asp .Glu.Gly.Ser
FIG.8. Internal regularities in the structure of the heme peptide from Chromatiurn
again in evidence. In some instances members of such a pair are set between identical or analogous di- and tripeptide sequences. The probability of chance occurrence of this type of analogy is rather high in comparison with the probability of recurrence of full identical or analogous sequences; however, they are of interest by virtue of their high incidence in structures such as that of hemoglobin and of cytochrome c. Some examples of this type of structural resemblance, which may be classed as a kind of repetition, are given in Fig. 9.
D. Symmetrical Arrangement of Peptide Chains A special case of linear analogy in peptide chains is the symmetrical development of sequences, in opposite sequence sense, in both directions from a single “central” amino acid residue (gorm, 1961a, b; Keil and gorm, 1962). Here again all the familiar structural variations such as standard interchanges, short-range rearrangements, and interpolated residues are met with. Examples have been found in all known protein structures in varying numbers; they appear to be particularly frequent in both chains of hemoglobin and in myoglobin but are rare in cytochrome c. I n the two
16
22
56
a-chain
59
L~s---
Gly-His
-Cly
19 85 Ala.Leu.Ser.Ala.Leu.Ser.Asp
b.
123 Ah-Ser-Leu-Asp
Hemoglobin A
a-chain
126
49 55 Lys.Asp(NH, ).Lys.Asp(NH, ).Ala.Asp.Thr
C.
95 1leu.Ala.Tyr. Leu. Ly s 75 Ileu -Tyr-
LYS
102 Ah--Thr
Cytochrome c
7
($ = 0.666 X 10- )
Ly s
89
- A r g---Lys.Thr
w
ti
LYS
Ileu.Phe .Ala.Gly .Ileu.Lys .Ly s.Lys.Thr -
k u . P h e -GGly
PJ E
72
81
d.
Hernoglobin A
Lys.Val.Gly.Ala.His.Ala.Gly
a.
40
Cytochrome c ($I = 2.03 X 10 -I1)
FIG.9. Peptide fragments recurring with the omission of certain residues.
REGULARITIES IN THE PRIMARY STRUCTURE OF PROTEINS
187
first-named proteins quite complex structures of this type can be made out; they are shown in Fig. 10 together with simpler examples from ribonuclease. In some cases such "symmetrical" structures extend over large regions of the peptide chain, as in the hemoglobin chain (gorm, 1962a; Keil and gorm, 1962) (Fig. 11). A symmetrical histidine residue (No. 20, a-chain) is exactly the junction of two a-helix units. Val Tyr Pro.Asp(NH,)
/
Pro
*-*-
'Val.His.Phe
Ribonucleaee
121 .ASP
40 /h.-Ala.LeLe-Lr.Glu Val 60 G 'u l (NH,). A l a . g . Cys. Se r. Glu (NH,)
Ribnucleaee
(
E
0.558 X 10
Myoglobin
e.
,s.Pro.Pz.Arg Phe '=.Pro.%.Arg
Bradykinin (Elliott etg., 1961)
FIQ.10. Symmetrical development of sequences along the peptide chain.
E. Regularities in the Spacing of Amino Acid Residues The fibrous proteins such as collagen and particularly silk fibroin, and the protamines (clupeine, salmine) appear to exhibit a high degree of regularity (periodicity) in the spacing of certain amino acid residues along the peptide chain. This phenomenon was in its time the basis on which Bergmann and Niemann (1937) built their theory of the regular periodicity of amino acid residues along the protein chains. Thus, salmine has afforded peptide fragments with arginine in 1, 4 spacing in high yield, and in silk
188
F. &ORM AND B. KEIL
fibroin the dipeptide sequence Ala.Gly is thought to alternate with the analogous Ser.Gly over large regions of the chain. However, even in the globular proteins some regularities in the spacing of certain amino acid residues can occasionally be made out. A particularly striking example of this kind is provided by the spacing of lysine and half-cystine in riboiiuclcase (Sorm, lutjla) (Fig. 12). In four instances, lysine residues are spaced 30 residues apart; in three cases alternate residues are involved; in the fourth, two residues are omitted. In two further instances, alternate residues are spaced 29 and 32 positions 136 128 Gly.Ala.Val.Va1. Lys.Glu.Tyr.Ala.Ala Ala. Gly -Val.
20
/
His(
Ala. Gly
<
12 Lys.Gly.Try.Ala.Ala 26 Glu. Tyr.Gly.Ala
Gly-Lys.Va1.-Glu.
His
(P
- chain)
( (Y
- chain)
( (Y
- chain)
53 Ala
65 Gly. Lys. Lys.Va1.Ala.Asp.Ala
56 Ala. Lys.Va1. Lys.PraAsp.Gly 70
His<
Gly. Lys.Lys.Val.Leu-Gly.Ala
-
( @ chain)
Fro. 11. Symmetrical development of the chains of hemoglobin A .
apart, and the remaining two lysine residues of ribonuclease are spaced 2 X 31.5, or 63 positions apart, with four other lysines in between. It therefore appears that the spacing of alternate lysine residues is very regular. Similarly, neighboring half-cystine residues in ribonuclease show spacings of 7 (two instances) and 2 X 7, or 14 (two instances). The remaining spacings (18, 12, 12) average out to 14 and thereby conform to the general regularity. It has further been shown by Morgan (1960) that ribonuclease shows a certain preference for identical amino acid residues in positions (n f 3) and n f 4) corresponding to one turn of an cr-helix. Deviations from this periodicity are shown particularly by serine. On the other hand, no marked regularities of this kind have been found so far in other proteins, although e.g., the alternation of short and long
REGULARITIES IN THE PRIMART WRUCTURE OF PROTEINS
189
190
F. ~ O R MAND B. KEIL
periods between the residues of certain amino acids can be made out. Similarly, a more detailed analysis has failed to confirm the suggestion made by the authors some time ago that a spacing of 1 , 6 occurred more frequently than others.
F. Simplified Structures Obtained W h e n Applying the Principle of A m i n o Acid Interchangeability Several of the characteristic groupings in the complete structure of the protein will become more apparent if the amino acid sequence is expressed in one-letter symbols (gorm el al., 1961). The following abbreviations were used by Keil and Sorm (1962) in their study interpreting protein structure by this approach: Gly,Ala: L Ser,Thr: S Asp,Glu,Amides: A Lys,Arg: I Ileu,Leu,Val: U
Phe,Pro: F Tyr,Try: T Met: M cys: c His: H
When using this system of symbols some of the regularities mentioned in previous sections will be more obvious. Thus hypertensin can be represented by the following formula: F-HU
I
F-HU-TUIA
Similarly, marked structural units are discerned in the formulas of two groups of hormones (MSH and ACTH). A characteristic feature of these units is both the recurrence of several sequences and also their symmetrical arrangement along the chain (Fig. 13). A particularly striking feature of the simplified formula of the tobacco mosaic viruss (Fig. 14) is the stereotype repetition of several simple structures (combinations UI, LS, AF) which in fact represent the cornerstones of the entire molecule. A phenomenon deserving special interest (apart from a number of symmetrical sequences occurring along the whole chain) is the symmetrical arrangement of the dipeptides of the hydroxyamino acid residues a t both terminals of the polypeptide chain. This relative concentration of the serine residues at the ends of the tobacco mosaic virus protein chain has been pointed out by Anderer et al. (1960) as a possible example of nonrandomness. 8 The graphical arrangement of different regions of the chain below each other used in this paper was chosen only for comparative purposes. No attempt was made to express the actual sterical arrangement of chains in the molecule.
REGULARITIES IN THE PRIMARY STRUCTURE OF PROTEINS
191
The simplified formulas may serve as a useful means in the search for regularities in larger polypeptide chains. It must, of course, be borne in mind that any simplification in symbolism will lead to structures which appear to be simpler and more regular than they are in reality. Any generalization on the basis of such simplified schemes alone, without reference to the original structures, is bound to be misleading.
rT I
FL-AA
B -(hog)
H-FITGSFFAI
A I
H-FITL-IFU
M I A
I
H-FITL-IFUL-II ALALA-F-TU -1UF-11 I ASALF-PU-AF
I
ACTH (beef)
I
0-ACTH (hog)
M I A
I
H-FITL-IFUG-11 AALLA-F-TU-IUF-II AULAL-F--PU-AF
FIG.13. Simplified formulas of the pituitary hormones. For the significance of one-letter symbols see text.
111. COMPARISON OF PROTEINS AND THE SEARCH FOR COMMON TYPEBOF STRUCTURAL REGULARITIES Mention has already been made of homologous protein structures. They represent examples of extensive similarities which are so conspicious and generally known that they do not require any further discussion, even though they are direct proofs of specific amino acid replacements. The fact that the same types of regularities as those which have already been
192
F. ~ O R MAND B. KEIL
demonstrated to exist in individual proteins can also be found when comparing different kinds of proteins (which, moreover, are sometimes phylogenetically far apart) was recently considered in detail by gorm (1961b,
1962a, b, c). Therefore only one example of such comparative analysis will be mentioned in this chapter. This analysis was facilitated by the recent success in the elucidation of the structures of heme proteins. The structure of human A-hemoglobin was solved by chemical methods
REGULARITIES IN THE PRIMARY STRUCTURE OF PROTEINS
193
by Braunitzer et al. (1961a, b) and Hill and Konigsberg (1961) (cf. also Konigsberg and Hill, 1961) with the same result. Almost simultaneously a nearly complete structure of sperm whale myoglobin was determined by a correlation of results of X-ray diffraction analysis (Kendrew, 1961) and chemical methods (Edmundson and Hirs, 1961). The amino acid sequence of another heme protein, horse heart cytochrome c, was elucidated by MarTABLEIV Pathological Substitutions in Human Hemoglobin
-
No. in Amino Substituchain acid tion 01 16 LYS Q 57 GlY His Q 58 Q 68 ASP Glu 86
rype o iuhsti.utiono SS
S
S SS
S
Hemoglobin
References
I Norfolk M-Boston Philadelphia, X
Murayama and Ingram (1959) Baglioni (1961) Gerald and Efron (1961) Baglioni and Ingram (1961a, b) Hunt and Ingram (1959, 1960), Baglioni and Ingram (1961a,
C,X
b) 86
Glu
(--)
P7
Glu
SS
G-San
P9 B 12 13 22 B 26
Ser Thr Glu Glu
S (--) SS S
6
B 50 B 63
Thr His His Val Glu
S
B 63 B 67 125
S
S (-1
S Jose
Hunt and Ingram 1959), Hill et al. (1960) Hill et al. (1960), Hill and Schwartz (1959)
6 6
,Ingram and Stretton (1961)
E
Ingram and Stretton (1961) H u n t and Ingram (1959, 1861) Ingram and Stretton (1961) Muller and Kingma (1961)
6 Zurich M-Emory M-Milwaukee Dp-Punj ab
,Gerald and Efron (1961) Baglioni et al. (unpublished)
a S denotes standard interchanges; SS, interchanges of the second order. Interchanges of a higher order are marked by dashes (-).
goliash and Smith (1961) and Kreil and Tuppy (1961). The last few years have also witnessed a considerable progress in the explanation of structural differences between pathological hemoglobins. The results of many studies in this field are summarized in Table IV. The investigation of the y-chain of fetal hemoglobin (Schroeder et al., 1961) permits a comparison of this protein with human adult hemoglobin. If this large body of factual material is extended by some of the older data on heme peptides from homologous cytochromes (Table I) and the structure of the recently isolated heme
194
F. ~ O R MAND B . KEIL
peptide from Chromatiurn (Dus et al., 1961) a large group of proteins is obtained which may be submitted to systematic analysis. A comparison of the two chains of hemoglobin with myoglobin was made by Watson and Kendrew (196l), Braunitzer et al. (1961a, b), Keil and gorm (1962), and gorm (1962a, b). Structural relations between cytochrome c and the remaining heme proteins were studied by gorm (1962~). The position of several amino acid residues at certain definite sites of the chain suggests that these residues play a specific role in the formation of the secondary and tertiary structure (Watson and Kendrew, 1961). Thus, e.g., phenylalanines in positions 43 and 46 (a-chain) and histidine in position 87 (a-chain) obviously participate in the stabilization of the heme. The hydrogen bond linking tyrosine in position 42 and aspartic acid in position 94 stabilizes two sections of the a-helix. At those sites where the chains 20
40
60
80
I00
120
140
FIG.15. Pattern of identical amino acid residues in the a- and @-chainof herno globin A and in the myoglobin chain. Identical amino acids are denoted by black vertical lines. Numerals denote the positions of residues in the chain.
pass close to each other are placed the glycine residues which take up the smallest amount of space (positions 25 and 59 in the &chain, 24 and 63 in myoglobin, and the same positions in the y-chain of fetal hemoglobin). A direct comparison of the two chains of hemoglobin with myoglobin shows that only a certain number of positions are occupied by identical amino acids (Fig. 15). In hemoglobin, 66 positions are occupied by identical amino acids in both chains. Considering hemoglobin together with the myoglobin chain, of the positions which can be matched in the three chains 22 are occupied by identical amino acids. The chains differ in the NH2and COOH-terminal regions. The transposition of the cysteine residues in the a- and P-chain (positions 104 and 112, respectively) can be accounted for by a mutual shift corresponding to one turn of the a-helix (Braunitzer, 1961a). A peculiar feature revealed by the comparison of the hemoglobin chains is the occurrence of gaps within certain limited areas of the molecule (“Sequenzliicken,” see Braunitzer, 1961b). The existence of one of these gaps (positions 50-51 in the a-chain and 50-56 in the &chain) which causes
195
REGULARITIES I N T H E PRIMARY STRUCTURE O F PROTEINS Ser.Ala. Leu.Ser.Asp -Leu
-His.Ala
Hemoglobin A, a-chain (No. 81-88)
Ah. !4’hr-Leu~Ser.Glu---
Leu-His. Pro T h r -Gly Asp-Leu-His.Gly. P h e -Asp-Leu. Glu -1leu.
Hemoglobin A, P-chain (No. 86-92) Cytochrome c
(NO. 28-34)
Ser.His.
Hemoglobin A, a-chain (No. 46-50)
Gly. HisoVal
Myoglobin
(No. 67-61)
Ala. Leu.Thr.Asp.AIa.Va1.Ala. His. Val.
Hemoglobin A, a-chain (No. 65-73)
Ala. Leu. Thr. LysAIa.Va1. Ala. His. Val
Hemoglobin Philadelphia, Hb X
Ser. Leu. Gly. AspAla.Ileu.
Hemoglobin F, y -chain Glu. Leu.
Lys.Gly. Leu-His. Ala. Gly. Val. Ala. Asp.Ala. Leu.Ala. His.
Ala. Phe.Ser.Asp.Gly. Leu.Ala. His. Leu.
x x
Phe.Thr.Pro.Ala.Va1. His.Ala.
Myoglobin
(No. 77-82)
Hemoglobin A, 0-chain (No. 135-143) Hemoglobin A, 0-chain (No. 70-78) Hemoglobin A, a-chain (No. 117-123)
Clu. Asp.Ala. Gly. Gly. Glu. Thr.Leu.Gly. Arg.Leu.Leu.
\ Asp. Ala. Val. Gly. Gly. Glu.Ala. Leu.Gly. Arg.Leu.Leu.
Hemoglobin F, y-chain Hemoglobin
Asp. Glu. Val. Gly. Gly. Glu. Ala. Leu.Gly. Arg.Leu.Leu.
Hemoglobin A, P-chain (No. 21-21)
Asp. Glu. Val. Gly. Gly. Lys.Ala. Leu.Gly. Arg.Leu.Leu.
Hemoglobin E
Asp. Glu. Phe.Gly.
Myoglobin (No. 119-122, No. 89-71)
Ala. Leu.Gly.
Glu. Tyr.Gly. Ala. Glu. Ala. Leu.Glu. Arg.Met Arg.Leu.Leu. Glu. Try.Ser-
GluJleu. Leu -Lys. Try Ala. Leu Gly. Lys.Va1.
&, 6-chain
Hemoglobin A, a-chain (No. 23-32) Hemoglobin A, P-chain (No. 104-106) Myoglobin
(NO. 4-10)
Hemoglobin A, P-chain (No. 13-18)
&a. Glu. Val. Lys.Gly. His. Gly. Lys.Lys.Va1. Ala.
Hemoglobin A, n-chain (No. 53-63)
Ala.Glu. Val. Lys.Asp.His.Gly. Lys.Lys.Va1. Ala.
Hemoglobin Norfolk
Ala. Glu. Val. Lys.Gly. Tyr.Gly. Lys.Lys.Va1. Ala.
Hemoglobin M-Boston
Gly. His. Gly. Lys-Leu.Thr. Leu His Gly. Lys.Lys.
Leu. LysGly.His.Gly. Gly. AspVal-
Myoglobin
(No. 21-26)
Myoglobin
(No.80-76)
Myoglobin
(No. 25-21)
Glu. LysGly. Lys.Lys.Ileu.
Cytochrorne c
T h r .Lys .Lys .Ileu.
Cytochrome c
FIG.16. Comparison of peptide sequences in different heme proteins.
a considerable shortening of the a-chain was also confirmed by X-ray diffraction analysis. If the hemoglobin chains are compared with respect to simple amino acid interchanges (within the definition given in the preceding section) the number of “matching” amino acid residues is increased to 92, ie., 65%
196
SORMAND
F.
B. KEIL
of the a-chain. Under this assumption the number of matching amino acid residues found a t corresponding sites in the a- and /3-chain and the myoglobin chain is 41. These similarities, which obviously reflect the differentiation of heme proteins during phylogenetic evolution, are more extensive than would be Glu.Val-
Hemoglobin A, p-chain (No. 22-24)
Gly -Gly Gly. Lys-Val. S e r . His. Ala. Lys -Leu.
Hemoglobin
4,p-chain
(No. 16-16) (No. 90-94)
Myoglobin
Pro. Lys.Va1. Lys.Ala. His. Gly. Lys.Lys.Va1. Leu.
Hemoglobin A, (3-chain (No. 58-68)
Pro.Lys.Va1. Lys.Ala. Arg.Gly. Lys.Lys.Va1. Leu.
Hemoglobin Zurich
Pro.Lys.Val. Lys .Ala. Tyr.Gly. Lys.Lys.Va1. Leu.
Hemoglobin M-Emory
Pro.Lys.Va1. Lys.Ala. His. Gly. Lys.Lys.Glu. Leu.
Hemoglobin M-Milwaukee Hemoglobin F, y-chain Myoglobin
(No. 96-69)
Asp.Leu.Lys .Val. His. Gly. Phe Leu.Arg.Val.Asp.Pro.Va1.Asp Lys. Phe Leu.His.Val.Asp.Pro.Glu. A s p Arg.
Myoglobin
(No. 58-63)
Val. His. Leu.Thr.Pro.Glu. Glu. Lys.Ser.
Hemoglobin A, p-chain (No. 1-9)
Hemoglobin A, a-chain (No. 91-99) Hemoglobin A, 0-chain (No. 96-104)
Leu Thr.Pro.Asp.Ala.. Val-
Hemoglobin A, p-chain (No. 48-53)
.
Leu.Ser Pro.Ala. Asp.Lys.Thr.
Hernoglobin A, a-chain (No. 1-8)
Val,His. Leu.Thr.Pro.Val.Glu. Lys.Ser.
Hemoglobin S
Val. His. Leu.Thr.Pro.Lys.Glu. Lys.Ser.
Hemoglobin C, Hemoglobin X
Val. His. Leu.Thr.Pro.Glu.Gly.
Lys.Ser.
Hemoglobin G-San Jose
Val. His. Leu.Thr.Pro.Glu.Glu.
Lya.Thr.
Hemoglobin
4,8 - chain
Gly. His. Phe.Thr.Glu. Glu. Asp.Lys.Ala.
Hemoglobin F, X - chain
Met.Leu.Thr -Glu.Glu. Lys. Ala Ser. His. Ala.Thr.Pro Glu.Glu. Leu.
Cytochrome c
(No. 65-60)
Myoglobin
(No. 90-62)
FIG.16. Continued.
judged only from a glance a t the basic chain. When comparing polypeptide fragments of the heme proteins we find, as in the case of the single chain, a number of characteristic groupings which either overlap, or differ in one single amino acid and interpolated structure, or in a gap (gorm, 1962a, b; Keil and 1962). Three polypeptide groupings of this type comprising 9, 11, and 13 residues are given in Fig. 16 as an example. Apart from the sequences of the a- and @-chainof hemoglobin and of myo-
germ,
197
REGULARITIES I N T H E PRIMARY STRUCTURE O F PROTEINS
globin, the sequences derived from cytochrome c and anomalous hemoglobins have also been considered. As may be seen from the given formulas a great number of fragments can be fitted into a scheme with a considerable degree of uniformity; the deviations from this scheme usually represent replacements of amino acids, mostly by closely related interchangeable residues. At the same time, the number of these deviations does not actually exceed the differences found when, for example, comparing the well-known homologous heme peptides of cytochrome c which are derived from the same region of the molecule. The importance of the similarities established in our case is even greater when taking into account the fact that hemoglobins, myoglobins, and cytochrome c have been compared, and that these proteins are from species phylogenetically far apart (man, whale, horse, respectively). The fragments given in three groups (Fig. 16) account for 55 % of the entire hemoglobin molecule.
d I
M
40
1
I
n
/ 3 1
M
20
o I
o
80
400
120
I
I
I
I
n
O
I
0
60
I
n
I
o
I (=I
I
I
0
I
o n
FIG.17. Peptide fragments of myoglobin (M) matched against identical peptide units in the 01- and p-chain of hemoglobin A.
If regions showing resemblance to myoglobin are selected in the schematically represented structures of the a- and &chains of hemoglobin, we find that these regions are confined only to certain definite and considerably limited areas of the molecules (Fig. 17), which moreover are very closely related in both hemoglobin chains. This leads us to assume that myoglobin represents a molecule formed at an earlier evolutionary stage. The data available, however, are as yet too meager to permit a definite statement as to whether the two chains of hemoglobin developed from the common structural prototype in parallel or successively. In his scheme representing the development of heme proteins, Ingram (1961) has also anticipated that myoglobin may be the fundamental structure from which the chains of both normal and pathological hemoglobins were formed. The principal solution of this topical problem, however, may be expected to emerge from a comparison of hemoglobin and myoglobin of the same species.
198
F.
BORM
AND B. KEIL
IV. MATHEMATICAL APPROACHTO THE EVALUATION OF STRUCTIJRAL SIMILARITIES BETWEEN PROTEINS Structural similarities of peptidic fragments contained in primary protein structures or structural relationships found for some protein pairs are in some cases of such a character as to be almost immediately obvious (cf., e.g., the heme protein from Chromatiurn). However, the question whether all similarities considered are real should be established by a rigorous application of the calculus of probability. Two attempts in this direction were made recently: the first one, by Williams et al. (1961), is experimental in nature while the second reported by Sorm and Knichal (1962) represents a theoretical approach. The former authors generated and analyzed random amino acid sequences of appropriate sizes and compositions using an electronic computer. They started with eight infinitely large pools of symbols the compositions of which corresponded to certain proteins of completely or partially known structure on the one hand, and to the average amino acid composition of some proteins on the other. A set of fifty random sequences of appropriate length was drawn from each pool and the data were evaluated statistically from the point of view of the repetition of di-, tri-, and tetrapeptidic fragments respectively. The average numbers of repeated peptides in the random sequences were compared with experimental results for some individual proteins; in addition the numbers of common fragments of random sequences drawn from different pools were compared with the numbers of common peptides obtained experimentally from different proteins. Some of the data published by these authors are reproduced in Table I1 (for discussion see Section 11, A ) . This very stimulating work of Williams and co-workers at present takes into account the lowest peptidic structures. The statistical insignificance of these structures was pointed out some time ago by the authors of the present article (8orm et al., 1961). 8orm and Knichal (1962) considered a general solution of the probability of occurrence of certain groups of symbols in long sequences of these symbols. The only assumption made in this treatment is the nonoverlapping of the peptidic fragments, the probability of occurrence of which is being considered; in other words, the symbol groups studied are assumed to have no common divisor. Let z denote the number of different amino acids occurring in the protein molt.cule (usually z = 20), the numbers of molecules of individual amino A, , and the total number of acids composing the protein being XI , X2 , all amino acids, N . The total number of possible structural variations of
-
a ,
REGULARITIES IN THE PRIMARY STRUCTURE OF PROTEINS
199
the protein (or of finite sequences composed from the element) is given by the well-known formula
Let us now consider, e.g., three groups (peptidic fragments), T , , T z , T 3, occurring kl-, kz-, and ks-times respectively in the protein sequence. The number of possible sequences containing such repeated sets of symbols will be denoted by the symbol P I C , k 2 / r 3 . In each sequence, we may assign the group TI possibly occurring there an index z 1, similarly T 2and T 3 may be assigned the indices z 2 and z 3 respectively. In this way a new sequence is formally obtained; for the total number of such sequences the following general formula was derived:
+
+
+
where $ i = X i - h,+lXI - X,+& - Xz+3X? (i = 1, 2, ..., z) is the number of the ith elements (amino acid residues) in the new sequence. The symbols X i ,X: , and A? denote the number of the ith elements in the groups (peptides) Tl , T z , and T3 respectively. Finally,
+ +
N = XI X++a (3) Application of this general formula leads to somewhat tedious computations even for repeated dipeptidic groups since it is found necessary to start with the maximum possible frequency of occurrence of the group in question; thereafter it is possible to calculate the probabilities of its low repetition numbers. As a particular case, let us consider the probability of sixfold repetition of the dipeptidic group Leu.Ser in the a-chain of hemoglobin; there are 18 leucine residues and 12 serine residues in the molecule. The dipeptidic group studied may therefore occur up to twelve times and we have to start the computation of the number of sequences containing twelve groups Leu.Ser. The probabilities of occurrence of some repeated structures obtained in this way are given in Table V. The general formula has to be used in calculations of the probabilities of occurrence of the lowest peptidic fragments. However, for the repetition of larger peptides beginning with tetrapeptidic structures the general formula may be considerably simplified and, to a good approximation, we may write for the number of sequences containing repeated groups T 1 ,
T,,
i
T M i
200
F.
BORM
AND B. KEIL
where
X, = A, - klA: - kzA: - - . -- kMAiM) (i = 1 , 2 , ,2 ) *
a
(5)
*
and 1V = X I
+ >;z +
+ X, + kl + kz +
+
Jcni
(6)
The probability of occurrence of such a sequence is then, to this approximation,
TABLE V Probability of Occurrence of the Repeated Sequence Lezder in the a-Chain of Hemoglobin A PIZ= 2.330392 X
lO-l3
P ~ =o 4.12145 3% = 1.84702 P8 = 4.98694 P7 = 0.86319 Pa = 0.99030 PF, = 0.76392 P , = 3.95608 P3 = 1.34798 Pz = 0.28814 Pi = 0.34821
1O-O 10-7 10-6 10-4
Pi, = 4.91379 X lo-” X X X X X X
10-3
10-2 10-2 X 10-1
x
This simplified formula was used in calculations of the probabilities of occurrence of random similarities in protein structures as discussed in the preceding paragraphs of this article. However, since the actual structures considered exhibit certain “regular perturbations” as e.g., rearrangements, standard interchanges of amino acids, and “gaps,” the probabilities calculated using the formulas given above had to be modified by some additional rules. With the aid of such formulas it is possible to obtain estimates of the probabilities of occurrence of various structures based on the over-all composition of the protein studied. However, these figures alone would not establish the question of significance of structural similarities in protein molecules since one cannot compare directly the experimentally found number of times a certain peptide occurs in the protein studied with the predicted probability of occurrence of a random sequence containing that repeated peptide. In this connection, two cases studied by both groups of
REGULARITIES I N THE PRIMARY STRUCTURE OF PROTEINS
201
authors may serve as an illustration of the methods used: Sorm and Knichal calculated the probability of occurrence of a sequence containing the sixfold repeated dipeptide Ileu.Ser found in a-hemoglobin as 0.99 X 11-3 while according to Williams et al. the frequency of occurrence of such a repetition is rather low (-0.1 f 0.3); on the other hand, for the repeated tetrapeptide Val.Glu.Lys.Gly in cytochrome c the latter authors find the same frequency of occurrence (0.1 f 0.3)6whereas Sorm and Knichal obtain the probability of 3.5 x 10-7. It should be noted that all the mathematical interpretations presented so far have approached the problem by analysis a t the level of individual phenomena. Even model structures drawn entirely at random may frequently exhibit regularities (symmetrical arrangement, repetition of sequences with interchange of individual elements, etc.) . Only a comprehensive mathematical treatment of all observed regularities within the structure of an individual protein and also between different proteins will make it possible to judge the significance of such regularities.
v. RELEVANCE OF STRUCTURAL
REGULARITIES TO PROTEIN SYNTHESIS
In the preceding sections of this review it has been demonstrated that the known protein primary structures, when considered from certain general aspects, exhibit definite resemblances in the construction of their peptide chains. These resemblances might be interpreted as accidental deviations from average randomness if they only concerned individual structures. Admittedly the protein structures available for detailed study are a t present rare and exceptional cases; on the other hand, there is evidence that the same structures, with some variation, recur in different forms of life and that therefore each of the proteins of fully known structure is representative of a whole family of related structures. These findings validate the working hypothesis on which these analyses were based-namely, that the structural differentiation of proteins proceeded in parallel with phylogeny, that is the evolutionary differentiation of the various forms of life. Moreover, even in their present tentative form these conclusions can serve as a guide in attempts to elucidate the actual mechanisms operative in the differentiation of protein structures, and as a stimulus to new ideas on the process of protein synthesis, particularly in its final phases involving interactions with the specific nucleic acid matrices (cf. Gamow and YEas, 1955, 1958; Gamow et al., 1956; YEas, 1960). The multiple recurrence of certain peptide sequences as such or in specifically modified form within the same protein molecule as well as in a 6 Values for the recurrence of peptide structures in a protein of average composition taken from Williams et al. (1961).
202
F.
SORM AND B. KEIL
number of different proteins (as set out for a group of heme proteins in Fig. 16 by way of example) in particular gives rise to speculation about the nature of primary protein structures in the prehistory of life. It seems legitimate to assume that the rarly forms of proteins, or more generally biopolymers, were much simpler in structure than the present-day, largely differentiated systems. Two extreme views could he put forward about the genesis of these early biopolymers. According to the first, the primitive proteins were already polymerized systems which had arisen by purely chemical polymerization reactions. Such systems, even if constituted from only a limited range of units, should possess completely random structures and it is difficult to imagine that in the course of further evolution they should have developed regions showing the observed degree of resemblance in their lower structures. Alternatively, the genesis of complex systems of high molecular weight may be envisaged as having proceeded by composition from simpler peptide structures, that is molecules of lower molecular weight, followed by further specific structural changes in the course of evolution. This mode of genesis of the multicomponent polymers might well have left traces in the structures of contemporary proteins in the form of characteristic repetitions such as linear analogies and “symmetrical” deployment of sequences. The second alternative is also supported by the marked resemblances found in the structures of functionally unrelated present-day compounds such as insulin on the one hand and the posterior pituitary hormones on the other (gorm, 1961b). These relations suggest that even at later stages of evolution the differentiation of new proteins or biologically active peptides proceeded under the influence of existing, phylogenetically older proteins or their structural fragments; or that such fragments might even have served directly as the basis for the genesis of new proteins. A relation of this kind admittedly presupposes a modification of the currently accepted unidirectional scheme of protein synthesis : DNA
--t
RNA -+ protein
to allow for some form of reverse transfer of information, which can by no means be excluded on present evidence:
- - -...,
,-
DNA
-+
-- -_.\.
,
y’
RNA
-, protein
Further processes of differentiation which gave rise to the gaps in analogous structures or to “abbreviated” analogies could only be discussed in terms of unsupported conjectures, with the single exception of the phenomenon of standard interchanges which appears to be very general and relevant to the genetic differentiation of living organisms even at the present time. Our tentative conclusions about these interchanges are summarized in the
REGULARITIES IN THE PRIMARY STRUCTURE OF PROTEINS
203
chart on Fig. 1 which has already been briefly discussed. As will be recalled standard interchanges are symbolized here by full lines. A further examination of the experimental data shows a relatively high incidence of interchanges between amino acid residues linked in the chart through a third, that is related by two successive standard interchanges (e.g. Gly/Glu, Ser/ Asp) ; these may be called second-order interchanges. Other interchanges are much less frequent; but among them, again, third-order interchanges (by way of two intermediate links) predominate, and interchanges of a TABLEVI Relations between Amino Acid Pairs When Comparing the Complete Structure of the a - and @-Chainof Hemoolobin A" Type of amino acid pair
Number
%
Identical amino acids Standard interchanges Second-order interchanges Third-order interchanges Interchanges of higher orders
67 36 16 11 5
50 27 12 8 3
The values in per cent were calculated without regard t o the existing gaps. TABLEVII Amino Acid Interchanges i n the Protein Component of Several TMV Mutantsat Type of Substitution
Amino acid
Ser/Thr Ala/Asp Pro/Leu Ser/Phe
S
SS (naturally occurring mutant)
ss (-)
The symbols used are: S for the standard interchange; interchange; (-) for an interchange of higher order. * Tsugita and Fraenkel-Conrat, 1961. 0
SS for the second-order
higher order are quite exceptional. A striking illustration of these relations is provided by a comparison of the full structures of the peptide chains of both normal and pathological hemoglobins. Table VI classifies the amino acid pairs in corresponding positions of the a- and @-chainsof normal hemoglobin (allowing for gaps) ; only 8 % of these pairs are found to be related by third-order interchanges, and merely 3 % by higherorder interchanges. A still more convincing picture emerges from an examination of the structural differences between normal and pathological hemoglobin chains (Table IV) : in the majority of cases the amino acid replacements responsible for these differences are standard interchanges or second-order interchanges, and only in one case, that of hemoglobin S, are
204
F. &ORM AND B. KEIL
the amino acids involved (Glu/Val) unrelated in this sense. Again, the structural differences between the proteins of mutant strains of TMV, although most of them are induced by chemical treatment of the virus (Tsugita and Fraenkel-Conrat, 1961), correspond in most cases to standard or second-order interchanges (Table VII) . The selective interchange of amino acids undoubtedly arises from certain peculiarities of the process of protein synthesis. In tracing their came it is relevant to consider the nature of the interchangeable pairs. It turns out that these generally involve amino acids which are similar in their chemical structure, and often also are related biogenetically (as shown by the four groups in Fig. 1). This is true not only of the groups around serine and valine and of the cyclic amino acids but also of the group including the acidic and basic amino acids: glutamine is sterically somewhat similar to lysine, and histidine, which is very probably interchangeable with arginine, resembles it both chemically and sterically. It therefore appears that in general the molecular shape and charge distribution are of decisive importance for the existence of a standard interchange relationship. The actual source of such interchanges might then be sought either in certain “specific” errors of the biosynthetic mechanism, or, more probably, specific changes in the matrix structures of the nucleic acids (see, for example, Ingram, 1961; Gierer, 1961). If it is accepted that the replacement of one amino acid by another originates from a certain change in the structure of a ribonucleic acid molecule then standard interchanges would appear to be due to individual readily occurring elementary structural changes in the relevant region of the matrix; second-order interchanges on this view would be caused by two successive elementary changes of this kind, and thirdorder interchanges by three successive changes, or by a smaller number of processes occurring less readily. These ideas can be developed in an obvious way by reference to the hypothesis which implicates a t least three nucleotide units of the nucleic acid matrix in the specific incorporation of any amino acid residue into a protein; with a standard interchange corresponding to a change in a single nucleotide residue, etc. (YEas, 1961). Since, however, this hypothesis is still not sufficiently comprehensive (e.g., there is no suggestion about the way in which the participation of a particular trinucleotide sequence of the overlapping sequence is secured), the more general formulation ascribing amino acid interchanges merely to a specific change in the ribonucleic acid still appears preferable. At the time of writing, rapid advances are being made in both the theoretical (e.g., Crick et al., 1961) and experimental (Nirenberg and Matthaei, 1961; Lengyel et al., 1961) approaches t o the coding problem. Whatever the final outcome of this work may be it is evident that the observed regularities of amino acid interchange will have
REGULARITIES IN THE PRIMARY STRUCTURE O F PROTEINS
205
to be taken into account in any theory of the mechanism of protein synthesis.
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Hirs, C. H. W., Moore, S., and Stein, W. A. (1960). J . A m . Chem. Soc. 236, 633. Huehns, E. R., and Shooter, E. M. (1961). Nature 189, 918. Hunt, J. A., and Ingram, V. M. (1959). Nature 184, 640, 870. Hunt, J. A., and Ingram, V. M. (1960). Biochim. et Biophys. Acta 42, 409. Hunt, J. A., and Ingram, V. M. (1961). Biochim. et Biophys. Acta, 49, 520. Ingram, V. M. (1961). Intern. Congr. Biochem., 6th Congr., Moscow, Symposium No. I. Ingram, V. M.. and Stretton, A. 0. W. (1961). Nature 190,1079. JollBs, J., JollBs. P., and Jauregui-Add61, J . (1960). Bull. soc. chim. biol. 43, 1319. Keil, B. (1961). Intern. Congr. Biochem., 6th Congr., Moscow, Symposium No. IV. Keil, B., and Sorm, F. (1962). Collection Czechoslov. Chert,. Cornmiin. 27, 1310. Keil, B., sorm, F., Meloun, B., VanEEek, 6 . , Kostka, V., nnd I’rusik, Z. (196211). Biochim. et Biophys. A d a 66, 595. Keil, B., Prusfk, Z., Morhvek, L., and germ, F . (19621,). (’olkction Czechoslov. Chem. Commun. 27, 2946. Kendrew, J. C., Watson, H. C., Strandberg, B. E., and Dickerson, R. E. (1961). Nature 190. 666. Konigsberg, W . , and Hill, R. J. (1961). Intern. Congr. Biochem., 6th Congr., Moscow, Symposium No. I. Kreil, G . , and Tuppy, H. (1961). Nature 192, 1123. Lanni, F . (1960). Proc. Natl. Acad. Sci. U.5’.46, 1563. Lanni, F . (1961). Proc. Natl. Acad. Sci. 77.S . 47, 261. Lee, T. H., Lerner, A. B., and Buettner-Janusch, V. (1961). J. Biol. Chem. 236, 1390.
Lengyel, P., Speyer, J. F., and Ochoa, 8. (1961). Proc. Nall. Acad. Sci. U . S. 47, 1936.
Li, C. H., Geschwind, I. I., Cole R. D., Rttnckc, I. D., Httrrix, J. I., and Dixon, .J. K. (1956). J. A m . Chem. Sac. 87, 5067. Li, C. H., Dixon, J. S., and Chung, D. (1958). J . Am. Chem. Soc. 80,2587. LokHina, L. A . , and OrechoviE, V. N (1960). Compt. rend. a d . sci. U . R. S . S. 133, 472.
Margoliash, E . , and Smith, E. L. (1961). Nature 192, 1121. Mikes, O., HoleyHovsky V., TomBHek, V., and sorm, F. (1961). Collection Czechoslov. Chem. Commun. 26, 1048. Morgan, R. S. (1960). J . Mol. Biol. 2, 243. Muller, C. J., and Kingma, S. (1961). Biochim. et Biophys. Acta 60,595. Murayama, M., and Ingram, V. M. (1969). Nature 183, 1798. Nirenberg, M. W., and Matthaei, J. H. (1961). Proc. Natl. Acad. Sci. U . S.47,1558. Ryle, A. P., and Sanger, F. (1955a). Biochem. J . 60, 535. Ryle, A. P., Sanger, F., Smith, L. F., and Kitai, R . (1955h). Biochem. J. 60, 541. Sanger, F . (1952). Advances in Protein Chem. 7,60. Schroeder, W. A., Jones, J. T., Shelton, J. R., Shelton, J. B., Cornick, J., and McCalla, K. (1961). Proc. Natl. Acad. Sci. U . S . 47, 811. Schwartz, D. (1959). Nature 183, 464. Shepherd, R. G., Wilson, S. D., Howard, K . S., Bell, P. H., Davies, D . S., Davies, 5. B., Eigner, E. A., and Shakespeare, N. E. (1956). J . Am. Chem. Soc. 78,5067. Sjtjquiet, J., Blomback, B., and WallBn, P. (1961). Arkiv K e m i 16,425. Smith, E., Kimmel, J. R., and Light, A. (1961). Intern. Congr. Biochem., 6th Congr., Moscow, Symposium N o . IV. Sorm, F . (1954). Collection Czechoslov. Chem. Commun. 19, 1003. Sorm, F . (1969). Collection Czechoslov. Chem. Commun. 24, 3161.
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Sorm, F. (1961a). Collection Czechoslov. Chem. Commun. 26, 1174. Sorm, F. (1961b). Collection Czechoslov. Chem. Commun. 26, 1180. germ, F. (1961~). Intern. Congr. Biochem., 6th Congr., Moscow, Closing Lecture. $or,, F. (1962a). Collection Czechoslov. Chem. Commun. 27, 303. Sorm, F. (1962b). Collection Czechoslov. Chem. Commun. 27, 994. sorm, F. (1962~). Collection Czeehoslov. Chem. Commun. 27, 1001. gorm, F., and Keil, B. (1958). Collection Czechoslov. Chem. Commun. 23, 1575. Sorm, F., and Knichal, V. (1962). Collection Czechoslov. Chem. Commun. 27, 1988. gorm, F., Keil, B., HoleySovskf, V., Knesslovh, V., Kostka, V., Masiar, P., Meloun, B., MikeS, O., Tom&Bek, V., and Vankhek, J . (1957). Collection Czechoslov. Chem. Comniun. 22, 1310. Sorm, F., Keil, B . , VanEEek, J., TomhBek, V., Mike&,O., Meloun, B., Kostka, V., and HoleySovskf, V. (1961). Collection Czechoslov. Chem. Commun. 26, 531. steinberg, D., and Mihalyi, E. (1957). Ann. Rev. Biochem. 26,373. Tsugita, A . , and Fraenkel-Conrat, H. (1961). Federation Proc. 81, 254. Tsugita, A . . Gish, D. T., Young, J., Fraenkel-Conrat, H., Knight, C. A., and Stanley, W. M. (1960). Proc. Natl. Acad. Sci. U . S. 46, 1463. Vaughan, M., and Steinberg, D. (1959). Advances in Protein Chem. 14, 115. Walsh, K. A., Kauffmann, D. L., and Neurath, H. (1961). Federation Proc. 20,385. Watson, H. C., and Kendrew, J. C. (1961). Nature 190, 670. White, W. F., and Landmann, A. A. (1955). J. Am. Chem. Soc. 77, 1711. Williams, J., Clegg, J. B., andMutch, M. 0. (1961). J. Mol. Biol. 3, 533. Y&s. M. (1958). I n “Symposium on Information Theory in Biology” (H. P. Yockey et al., eds.), p. 70. Pergamon Press, New York. Y6as, M. (1960). Nature 188, 209. Y6as, M. (1961). J. Theoret. Biol. 2, 244.
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CROSS-LINKED DEXTRANS AS MOLECULAR SIEVES By JERKER PORATH Institute of Biochemistry, University of Uppsala, Uppsala, Sweden
I. Int>roduct,ion.. . . . . . . . . . . . . . . . . . ............................. 209 11. Principle and Mechanism. . . . . . . ..................................... 211 111. Simple Fractionations Employing Highly Cross-Linked Gels. . . . . . . . . . . . . . 211 A. Separation of Salts and Other Small Molecrilnr Size Solutes from Proteins and Peptides.. . . . . . . . ........................... 211 B. Methods Based on Complex Formation and Dissociation.. . . . . . . . . . . . . 213 C. Methods Based on Solubility E q ........................... 216 IV. Separation of Peptides and Proteins lar Sieving., . . . . . . . . . . . . . . 216 A. Procedures Based on the Interplay between Several Sorption Factors. . 218 B. Gel Filtration in Mixed Solvents. .................................... 221 C. Graded Molecular Sieving of Proteins. . . . . ...... V. Concluding Remarks ................... ..................... 224 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I. INTRODUCTION The term “molecular sieving” was introduced by McBain in 1926. Molecular sieves have found widespread use particularly in petroleum chemistry through the natural and synthetic zeolites. Molecular-sieve phenomena were observed by Deuel and Neukom (1954), Lindqvist and StorgArds (1955), and Lathe and Ruthven (1955) in connection with the passage of solutions of peptides and proteins through beds of uncharged granular materials. Furthermore, molecular sieving has been known to participate in ion-exchange chromatography (for references see Helfferich, 1959) and in electroosmotic and electrophoretic migration through gels (Synge and Tieelius, 1950; Smithies, 1955). It was the introduction of cross-linked dextran gels, however, that made molecular sieving a valuable tool in preparative chemistry of polymers such as proteins. The chromatographic properties of these new materials and their use in protein chemistry are the topic of this paper. Selected examples will serve to illustrate fractionation principles. An exhaustive review of published work in the field is not attempted. Several gel-forming cross-linked hydroxylic substances have been synthesized and tested for their molecular-sieve properties. The list includes cross-linked products of locust bean gum (Deuel and Neukom, 1954) polyvinyl alcohol, sorbitol, cellulose, starch, and partially hydrolyzed 209
210
JERKER PORATH
dextran (Porath and Flodin, 1959). The last-mentioned types of substances are produced commercially for chromatographic use under the trade name of Sephadex. Epichlorohydrin serves as a cross-linking agent. The essential features of Sephadex can he visualized from the following schematic formula: 0-
\
CHz
\
-0.
The position of the preferentially substituted hydroxylic groups is not known. This is probably a matter of little importance, as is, perhaps, the presence of some non-l,6-glucosidic linkages. An unknown number of one-sided bound glycerol residues is also attached to the network. The carboxylic group content of the cross-linked dextran is very low but will increase on exposure to alkali in the presence of oxygen. The amount of cross-linking agent, the chain length of the dextran monomer units, and a number of other parameters must be carefully controlled in the synthetic process in order to yield a product of specified ability to absorb water and accommodate solute molecules within a specified size range. Sephadex G-25 is a strongly cross-linked dextran, whereas G-200 is characterized by its low degree of cross-linking. More information on synthesis, and physical and chemical properties is given in a recent publication by Flodin (1962). Recently synthesis and molecular-sieve properties of other kinds of polymers have been described. Cross-linked polyacrylamide, polyvinyl pyridine, and polyvinyl carbitol may turn out to be very useful (Lea and Sehon, 1962; H j e r t h and Mosbach, 1962). The chains need not be interconnected with homopolar bonds. In starch, for example, the polyglucose chains are kept together by hydrogen
CROSS-LINKED DEXTRANS AS MOLECULAR SIEVES
211
bonds. The same is true for agar and agarose. These latter polymers can also be used for gel filtration (Polson, 1961; H j e r t h , 1962).
11. PRINCIPLE AND MECHANISM Consider a bed of granular uncharged gel subst,ance in equilibrium with surrounding interstitial liquid. When nonionic solutes are allowed to pass through the bed they will migrate with a speed relative to the moving liquid which is determined by molecular size and shape and the solid content of the gel. The substances migrate in symmetrical, slowly expanding zones if the bed is uniformly packed. The rate of development is not very critical and may be higher than in other chromatographic procedures in comparable beds. The migration of peptides, proteins, and other polyelectrolytes may also be governed solely by steric factors under appropriate conditions. Often, however, adsorption will contribute to solute-gel interaction. Lathe and Ruthven (1956), in a discussion of the penetrability of solutes in starch grains, suggested that solute molecules may penetrate only to the depth a t which their diameter is equal to the distance between the amylopectin chains. This should explain why the stationary volume available for a solute is determined by its molecular size. They expressed the viewpoint that the graded penetration of solute according to this mechanism might be unique to starch. In view of present knowledge i t appears that most, if not all xerogels may act as molecular sieves. Flodin (1962) has advanced the idea that extensive cross-linking may occur in scattered regions within the gel grains. Thus clusters of high density of glucose residues are formed. Solute molecules are screened according to molecular size when they approach the interior of the clusters. Based on simple models for screening a relation between K D, molecular size and water regain may be derived (Porath, 1962b). Craig (1962) has observed that molecular size and shape affect gel filtration and dialysis through porous membranes in a parallel manner indicating a relationship between the two procedures. 111. SIMPLEFRACTIONATIONS EMPLOYING HIGHLYCROSS-LINKED GELS A . Separation of Salts and Other Small Molecular Size Solutes f r o m Proteins and Peptides I n 1954 Deuel and Neukom briefly pointed out that beds of granulated cross-linked locust bean gum could be used to separate saIts from highmolecular weight solutes (this fact was unknown t o Dr. Flodin and me until 1961 when Professor Deuel kindly informed us of it). Desalting of proteins was one of the first applications of Sephadex (Porath and Flodin, 1959). Distilled water was first used as a developer for the serum proteins
212
JERKER PORATH
which are soluble in 50 % saturated ammonium sulfate solution. However, many proteins are precipitated or adsorbed at low concentration of salts. In most cases (but not all; see below) this is a complication. Sephadex is particularly useful for buffer exchange. When exhaustive removal of salts is required Sephadex may be used in a two-step procedure. The original buffer is exchanged for a volatile buffer system and the latter is subsequently removed by lyophilization. Gel filtration compares very favorably with dialysis through membranes (Kisliuk, 1960). It is rapid and can be performed on either a micro- or a macroscale. The desalting of virus by this procedure has been reported by Matheka and Wittmann (1961). Eluents of ionic strength in the range 0.01-0.05 often yield very satisfactory results, but for highly charged proteins (and other polyelectrolytes) high concentration, sometimes very high concentration of salts may be required for satisfactory results. Conditions for desalting protein solutions have been studied by Flodin (1961). Figure 1 illustrates how the amount of applied sample affects the separation efficiency. The salt maxima appear in the same place. The performance of Sephadex chromatography, and gel filtration in general, is remarkably reproducible at moderate or high solute concentrations. The sorption isotherms of ions are curved at extremely low solute concentrations which explains some anomalies reported in the literature, e.g., those of Spitzy et al. (1961). In the case cited radioactively labeled iodide in quantities of the order of 10-9 gm was used. No retention was observed in distilled water. This is in agreement with earlier findings for acids and negatively charged compounds (Gelotte, 1960; Porath, 1960). A progressively increased retention of iodide was found in a series of experiments with increasing concentrations of sodium chloride in the developing solvents. Thus a small but significant increase in elution volume was found even between 1 and 10 % sodium chloride concentrations. Based on these findings a very satisfactory method was devised for analysis of proteinbound iodine. The method was considered to be much superior to those employing trichloroacetic acid precipitation or dialysis. Bill et al. (1960) used Sephadex to remove excess iodide from iodinated albumin. A sample as small as 0.02 ml was added to the top of a 0.3 X 25-cm column. During the development in distilled water each effluent drop was assayed for radioactivity and protein. Among other similar applications the convenient removal of excess reagent after protein modification should be mentioned (Anfinsen and Haber, 1961; Killander et al., 1961; Zwaan and van Dam, 1961; Lipp, 1961; Curtain, 1961; Rinderknecht, 1962).
CROSS-LINKED DEXTRANS AS MOLECULAR SIEVES
213
Effluent (ml)
Effluent (mi) FIG. 1. Gel-filtration diagrams obtained in two similar experiments, the difference being the sample volume applied t o the column: A, 10 ml; B, 400 ml; 00--0 =hemoglobin; X--X--X = sodium chloride. (Flodin, 1961.)
B. Methods Based on Complex Formation and Dissociation Consider the problem of isolating a low-molecular weight substance from a mixture of similar compounds. This problem can be solved in a very elegant manner with Sephadex if some suitable polymer can be combined with the substance under study to form a specific soluble and stable
214
JERKER PORATH
complex. A subsequent filtration through a Sephadex column will then remove all free low-molecular weight solutes (Fig. 2a). After dissociation the polymer is separated from the desired substance(s) by another filtration this time in a solvent that will not allow reassociation to occur (Fig. 2b). Solute concentration
Effluent volume
a
Solute concentrafion A'
Effluent volume
b FIG.2. Gel-filtration diagram illustrating how formation and dissociation of a complex can be used for selective fractionation. Further explanation in the text.
An excellent example of application of the first step of this technique has been reported by Daisley (1961). The vitamin Blz content of oceanic waters cannot be determined at sea water salinity. A binding agent prepared from pig pyloric mucosa may be added to the sea water before filtration through a column of Sephadex suspended in distilled water. If the binding agent is present in excess, vitamin B12 will be quantitatively com-
CROSS-LINKED DEXTRANS AS MOLECULAR SIEVES
215
bined as a complex that can easily be freed from salt when passed through the column. The fractions predicted to contain vitamin B,, are combined, concentrated with dry Sephadex, and assayed (Flodin et al., 1960). It should be possible to find many similar applications in peptide and protein chemistry. Wilcox and Lisowski (1960), for example, have shown how Sephadex can be used to study complexes between metal ions and proteins. Figure 2a and 2b may also serve to illustrate the principle of a very efficient method for the purification of a low-molecular weight compound of a natural complex. It has been used to concentrate the peptide hormdnes oxytocin and vasopressin (Lindner et al., 1959). An active proteinpeptide complex is extracted under mild conditions from pituitary gland powder. The extract is filtered through a Sephadex column to remove free peptides, amino acids, and other small molecular size solutes. The complex in peak A, Fig. 2a, is split in strong acid solution and subjected to another filtration. This time the protein peak will be inactive (A’, Fig. 2b) and all activity is recovered to peak B’. It is a rapid procedure which yields excellent recoveries. The chromatographic pattern is often a function of pH and ionic strength. This is not to any appreciable extent caused by alteration in the swelling of the gel since the molecular sieving of neutral substances like polysaccharides is essentially unaffected except occasionally a t very high salt concentration. The dependence on the medium, however, reflects breaking or formation of aggregates or changes in the state of ionization. The choice of buffer system is therefore a matter of considerable importance. Bassett et at. (1961) have described purification of galactosyl-serum albumin antibody. Sephadex G-25 was used to remove residual antigen and p nitrophenyl-P-D-galactoside. Urea affects the gel as well as the state of aggregation of solutes. Stepanow et al. (1961) have shown that formation of peptide-peptide complexes may be avoided in phenolate or alkaline urea solutions. Two peptides derived from tobacco mosaic virus protein could be separated with Sephadex only in the presence of 8 M urea. Urea need only be included in the sample solution, not in the eluting solvent (0.01 d l sodium hydroxide). Upon filtration on a Sephadex G-50 column, urea and the two peptides moved as well-separated zones. The authors did not comment on the choice of G-50. It was probably found to be superior to G-25 since urea seems to close the pores and meshes of a Sephadex gel. The reduction in effective pore and mesh size is perhaps caused by urea being bound to the carbohydrate network since the swelling is in fact increased in strong urea solutions.
2 16
JERKER PORATH
C . Methods Based on Solubility Equilibria Protein mixtures can be fractionated on Sephadex G-25 despite the fact that the proteins are excluded from the gel phase. Fractionation may be based on differences in protein solubility. Consider, for example, what happens if serum is allowed to pass through a gel column in distilled water. Globulins are at first precipitated in the column because of the progressive removal of ions from the protein solution. When brought in contact with the advancing salt front the precipitate will dissolve. Two peaks, therefore, appear in the chromatogram. Two cases have been described, both dealing with human serum. Epstein and Tan (1961) reported the occurrence of four peaks when human serum dissolved in 0.2 M sodium phosphate buffer p H 8, was chromatographed on Sephadex G-25. Only the first two peaks contained proteins. The protein separation was proved to be dependent on solubility characteristics. Euglobulins were retarded and among them rheumatoid macroglobulin was recognized. Fjellstrom (1962) demonstrated that the method can be used t o separate complement factors. When filtration is made in acetate buffer, ionic strength 0.02, pH 5.1-5.6, the complement factor C’2 moves together with albumin and pseudoglobulins while factor C’1 and other euglobulins trail behind. The method is claimed to be superior to other methods heretofore described for the preparation of complement reagents. Salting out and subsequent extraction on a gel column is also possible and can be performed in the following way. A gradient of a low-molecular weight precipitating agent is introduced in the column, followed by the protein mixture. In the first stage of the development (with buffer) the proteins move faster than the gradient and regions will be reached where precipitation occurs (Fig. 3). As development proceeds the precipitated proteins will dissolve and move again. This is an extremely interesting procedure which unites the characteristic features of chromatography and crystallization. This principle will be discussed elsewhere (Porath, 1962a). Rapid and reversible precipitation and dissolution is essential for successful outcome of the above-mentioned methods.
IV. SEPARATION OF PEPTIDES AND PROTEINS BY MOLECULAR SIEVING Distribution coefficients obviously cannot exceed unity when they are solely determined by molecular-sieve equilibria. This fact does not seriously detract from the value of the gel-filtration method. Even substances with K Dratios close to unity can often be separated in long columns or by recycling procedures (Bennich and Porath, 1962). When filtrations are performed under appropriate conditions Sephadex and similar gel substances act as almost ideal molecular sieves. These gels,
CROSS-LINKED DEXTRANS AS MOLECULAR SIEVES
217
therefore, are unique among the sorbents used in chromatography. Given other conditions, however, additional factors come into play. They can easily be controlled and used to advantage, for example by employing gels concentration precipitating column agent 4+a
concentration precipitating column agent
distance from column head
"
"
a
b
E'io. 3. Diagrams illustrating the use of a concentration gradient of a precipitating agent in a gel column t o achieve separation of two components A and B according to their solubility. The position of the concentration gradient (left) and t h e samplecontaining zone a t the s t a r t of filtration is indicated in diagram a. During filtration the high-molecular weight substances A and B move faster than the gradient substance (diagram b ) . When they have traveled to a position in the gradient where precipitation occurs the speed will be reduced. If A and B precipitate a t different concentrations of gradient substance they will move in separate zones a t a final speed equal to t h a t of the gradient.
of different swelling capacity or by use of suitable solvents. Peptide and protein separations are therefore possible even in cases where high selectivity is required. Gel filtration is a good complement to other methods. It may be used for separation of complex peptide or protein mixtures before
218
JERKER PORATH
or after application of ion-exchange or paper chromatography (Pierce and Webster, 1961). Craig and collaborators have extensively supplemented countercurrent extraction with gel filtration (Hill and Konigsberg, 1960; Rasmussen and Craig, 1961). Occasionally the more laborious countercurrent extraction method can be replaced by Sephadex filtration (Rasmussen and Craig, 1962).
‘I
A . Procedures Based on the Interplay between Several Sorption Factors Aromatic and basic substances are adsorbed on Sephadex columns. Aromatic sorption is usually completely reversible and if electrolyte concentration is high enough this is also the case for adsorption due to electrostatic interaction. Negative adsorption or ion exclusion is encountered for acidic substances in sufficiently weak electrolyte solutions. The conditions for molecular sieving can be altered by changing the degree of cross-linking of the gel. Electrostatic interaction can be depressed or eliminated by increase in ionic strength or by proper choice of pH. Likewise, sorption due to aromaticity can be altered by p H change or by including urea in the solvent. Such alterations in sorption characteristics deserve particular attention since they permit the utilization of independent “separation parameters.” Extensive use of combinations may be very profitable for fractionation of complex mixtures. The potentialities in this field may perhaps be surmised from the following examples. Viscotoxin, a basic peptide of molecular weight ca. 9000 (Samulsson, 196l), moves unretarded in strongly cross-linked gels such as Sephadex G-25 (Fig. 4a) (in phosphate buffer, ionic strength 0.05, pH 6.8. When filtered under similar conditions in weakly cross-linked dextran, viscotoxin behaves quite differently (Fig. 4b). In fact it moves behind isoleucine. The gel of the first kind can be used to remove solutes of lower molecular size, the purification being based on molecular exclusion. Filtration in the second kind of gel may be used not only for separating solutes of different molecular size but also to separate peptides and other substances of similar molecular size when they differ in certain structural features. I n a study on gel filtration of tryptic hydrolyzates of a-casein, Bennich (1961) presented results of interest in this connection. On Sephadex G-25 he obtained a single peak containing bound hexoses. A gel as expanded as Sephadex G-75 was necessary to reveal the presence of several hexosecontaining peptides. They are probably of different molecular size. The intermediate swelling grade of Sephadex, G-50, gave optimum conditions for separation of phosphopeptides. Because of their strongly acidic properties these peptides are probably subjected to some kind of ion exclusion
CROSS-LINKED DEXTRANS AS MOLECULAR SIEVES
219
E 1. 5
lsoleucine
.. .. ..
I.c
0.5
i
..
i
.. 300
400
500 MI effluent
(a)
b) FIG.4. Elution diagrams showing the behavior of a basic polypeptide on columns of gels of different degree of cross-linking. Further explanations in the text. (Porath, 1960.)
220
JERKER PORATH
superimposed on molecular sieving. The most distinct separation of ninhydrin-positive materials was achieved with Sephadex G-25. Each kind of Sephadex possesses sorption properties that in some respect makes it 1.8-
-1.8
1.6-
- 1.6
1.4-
- 1.4 10
I-J
c
0
1.2-
-1.2
5Q)
1.0
A1 cm. 275mp 0.8
0.6
0.4
0.2
0
20
40
60
80
100
Effluent milliliters FIG.5. Fractionation of tryptic digest of 22.6 mg of oxidized ribonuclease A on Sephadex G-25 in 0.2 M acetic acid. Column size, 0.9 x 150 cm; flow rate, 6.3 ml/ hr; fraction size, 1.0 ml; -O--O--O-= absorption at 275 ms; --0-0-0= ninhydrin analysis. (Eaker, 1962.)
superior to the others. When employed in appropriate order these filtrations can be expected to yield very effective fractionations. Eaker (1962) has recently studied the behavior of tryptic digests of oxidized ribonuclease A and delysylglutamyl-ribonuclease A in narrow G LnhoAnv UGpllCW-LGA
nnhimno
CIVIUIIILIU.
Uo Fnirnrl tha+ thn n n n t t r l n m nf tI,llG L n hl lvJTU r ll-U n ll.JT~n c n t% n M ;nf LLG l W U I l U UIIC2U U l l G p G p U l U G D Ul UI
oxidized ribonuclease grouped themselves into three sharply separated zones in Sephadex G-25 in 0.2 M acetic acid (Fig. 5 ) . The first zone con-
CROSS-LINKED DEXTRANS AS MOLECULAR SIEVES
22 1
tained 19-22 amino acid residues; the second zone contained tetra- and heptapeptides. The third zone contained, together with di- and tripeptides, one hepta- and one decapeptide, each of the two latter containing two tyrosine residues. This distribution of peptides is in accord with expectations based on earlier findings.
B. Gel Filtration in Mixed Solvents As mentioned in the previous section a gel of proper swelling capacity, i.e., degree of cross-linking,permits considerable selectivity in the molecular sieving. Sometimes, however, it is difficult to find a suitable gel substance. In such cases favorable conditions may be found with mixed solvents (Porath and Lindner, 1961). Oxytocin and vasopressin cannot easily be separated from the common aliphatic amino acids on Sephadex G-25 in spite of a considerable difference in molecular size. In a solvent consisting of acetic acid, pyridine, and water (60: 15:25 parts by volume) the separation is an easy matter. The explanation is very simple. The gel is not capable of extensive swelling in this solvent and consequently the pores are smaller and the peptides are therefore excluded. This technique has also been used to separate a- and @-meIanophore-stimulating hormones of the hypophysis (Porath and Schally, 1962). Gel filtration in mixed volatile solvents provides a convenient way to desalt peptide mixtures; a problem often difficult to resolve. Synge and Youngson (1961) have described filtrations on Sephadex G-75 in a mixture of phenol, acetic acid, and water (1:l:l by weight). Complexes between proteins, peptides, and amino acids are not formed in such a solvent. Of course, liquid-liquid partition may play a role. In this connection the possible use of Sephadex for liquid-liquid chromatography should be mentioned. According to Moore (1961) Sephadex may offer certain advantages over cellulose as a support material for the stationary phase.
C . Graded Molecular Sieving of Proteins A great number of proteins have been purified on Sephadex G-75 (Bjork and Porath, 1959; Hanson and Johansson, 1960; Pr6aux and Lontie, 1961; Bjork, 1961; Palmstierna, 1961; Ames et al., 1961; Pettersson et al., 1962). Considerably improved gel types have been introduced recently thanks to pioneering work by Flodin. These gel substances, named Sephadex G-100 and G-200, are produced in the form of spherical beads. They can be packed in beds with excellent filtering properties; this is particularly true for G-100. The behavior of serum proteins has been studied by Flodin and Killander (1962). As is seen in Fig. 6, 7 S and 19 S 7-globulins can be separated on Sephadex G-200. When Pettersson (1962) filtered a commercial sample of Aspergihs
222
JERKER PORATH
500
600
700
800
900
1000
1100 1200 MI effluent
t
FIG. 6. Fractionation of proteins in 50 ml of human plasma on Sephadex G-200. 1 M NaCl, pH 8.0; elution rate, 68 ml/hour; column size, 7 X Eluent, 0.1 M Tris 50 cm; absorption measured with LKB Uvicord recorder. Region A contains ~ Z M (19 S 7.) and 01~~-gIobulins, 01- and 0-lipoproteins, and fibrinogen; B, 7 S ~ - g l o h u l i n ; C, transferrin; and D, albumin. (Flodin, 1962.)
+
CROSS-LINKED DEXTRANS A S MOLECULAR SIEVES
223
cellulase through a small column (1.5 x 38 cm) of Sephadex G-100 he obtained four separate zones with cellulolytic activity and two well-separated zones of 0-glucosidase (Fig. 7). The recovery of activity was excellent. The same type of gel has also been successfully applied to highly
2.5
2.a
P
f a
1.5
1.0
0.5
Fraction number
FIG.8. Gel-filtration diagram of serum albumin prefractionated by ammonium sulfate precipitation. Gel, Sephadex G-200, 20G400 mesh; buffer, 0.1 M Tris, 0.2 M NaCl, p H 8.0; column size, 5 X 110 cm; elution rate, 40 ml/hr; sample volume, 7 ml; fraction volume, 10 ml. Ordinate, absorbancy a t 278 mp; abscissa, fraction number. (Pedersen, 1962.)
purified bovine thyrotropin (Condliffe and Porath, 1962) and pancreatic enzymes (Gelotte, 1962). The molecular-sieving effect on proteins has been very clearly demonstrated by Pedersen (1962). Bovine serum albumin, obtained by fractionated ammonium sulfate precipitation, was filtered through a column of cross-linked dextran of water regain 15 gm/gm (Sephadex G-200). Monomer and dimer forms were easily separated from each other and from other components of larger sedimentation constants (Fig. 8). Heterogeneity in the monomer as well as in the dimer preparation was indicated
224
JERKER PORATH
by the asymmetric shape and spreading of the distribution curves. By appropriate cuts and refiltration the presence of small amounts of contaminants of intermediate K n values was confirmed. Very good preparations of monomer and dimer could also be obtained. The reproducibility of the gel filtration is extremely good and the spreading of the monomer zone can be used for checking the quality of a n albumin preparation. The methods described in earlier sections can also be applied to proteins. Since gel filtration can be carried out under conditions that favor aggregation or dissociation (for instance in urea solutions) it should be a useful tool in studies of protein-protein interaction. The following examples may be mentioned. Polymers of ribonuclease can be separated with Sephadex G-75 in 0.2 M phosphate buffer of p H 6.47 (Crestfield, 1962). Cruft (1961) has reported that histones can be separated on G-75 in 0.02 N HC1. Since a-histones were eluted first they seem to be present as polymers. Partially purified acid phosphatase from h’scherichia coli (v. Hofsten and Porath, 1961) moves as a complex at and above pH 4 but as a lowmolecular size protein in acetic acid. An observation made in this study is worth mentioning. When freed of metal ions the enzyme can be stored in the cold for long periods of time, but the activity disappears within a few seconds when traces of sodium chloride are added. The use of Sephadex was indispensible for transferring the enzyme from extract to acid medium without losing activity.
V. CONCLUDING REMARKS Gel filtration in its various forms is an efficient tool for preparations in protein chemistry. It can also be used to test the supposed homogeneity of a protein or peptide preparation. A gel column calibrated with respect to its molecular-sieving properties may also be used to estimate the molecular size of biologically active or otherwise labeled substances before their isolation has been accomplished. Needless to say, the result of such an estimation must be cautiously interpreted. Dextran gels and other gel substances mentioned can be used as support in electrophoresis. It should be of great value to find out if similar separations can be obtained with gel filtration supplemented with “pure” zone electrophoresis as with starch-gel electrophoresis according to Smithies (1955). This survey of the present stage of gel-filtration methods as applied to proteins and peptides has been confined mainly to the dextran gels. It should be realized that the methods outlined should be applicable also to other gels with similar sieving properties.
REFERENCES Ames, B. N . , Martin, R . G . , and Garry, B. J . (1961). J . Biol. Chew&.236, 2109 Anfinsen, C . B., and Haber, E. (1961). J . Biol. Chew.. 236, 1361.
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Basset, E., Beiser, S. M., and Tanenbaum, S. W. (1961). Science 133, 1475. Bennich, H. (1961). Biochim. et Biophys. Acta 61, 265. Bill, A., Marsden, N., and Ulfendahl, H. R . (1960). Scand. J . Clin. & Lab. Invest. 12. 392.
Bjork, W. (1961). Biochim. et Biophys. Acta 49, 195. Bjork, W., and Porath, J. (1959). Acta Chem. Scand. 13, 1256. Condliffe, P., and Porath, J. (1962). To be published. Craig, L. C. (1962). Personal communication. Crestfield, A . M. (1962). Personal communication. Cruft, H . J. (1961). Biochim. et Biophys. Acta 64, 611. Curtain, C. C. (1961). J . Histochem. and Cytochem. 9, 484. Daisley, K . W. (1961). Nature. 191, 868. Deuel, H., and Neukom, H . (1954). Natural Plant Hydrocolloids 11, 51. Eaker, D . (1962). To be published. Epstein, W. V., and Tan, M. (1961). J . Chromatog. 6, 258. Fjellstrom, K . E. (1962). Acta Pathol. Microbial. Scand. I n press. Flodin, P. (1961). J. Chromatog. 6, 103. Flodin. P. (1962). “Dextran Gels and Their Application in Gel Filtration.” Thesis, University of Uppsala. (Available from AB Pharmacia, Uppsala, Sweden.) Flodin, P., and Killander. J . (1962). Biochim. et Biophys. Acta. I n press. Flodin. P., Gelotte, B., and Porath, J. (1960). Nature 188, 493. Gelotte, B. (1960). J. Chromatog. 3, 330. Gelotte, B. (1962). To be published. Granath, K. A., and Flodin, P. (1961). Makromol. Chem. XLVIII, 160. Hanson, L. b..and Johansson, B. G. (1960). Nature 187, 599. Helfferich, F. (1959). “Ionenaustauscher” Vol. I, p. 122. Verlag Chemie, Weinheim, Germany. Hill, R. J., and Konigsberg, W. (1960). J . Biol. Chem. 236, PC 21. HjertBn, S. (1962). In press. Hjertkn, S., and Mosbach R . (1962). Anal. Biochem. 3, 109. Killander, J., Pontkn, J., and Rodkn, L. (1961). Nature 192, 182. Kisliuk, R. L. (1960). Biochim. et Biophys. Acta 40, 531. Lathe, G. H., and Ruthven, C. R, Y. (1955). Biochem. J . 60, XXXIV. Lathe, G. H., and Ruthven, C. R. Y. (1956). Biochem. J . 62, 665. Lea, D . J., and Sehon, A. H. (1962). Can. J. Chem. 40, 159. Lindner, E. B . , Elmqvist, A., and Porath, J. (1959). Nature 184, 1565. Lindqvist, B., and Storghrds, T. (1955). Nature 176, 511. Lipp, W. (1961). J. Histochem. and Cytochem. 9, 458. Matheka, H. D., and Wittmann, G. (1961). Zentr. Bakteriol. Parasitenk. 182, 169. Moore, S. (1961). Personal communication. Palmstierna, H . (1961). Science Tools 1, 29. Pedersen, K . (1962). Arch. Biochem. Biophys. Suppl. 1, 157. Pettersson, G. (1962). To be published. Pettersson, G., Cowling, E., and Porath, J. (1962). Biochim. et Biophys. Acta I n press. Pierce, J. V., and Webster, M. E. (1961). Biochem. Biophys. Research Commun. 6. 353.
Polson, A. (1961). Biochim. et Biophys. Acta 60, 565. Porath, J. (1960). Biochim. et Biophys. Acta 39, 193. Porath, J., and Bennich, H . (1962). Arch. Biochem. Biophys. Suppl. 1, 152. Porath, J. (1962a). Nature (in press).
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Porath, J. (196213). Chem. Prod. Aerosol News. Inter. Symposium on Pharmaceut, Chem., Florence, 1962. I n press. Porath, J., and Flodin, Y . (1959). Nature 183, 1657. Porath, J., and Lindner, E. B. (1961). Nature 191, 69. Porath, J., and Schally, A. (1962). Endocrinology 70, 738. PrBaux, G., and Lontie, R. (1961). Arch. intern. phusiol. biochem. 69, 100. Rasmussen, H., aiid Craig, L. C. (1961). J . B i d . Chem. 236,759. Rasmussen, H., and Craig, L. C . (1962). Biochim. et Biophys. Acta 66, 332. Rinderknecht, H. (1962). Nature 193, 168. Samulsson, G. (1961). Svensk Farm. Tidskr. 66, 481. Smithies 0. (1955). Biochem. J . 61, 629. Spitzy, H., Skrube, H., and Muller, K. (1961). Microrhim. A c t a p. 296. Stepanov, V., Handschuh, D., and Anderer. F. A. (1961). Z.Naturforsch. 16b. 626. Synge, R . L. M., and Tiselius, A. (1950). Biochem. J . 46, xli. Synge, R. L. M., and Youngson, M. A. (1961). Biochem. J . 78, 31 P. v. Hofsten, B . , and Porath, J. (1961). Acta Chem. Scand. 16, 1791. Wilcox, P. E., and Lisowski, J. (1960). Federation Proc. 19,333. Zwaan, J., and van Dam, A. F. (1961). Histochernica 11, 306.
ELASTIN By S. M. PARTRIDGE Low Temperature Research Station. Downing Street. Cambridge. England
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Occurrence and Morphological Struetiire . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Elastin as :t Component of Animal Connective Tissue . . . . . . . . . . . . . . . . I3 . Microscopy and Histochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Structure of Elastin Fibers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 ) . Submicroscopic Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Age Changes in Elastic Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F . Relationship between Collagen and Elastin . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Physical Properties of Elastin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Elast.ic Yield of t h e Wet Fiber . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Hydration, Swelling, and Solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV . Isolation and Analytical Characterization of Elastin . . . . . . . . . . . . . . . . . . . . . A . Purification Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . €3 . Composit.ion of Elastins from IXfferent Tissues . . . . . . . . . . . . . . . . . . . . . . . ociation with Mucopolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Association with Lipids arid Lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . E . Determination in Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Elastolyt.ic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Occurrence of Elastases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Properties and Mode of Action of Elastases . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Chemical Structure of Elastin., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Soluble Prot.eins J>erived from Elastin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . Ultraviolet Absorption and Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Composition of Peptides from Enzymatic Hydrolysis . . . . . . . . . . . . . . . . . VII . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
1.
227 228 223 2211 232 236 212 247 251 251 25 257 257 262 2(17
270 272 276 276 280 23f
285 2'30 292 297 237
INTRODUCTION
The load-hearing prot.ein fibers of animal connective tissue are of two types, collagenous and elast.ic in different tissues these two proteins a r r present in diffcrent proportions, and in all connective tissues the protein fibers arc embedded in an amorphous ground substance which varies in composit.ion from tissue to tissue . I n mammalian skin, tendons, adipose tissue, arid loosc connective t.issue elastin is a very minor component, but. in certain tissues, particularly the ligaments of the vertebrae and the walls. of the large arteries, clastin forms a large part of the dry matter . Elastin fibers were recognized early by their morphological form and characteristic 227
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S. M. PARTRIDGE
staining reactions, and by their easy yield to stretching when wet; from the outset they were regarded as being composed of a substance quite different from collagen. However, in recent years doubts have been expressed, mainly by physiologists and pathologists, as to whether the protein of elastic fibers is a distinct chemical entity or even as to whether there is a real distinction between collagen and elastin. These doubts are in the main due to the unfortunate circumstance that the chemistry of elastin has been little studied by comparison with collagen,’ and one of the purposes of the present review is to set out the available evidence bearing on this. The task is regarded as urgent a t this time because of the present great interest in the chemical constitution of connective tissue components arising from the study of rheumatism, atherosclerosis, and age degeneration. In a broad sense, the protein of the elastic fiber is readily characterized by chemical methods since this protein, or family of proteins, is among the most chemically inert of any. The resistance of elastin to acid and alkaline hydrolysis approaches that of the keratins. It is insoluble in a wide range of hydrogen bond-breaking solvents at temperatures up to 100°C and swells, but does not dissolve, in phenolic solvents. It appears not possible to bring elastin into solution except by hydrolytic reagents capable of rupturing peptide bonds. Purified elastin has a pale yellow color and a characteristic bluish fluorescence in ultraviolet light. The most ready source of elastin of high purity is the ligamentum nuchae of the larger ruminants, and from this source a protein of constant composition can be isolated by a variety of methods depending on the solubilization and removal of other less inert tissue components. Because of the ease with which i t may be isolated the elastin of bovine ligamentum nuchae has come to be regarded as the type standard, but it is by no means certain that elastins from other mammals or other tissues in the same mammal are of identical constitution. The physiology and morphology of the fibrous components of connective tissue has been reviewed recently by Hall (1959) with special reference to the elastic fiber and its relationship to collagen, in the present review the intention is to bring together recent information on the chemical nature of elastic fibers, and the chemistry of elastin as a protein. 11. OCCURRENCE AND MORPHOLOGICAL STRUCTURE
A. Elastin as a Component of Animal Connective Tissue The specific elements of Connective tissue called elastic fibers are characterized by their high refractivity, their homogeneous filamentlike or ribbonA comprehensive review on collagen by W. F. Harrington and Peter von Hippel appears in Volume 16 of Advances in Protein Chemistry.
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like character, and their easy yield to stretching, but since the introduction of the orcein stain by Tanzer in 1891 and the resorcin-fuchsin method of Weigert (1898) they have usually been identified almost entirely on the basis of these and other selective staining reactions. In loose connective tissue or in skin or tendons, elastic fibers are scarce, but they are highly concentrated in other structures where the situation demands high deformation under small loads and complete elastic recovery after remova1 of the deforming force. These tissues usually have a marked yellow color and are referred to collectively as elastic tissue or sometimes “yellow connective tissue.” The form in which elastin is laid down varies considerably in the different types of elastic tissue. Membranes with a very high elastin content are found in the walls of the larger arteries, in some parts of the heart, and in the trachea and bronchi. In the larger arteries the structural units of the elastic tissue formation are concentric lamellae which are often of rather variable thickness and always contain many irregular openings. In the ligamentum nuchae of some animals, particularly the ox, the structure is quite different and thick longitudinal elastic fibers, of almost circular cross section, form most of the tissue. I n mammals the supporting member of the external ear is formed from a specialized variety of cartilage which has great flexibility and elasticity. It has a yellowish color and the intercellular matrix, which is cartilaginous in character, is penetrated by a lacelike network of fine branching fibers which give the staining reactions for elastin. This tissue is usually described as elastic cartilage and is found also in the walls of the eustachian tubes, the epiglottis, and in parts of other cartilaginous structures.
B. Microscopy and Histochemistry The intercellular material of connective tissue consists of fibers embedded in an amorphous ground substance. The fibers are of three main types: collagenous, elastic, and reticular; each of the three has different staining properties and in healthy tissue there is ordinarily no special difficulty in identifying them by normal histological procedures. Thus collagen has a high content of amino acids with acidic and basic side chains and reacts with acidic and basic dyes under conditions of p H and ionic strength which will allow saltlike linkages to form between the protein macromolecule and the dye ions (Singer, 1952). Reticular fibers appear to be composed largely of collagen but have a high content of bound lipid containing a large proportion of myristic acid (Windrum et al., 195.5). The fibers form characteristically fine networks and are stained densely black by ammoniacal silver nitrate solution (Bielschowasky, 1905). Elastin, in contrast to collagen, has a very low content of amino acids with charged side chains
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{Section IV, R ) and its affinity for dyes with a predominantly acidic or haqic function is low, regardless of the pH of the solution. The staining reactions of elastic fibers have been studied extensively by Lansing and Dempsey and their colleagues and have lieen reviewed by these authors (Dempsey and Lansing, 1954) and also by Hall (19.69). Elastic fihers were found early to stain selectively with orwin and Weigert’s resorcin-fuchsin. They also take up a number of other stains such as Verhoeff’s hematoxylin, Nile blue sulfate and to a lesser extent basic fuchsin, ohmic acid, arid Sudan black (Lansing, 1951). The McManus periodic at.id-Schifl reagent usually gives a light reaction with elastic. fibers and >ometimes selectively stains the periphery of the structures. Mallory’s aniline hlue (1936) stains collagen blue and elastic tissue red. Irseful combined stains for demonstrating elastin, reticulin, and ciollagrn in the same tissue sections havr been developed by 1,ewis and *Jones(19.51) and Humason and Lushbaugh (1960). The technique is based on the use of silver stain for reticulin, orcein for elastin, and picroaniline blue or fastJ green for collagen and other tissue structures. A similar differential staining method for elastic fibers, collagenous fibers, and keratin was proposed by Margolena and Dolnick (39,51). This used orcein for elastin, aniline bliie for collagen, and orange G for keratin. The reaction of orcein with elastic fibers has been studied hy Dempsey r t nl. (1932), Engle and Dempsey (1954), and Weiss (1954). The affinity of elastic fibers for orcein is unaffected by pH over a fairly wide range, but nhove pH 8.,5 the dye changes to a blue color and staining fails. Staining i5 intensified by the addition of alcohol to the staining bath, but is suppressed if the tissue s ions are previously exposed to phenol or naphthol. Orcein is not a pure substance and both the natural and synthetic materials coiitain a mixture of dyes which may be separated by chromatography. I’our colored dyes and one colorless but fluorescent substance have heen isolated (Engle and Dempsey, 1954) and when tested by paper elertrophoresis for net charge, one fraction having a blue color migrated to the negative pole, while other fractions migrated to the anode. In staining tests elastic tissuc stained equally well by the fractions carrying the negative or the positive charge. These observations suggest that ionic forces are not critically involved in the staining of elastic fibers with orcein and the adsorption must be due to secondary short-range forces not involving :I difference in iiet charge. Orcein and resorcin are hoth phenolic derivatives which would bc exprcted to take part in hydrogen-bond formation, hut although hydrogcn bonding has been suggested as the basis for orcein st:Lining there appears to he no factual information concerning the number utid kind of hydrogen-bond sites engaged in the protein molecule. Tn view of the specificity of the reaction it has been thought that orcein staining
ELASTIN
23 1
may be due to some non-amino acid prosthetic group in elastin. However it may equally be due to the unique configuration of the peptide chains in this protein. As will be discussed later, it appears that in elastin the hydrogen-bond forming sites along the peptide barkbone are not involved in interchain bonding. These sites are therefore available for loose bonding with water molecules which may readily be exchanged for phenolic dyes or other hydrogen-bonding solutes. If this view is correct it would be expected that dyes such as orcein might react with denatured collagen and perhaps other denatured proteins. When collagen fibers are treated with strong solutions of rertain salts or are heated in water to temperatures above the critical point, interchain hydrogen bonds are broken and the fibers contract. In their new state the fibers show a rubberlike elasticity similar to that of elastin and the peptide chains may thus be assumed to be kinetically free aiid available for hydrogen bonding with suitable dyes. As will appear later (Section 11, F ) , denatured collagen fibers readily take up the orcein dye under suitable conditions of pH and ionic strength and in stained scctions degenerated fibers of collagenous origin have sometimes been mistaken for fibers of elastin (see (Gillman et al., 19.54). It should be noted that variations in staining reactions between the same type of fiber in different kinds of connective tissue may be caused by differenres in the environmental ground substanre. Thus elastic fibers are usually in close physical association with the ground substance and collagenous structures. In some situations they may be actually coated with components of the ground substance giving rise to the appearance of a sheath. Thus Rhinehart arid Abul-Haj (1911) using a modification of Hale’s (1946) method, showed the presence of an outer layer in intact elastic. fibers which was rich in acidic polysaccharides. Bahr and Huhii (1952) carried out an extensive histological investigation of elastic fibers that had been treated with pepsin in acid solution at 37°C. They noted many differences in the staining behavior of the treated fibers as compared with native elastica and attributed this to the removal of ground substance. Elastic fihers show alterations in staining properties if they are damaged by exposure to enzymes or chemical treatment. Thus Sad (1957) showed that elastic fibers treated with proteolytic enzymes still retained their structure after they had ceased to take up elastic stains. Partridge aiid Davis (1953) s h o w d that if elastin is treated with elastasc, new a-amino and acarbovyl groups are produced prior to complete dissolution. Under suitable conditions of pH and ionic strength these new charged groiips may inhibit the uptake of phenolic dyes, due either to a gross change in net charge or an alteration in charge distribution. In connective tissue diseases, particularly skin carcinoma and degenera-
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tive vascular diseases, there are often accumulations of morphologically unusual fibrillar material which may derive either from collagen or elastin, and the identification of the origin of such fibers by normal histological techniques is difficult. “Elastotically degenerated” fibers have been studied by Gillman et al. (1954). These authors showed that while such fibers stain with orcein and other typical elastic stains they may be distinguished from elastin by a series of specialized staining reactions. They observed that some time after grafting trypsin-treated dermal collagen in man, extensive beds of elastoticalls degenerated tissue can usually be found in the dermis of the host area, and suggested that the elastinlike fibrous masses may have been derived from the original collagen of the graft. I n the study of such degenerated or morphologically unusual fibers, chemical isolation would obviously be desirable as a means of identifying the structures unambiguously on the basis of their amino acid composition. Such a procedure is not always possible, but because of the remarkable stability of elastin it should be readily possible to make the distinction between true elastin and any fiber formation derived from collagen. Isolation, as an aid to histological identification was applied in an elegant way by Carton ~t al. (1960). These authors found that the extensive elastic tissue network in the human lung could not be well visualized under the microscope in conventional thin sections and reported a n isolation method whereby the architecture of the pulmonary elastic network is retained. Blocks of tissue 1.5 cm in all dimensions are extracted in concentrated formic acid a t 45OC until free from collagen, and then washed in running water. The tissue is stained with basic fuchsin, and the blocks are then immersed in warm gelatin solution. When the gelatin has solidified the blocks are hardened in formalin and sectioned. A rather simpler technique is used in the present author’s laboratory for routine examination of homogenates of elastic tissues. The suspensions are autoclaved to remove collagen and most of the ground substance and the remaining clumps of elastic fibers are recovered by centrifuging. The clumps still retain the characteristic morphological features of the original structures and may be embedded in in wax and sectioned. Figure 1 (Partridge and King, 1961) shows preparations made in this way from bovine aorta, bovine ligamenturn nuchae, and bovine ear cartilage.
C. Structztre of Elastin Fibers 1. The Fibers of Ligamenturn Nuchae
Probably because of the ease with which they can be prepared, purified fiber preparations from bovine ligamenturn nuchae have formed the starting point of most chemical investigations of elastin. These preparations how-
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233
ever are by no means typical of the elastic fiber as it occurs in other tissues such as skin, lung tissue, elastic cartilage, or the vascular elastic membranes. I n the ligamentum nuchae of the ox, thick elastic fibers make up the greater part of the tissue. In the spaces between them fine collagenous fibers and a few cells can be seen. These cells are embedded in the amorphous ground substance which contains acidic mucopolysaccharides and fills the remainder of the space between the elastic fibers. The character of the thick elastic fibers is demonstrated very clearly by microscopic examination of purified fiber preparations in which the collagen and mucopolysaccharide elements have been removed by autoclaving. Under the microscope the preparations appear as short, smooth, rodlike fibers of almost circular crosssection (Fig. le). The fibers appear to be remarkably uniform in thickness; measurement of 50 fibers gave a mean value of 6.5 p for the diameter with a variation from 3.6 to 9.9 p (Partridge et al., 1955). 2. Elastic Tissue of the Arteries and Veins
The elastic formations in the arteries and veins are very different in character from the long, thick, relatively homogeneous fibers found in ligamentum nuchae. In the aorta, and the carotid or other large arteries, the elastic structures appear to be very closely associated with collagen and polysaccharide. The structures contain in addition smooth muscle cells which are present in varying amounts in arteries of different kinds. Probably because of the presence of muscle proteins, elastin of high purity cannot be obtained from arteries by autoclaving alone, and additional extraction with dilute alkali is necessary to remove a residue of noncollagenous protein impurity. Harkness and his co-workers (1957) studied changes in elastin content along the length of the aorta system. Elastin was estimated by autoclaving the tissue with water to remove collagen followed by boiling with 0.1 N alkali to remove noncollagenous, alkalisoluble proteins. They reported that in adult dogs the ratio elastin/elastinplus-collagen remained roughly constant at 50-60 % from the aortic valve to within about 5 ern of the diaphragm. The proportion of elastin then decreased rapidly and remained a t about 25-30 % throughout the abdominal aorta and the iliac, femoral, and saphenous arteries. The large elastic arteries consist of three layers which, after the artery has been opened longitudinally, may, with care, be stripped more or less cleanly one from the other. The inner coat has been named the tunica intima or interna, the middle coat tunica media, and the outer coat tunica adeventitia or externa. In the aorta of an adult man the tunzca intima consists of an endothelial layer backed by a layer containing longitudinally oriented branching elastic fibers. Between these fibers are collagenous fibers, fibroblasts, and
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ELASTIN
235
smooth muscle cells. The tunica media accounts for the greater part of the weight of the whole structure and consists mainly of elastic tissue. In the human aorta it takes the form of fifty or sixty concentric elastic membranes about 2.5 p thick between which the interspaces measure 6 to 18 p (Maximotv and Bloom 1953). Between the elastic membranes are thin layers of tissue containing collagenous and elastic fibers, lipid material, smooth muscle cells, and fibroblasts, embedded in a ground substance which appears to contain much acidic polysaccharide. The elastic membranes take up a characteristically wavy form and appear to be penetrated by irregular gaps. The tunica adventitia is rather sharply defined from the tunica media but its outside boundary is less distinct and it, appears to fuse into the surrounding loose connective tissue. It is composed of much the same elements as the surrounding tissue but collagen and elastic fibers are more densely packed in the region adjacent to the tunica media. In Fig. l c is seen a preparation made by repeatedly autoclaving material of the tunica media of bovine aorta. The particles of purified elastin were dried, embedded in wax, and then sectioned. The sections were mounted and stained with Verhoeff’s hematoxylin. Examination of the sections showed that much of the membranous elastic material had been resolved into flat bundles of fine fibrils, arranged parallel, and in registerlike locks of wavy hair. It is common observation that in the aorta and other large vessels the FIG. 1. Bovine preparations stained with Weigert’s resorcin-fuchsin (Partridge and King, 1961). a. Elastin from the elastic cartilage of the external ear. (Magnification: X105.) The tissue was disintegrated in a homogenizer, extracted with cold dilute NaOH, and then autoclaved. The clumps of fibrous elastin were embedded in wax, seetioned, and stained. The mat of elastic fibers retains much of its original structure and the holes left by removal of the chondrocytes can be seen in the purified preparation. b. Ear-cartilage elastin prepared as in (a) after further treatment with 0.1 ,V NaOH a t 98°C for 45 min. The fibers have separated and some appear t o have split longitudinally. (Magnification: X158.) c . Elastin from bovine aorta prepared as for (a). (Magnification: X105.) The fibers are rather thicker than those from bovine ear cartilage and are frequently arranged in parallel wavy bundles. d. Bovine aorta elastin prepared as for ( c ) after further treatment b y hot digestion with 0.1 N NaOH for 45 min. (Magnification: X158.) The fibers are free from surface contamination and the formation of membranous mats by roughly parallel fibers can be seen. e. Elastic fibers from ox ligamenturn nuchae. These are much thicker than fibers from other tissues and remain unchanged after boiling with dilute sodium hydroxide. (Magnification : X 105.) f . Elastin from ox aorta prepared by digesting a t 98°C with dilute NaOH. (Magnification : X343.) The formation of “fenestrations” is clearly illustrated.
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elastic membranes and fibrous structures are structurally closely interrelated with the surrounding reticulin, collagen, and polysaccharides. It is perhaps this close relation with surrounding materials that accounts for apparent slight differences in st,aining properties observed between the elastic structures of vascular and arterial tissues and elastic fibers found elsewhere. Thus Gillman et al. (1957) report that the elastin of the vascular elastic membranes differs tinctorially, morphologically, and histochemically from elastin fibers found in skin, loose arcolar connective tissue, and elast,ic cartilage. 3. Elastic Cartilage
The elast,ic cartilage from the ext,ernal ear of mammals is characterized by the presence of large numbers of cells which are generally similar in form to the chondrocytes of hyaline cartilage. I n elastic cartilage the cells are characteristically round and regular and in the ear cartilage of rabbits they appear to account for about 50% of the tissue. The cells are surrounded by very regular halos or capsules of metachromatic material which resembles the ground substance of hyaline cartilage. In the spaces between the capsules collagen fibers can be observed and also a lacelike network of fine fibrils which give the staining reactions for elastin. The metachromatic capsules appear to prevent the approach of the elastic fibrils to the cells so that around the periphery of the capsules there is a concentration of elast,ic fibers which somet,iincs gives the appearance of a membrane. The cells and ground substance can be removed from elastic cartilage by cutting it in small pieces, suspending in saline solution, and disintegrating the tissue in a homogenizer. The clumps of fibrous mat,erial are collected by gentle cent,rifugation and shaken with further saline until the supernatant is clear. The suspension is then autoclaved in water until no more soluble protein is extracted. The dried clumps of fibers are then embedded in wax and sectioned. The appearance of the preparation after staining with Verhoeff's hematoxylin is shown in Fig. la. It will be seen that the characteristic lacelike structure is st>illretained and the holes that originally contained the cells with their metachromat,ic capsule have survived in spite of the severity of the purification t,reatment. The question of the chemical nature of the protein comprising the clast,ic fibers and its identity or otherwise with elastic fibers from other tissues will be discussed later (Section IV, B).
D. Submicroscopic Structure Early att,empt,s to s h d y the fine structure of elastic membranes and fibers both by X-ray diffractlion (Kolpak, 1935) or electron microscopy revealed no distinctjive patterns of st,ructure (Wolpers, I944), and the for-
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mations were thought to consist of homogeneous filaments of amorphous material. Astbury (1940) and Bear (1952) noted that the X-ray diffraction pattern of elastin from ligamentum nuchae showed only amorphous rings after purification by autoclaving. Untreated ligament, which is known to contain some 20% of collagen, gave a typical collagen X-ray diffraction pattern when stretched. The use of the electron microscope for the study of elastic fibers is difficult because in most tissues the fibers are too thick to be penetrated by the electron beam. Most workers have attempted to split the fibers longitudinally by various means in order to produce fragments thin enough to show details of structure. Early attempts to do this were usually based on partial dissolution of the fibers with enzymes, b u t the resulting electron micrographs showed evidence of extensive damage attributable to the enzyme treatment and the fine structure could not be revealed with clarity. Wolpers (1944) attempted to obtain suitable preparations by treating elastic tissue with pepsin in acid. The preparation from ligamenturn nuchae revealed fibers varying in width from 250 mp to 20 mp, the very smallest seen being 8 mp. No axial periodicity was observed. Fixation in osmic acid resulted in the appearance of fine longitudinal fibrillation, but Wolpers considered that, this was due to the action of the fixative and not intrinsic to the actual structure. The elastica of the mouse aorta, after acid pepsin digestion, osmic acid fixation, and sonic fragmentation appeared as thin fenestrated laminae with many short stubby fibers protruding from the surface. Similar findings were reported by Bahr (1951) who studied elastic tissue from the rat-tail sheath. Like Wolpers he observed a fibrillar structure with a terminal fibril about 8 mp in diameter. The fibrils appeared to be progressively aggregated into bundles and larger bundles, some of the bundles lying straight and others loosely twisted. Gross (1949) used the swim bladder of the carp, the aortas of the adult rabbit, 2-day-old rat, and 5-year-old human and also the ligamentum nuchae of ox as sourres of elastin for electron microscope study. The swim bladder proved a particularly convenient source of material because the collagen was readily removed by heating in dilute acetic acid a t G0"C. Mild fragmentation procedures then reduced the elastic fibers to a suitable size. The elastic formations from swim bladder were revealed as long, contorted, branching fibers ranging from about 30 mp to 500 mp in width and often measuring many microns in length. Elastica from the aorta preparations was much more difficult to disintegrate and fragmentation by freeze-sectioning and high-speed homogenization produced very few tissue fragments small enough for electron microscopy. However, strong sonic vibrations disrupted the strongly cohcrent elastic membranes producing fibers of a size suitable for study. These closely resembled the elastic fibers found in
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the tissue of the swim bladder. Whether the fibers observed were part of the laminae or actually represented the interlamellar fibers could not be determined arid considerable amounts of granular material was also present. The long thick fibers from Zigammtum nuchae were more resistant to fragnieiitation than the fibers of the aortic e1astic.a. Prolonged treatment with 9-kc sonic waves was required to produce even a few suitable fibers, and the fragments were usually small irregular chtunks rather than fibrous units. Apparently these fibers are as strongly coherent in the lateral dirertiori as they are longitudinally, and the few fibers obtained were too thick and dense to reveal any detailed structure. On treatment with crystalline trypsin (Armour) the fibers from the swim bladder and aorta both appeared to suffer partial digestion and an internal fibrillar structure was revealed. The enzyme appeared to remove a matrix sribstaiicc from the surface of the fibers revealing many fine fibrils of almost constant cross section. I n his 1949 paper Gross also reported the presence of regularly (.oiled helical threads in elastics but this was discounted by Franchi and de Robertis (1951) and by Gross himself in 19.51. However there seems little doubt from Gross’ electron micrographs that elastic fibers are riot homogeiieous filaments and that a tendency for the material to be laid down as bundles of closely adherent fibrils is discernible. I n 19rt2 Lansing and his colleagues published an account of a careful investigation of the microscopic and submicroscopic structure of elastic fihers from the ligamenturn nuchae of the horse. The fibers were isolated by extracting the ground, defatted tissue for 4.5 mi11 with 0.1 N NaOH a t 98°C. Like preparations from ox ligamenturnnuchae the purified fibers were glassy, optically homogeneous cylinders with a very high refractive index (1.536). No structure could be observed with the light microscope and because of their excessive thickness (4.5 to 5 p ) the intact fibers caould riot be viewed in the electron microscope. Recrystallized trypsin (Armour) had no effect on the purified fibers, but, commercial grade trypsin effected the dissolution of the fibers when incubated overnight a t 37°C. This was apparently due to the presence in the commercial trypsin preparations of pancreatic elastase (Ba16 and Banga, 1949). When treated for a short time with a partly purified preparation of elastase, obtained from pancreatin, the elastic fibers were eroded to a degree that the smooth cylindrical structure was lost and under the light microscope the fiber appeared to be composed of a pair of intertwined fibrils each approximately 2 p in diameter. The process of dissolution then accelerated, and in favorable specimens each fibril was seen to resolve into a pair of similarly intertwined members, each with a diameter of approximately 1 p . These 4 strands were very friable and under Brownian motion rapidly broke up into short beadlike segments and soon disappeared.
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Samples of elastic fibers in various stages of elastase trratment were examined with the electron microscope. Intact elastic fibers, because of their considerable thickness, appeared as opaque cylinders with smooth walls. The surfaces a t the brokrn ends were smooth and sharply angular. The largest elastic fibrils that could he effectively penetrated by the electron beam were 1 p in diametrr and corresponded to thr fibrils described above as making up one-fourth of the wholr elastic fiber of ligamenturn nuchae. These fibrils npppared as fairly smooth cylinders with slightly convoluted ~ v d l s ,which sometimrs bifurcated into two smaller fibrils or threw out still smaller fibrils sometimes in the form of loops. Since elastase seems to act initially on the ends of the fiber the internal structure was most clearly revealed by the study of such sites, and chromium-shadowed micrographs of the frayed ends revealed typical appearances. These ends appeared to be extensively frayed out into a brush of many minute threads of fairly uniform thickness. These were approximately 25 mg and were regarded as the elementary unit of the elastic fiber in ligamentum nuchae. Varying numbers of these threads seemed to be twisted together in several areas to give a corded appearance. The appearance of a fibrillar structure after a short treatment with elastase was interpreted by Lansing and his colleagues to indicate that the structure of the intact fiber consisted of fibrils embedded in and covered by a homogeneous matrix, but they observed that since the stained or unstained whole fiber is optically homogeneous, this would imply that the matrix has physical and chemical properties nearly identical with that of the fibrillar substance. A recently developed technique for cutting ultra-thin sections (SO-100 mH thick) by means of glass knives was employed by Yokota (1937) in a n examination of bovine ligamenturn nuchae. The sections revealed the presence of a moderately electron-dense substance surrounding the elastic fiber. Elastase treatment of the ultra-thin sections resulted in a stepwise degradation of the elastic component. Pitlike depressions occurred on cross-cut elastin surfaces together with corrosion of the outer part of the fiber. With longer elastase treatment the elastic fibers split longitudinally into three, four, or more component fibrils with swelling and loss of electron density. These observations were extended by Usuku (19.58) who fixed tissue from bovine ligamenturn nuchae in Luft’s (19.56) potassium permanganate fixative and cut ultra-thin sections. Electron micrographs of the cross section of the elastic fiber gave the appearance of a dense reticulum of minute fibrils (10-20 xnp in width) which was surrounded and penetrated by collagen microfibrils. In longitudinal sections no regular axial periodicity was observed, and the beaded fibrils appeared to run in a random way, connect with each other, and form a complicated reticular network.
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The contributions of the Japanese school have been reviewed in detail by Kawase (1959). A fibrillar st,ructure was also noted by Laitinen (1960) in elastica from the alveolar walls of human lungs. He observed osmiophilic fibrillar formations and concluded that these were macromolecular structural units of the elastic fibers. The fibrillar structure showed no periodicity; longitudinal sections showed that there is a fibrillar structure along the fiber lengt.h, but t,he fibrils did not lie quite parallel nor were they continuous over the entire length of the fiber. It has already been noted that the use of elastolytic enzymes to secure the separation of the fibrils is objectionable on the grounds that the fibrils themselves are at,tacked. The firsb attack of elastase is characterist,ically local in nature and the enzyme usually begins by producing pitlike corrosions a t close intervals. It is possible that the beaded appearance of the elementary fibril, stressed by some workers, may be an artifact due to this local mode of action of the enzyme. In recent work Gotte and SerafiniFracassini (1962) have been successful in liberating the elementary fibrils from fibers of elastin without the use of enzymes. The elastin fibers were prepared from bovine ligamenturn nuchae by cutting with a freezing microtome and removing the fat by extraction with acetone followed by ethanolether. The fat-free tissue was treated with 0.1 N sodium hydroxide at 93°C for 1 hr, washed with hot water, and homogenized. The larger fiber bundles were eliminnt,ed by centrifuging a t 2000 g and the supernatant. was then treat'ed ultrasonically (piezoelectric quartz, 40 kc) for three separate periods of 10 min, allowing time for the suspension to cool. Figure 2 shows :In electron micrograph of the suspension, chromiumshadowed a t 20 degrees. The magnification is 63,000 X and the individual fibrils are 10 f 2 mfi in diameter. It will be noted that the long fibrils are very uniform in structure throughout their lengt,h and do not appear to be cont,aminated by amorphous deposits arising from the drying of matrix material. This may be regarded as an indication of the chemical purity of ligarnentum nuchae elastin as prepared by hot alkali treatment tinder controlled conditions. Ligamentum nuchae elastin, prepared by aut,ochving, contains not more than 1-2 % of material that can subsequent,ly be extracted by mild alkali treatment; its chemical composition is very similar to that of the alkali-prepared material, but nevertheless the fibers resist disintegration by ultrasonic treatment and the lateral cohesion of the elementary fibrils is apparently greater. Owing to differences in approach and technique it, is still difficult to obtain an entirely unambiguous picture from the observations and interpretations of the various workers who have examined the fine structure of elastic fibers from different tissues. However it is clear that unlike collagen
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and the muscle proteins, elastin forms no system of regular cross striations when it is laid down as fibers; on the contrary the molecules of the fibrous protein do not seem to be arranged in any strict orientation or true register.
FIG. 2. Electron micrograph showing the fibrillar structure of an elastic fiber from bovine ligamentum nuchae. (Gotte and Serafini-Fracassini, 1962.) The elastin fibers were treated with dilute sodium hydroxide a t 98°C for 1 hr, washed with hot water, and disintegrated ultrasonically a t 40 kc. The magnification is 63,OOOX and the individual fibrils are 10 f 2 mM. The specimen was shadowed with chromium a t 20 degrees.
They form a network of long, fine elementary fibrils about 10 mp in diameter which seem to be almost as strongly adherent in the lateral direction as they are along the fiber axis. Partial digestion with elastase or ultrasonic treatment reveals systems of interconnected fibrils which are tangled and loosely twisted together to form dense cordlike fibers in ligamentum nuchae or thick membranes in arterial walls.
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In different tissues the microscopic and submicroscopic organization of elastica takes different and highly characteristic forms and it is of interest to speculate if this differentiation springs from differences in the chemical coiistitution of the elastin molecule or if it is induccld b y environmental conditions a t the site of growth. Further constitutional studies on purified elastins isolated from the different tissues may help to resolve these cluestions. E. Age Changes in Elastic Tissue 1, Metabolic Turnoaer
There have been numerous studies concerning the growth and replacement of collagen in mammalian tissues but little is known about the metabolism of elastin, its synthesis, or the means by which formed fibers may be replaced by new tissue. The mature collagen fibers of adult connective tissue are known to have a very slow rate of metabolic turnover (Neuberger and Slack, 1953) but recently evidence has accumulated showing that the first product of collagen synthesis is a soluble precursor of relatively high tarnover rate (Harkness et aE., 1954). No such precursor of elastin formation has yet been identified, but it must be assumed that, in the case of elastin also, the extracellular fiber formations must arise initially by the migration of some soluble precursor from the cell to the surface of the growing fiber. The turnover rate of elastin from adult animals was investigated by Slack (1954) by injecting adult male rats interperitoneally with (214-labeled glycine. Half the animals were killed after 1 day and the remainder 9 days after the injection. Purified preparations of elastin were then made from the aortas of each group. These preparations were hydrolyzed and the glycinc isolated chromatographically as the dinitrophenyl derivative. The radioactivity of the crystalline samples was then determined. It was found that there was no significant fall in specific activity during the 9 days of the experiment. This time interval is rather short, but is long enough to have allowed demonstration of an active metabolic turnover if this occurs in adult aorta. It therefore seems probable that aortic elastin, once it, has reached maturity, is replaced slowly, if a t all. Variations in the growth rate of elastin with age of the animal were investigated by Viola and his colleagues (1960). These authors gave interperitoneal injections of C14-glycineto three groups of rats aged 4 weeks, 8 months, and 3 years respectively. The radioactivity of isolated aortic elastin was measured after 2 , 10, and 15 days. There was a progressive decline in the uptake of glycine with age. The maximum specific activity of elastin was reached about 1 week after injection and the counts measured
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were in the ratio: 20 for the youngest rats, 1.5 for mature rats, and 7 for old rats. Similar conclusions were reached by Kao et al. (1961) who injected female Wistar rats, 5 weeks, 8 months, and 2 years old with uniformly labeled C14-lysine. The animals were sacrificed at intervals up to 40 days after injection. Elastin and collagen were isolated from aortas, tendon, uterus, and skin and examined for radioactivity. The results showed that with the exception of the uterus, insoluble collagen and elastin were synthesized at a significantly higher rate by 5-weeks-old rats than at 8 months or 2 years, but a t all ages the turnover rate relative to other proteins was low. In agreement with the results of Slack, the elastin of aorta did not decay in activity in any age group above 5 weeks old. The experiments with labeled amino acids so far completed are too narrow in range to permit a general conclusion, but from the limited evidence available i t seems that elastin is synthesized a t a rapid rate by growing animals and a t a much slower rate throughout adult life. The breakdown of mature elastic fibers is clearly very slow, if it occurs under normal circumstances a t all. Removal of elastic elements that are damaged by disease or displaced by mechanical stress is no doubt the function of the normal defense and repair mechanisms and is probably mediated through the action of phagocytic cells. As will appear later (Sheldon and Robinson, 1960) if elastic formations are removed from the tissues of young animals by artificial means, regeneration may be rapid and complete. 2. Age Changes and Disease
The work on metabolic turnover underlines the biological and chemical unreactivity of elastin which is perhaps its most characteristic property. Apart from the destructive effects of recognizable disease it has seemed to many authors that the mature elastic fiber, once laid down, is retained for life. In this situation it may be expected that changes may occur in elastic fibers which could be ascribed to the results of aging alone. Such changes have been looked for in a number of laboratories. Diseases of elastic tissue are few compared with those that affect collagen, and in these destruction of preformed elastic fibers appears to occur only in localized areas, particularly in the walls of blood vessels and in the skin. In arteriosclerosis loss of elasticity and breakdown in the structure of the elastic elements in the media of arteries is accompanied by calcification of the media and the development of calcified plaques in the intima. Since calcification of the media may he seen to occur without the development of atheromata, it is thought that this change may be associated in the first place with age. Other age-related changes looked for have been changes in the gross content of elastin in the media and changes in the amino acid
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content of the elastic protein itself. Much of the literature concwning the pathological involvement of elastic tissue has been reviewed by Hass (1939) and Hall (19.59). 3. Changes in the Elastin Content of the Timica Media of the Largr Blood Vessels
In the human arota or human pulmoiiary artery very little change is observed in the elastin content of the media throughout life. Lansing (1954) found that the average elastin content of the media of the aorta during the first two decades of life is slightly over 48 gm per 100 gm of dry fat-free tissue, while the average elastin content in the third decade arid thereafter drops to values ranging between 41 and 44 %. Thr data for the pulmonary arteries followed a slightly different pattern. During the first three decades of life the media of the pulmonary artery contained slightly less than 31 9% of elastin. This increased slowly throughout life to 34% in the seventh decade and to almost 37% in the eighth decade. It appears that a t least after the second decade of life the elastin content of the large arteries either remains constant or actually increases. The loss of arterial elasticity with age cannot be attributed to a geiieral loss of elastin arid is more probably due to a change in the properties of the elastic tissue itself. The development of collagen and elastin in conrirctive tissue is influencrd by sex and by hormones. McGavack and Kao (1960) examined tissues from the tail tendon, aorta, skin, uterus, lung, musck, and heart of male and frmale Wistar-strain rats age 3-5 weeks, 8 months, and 2 years. Insoluble collagen increased with age in the tail tendon, aorta, skin, and abdominal muscle of both male and female rats, in the lower leg muscle of the male, and in the uterus. In the tendon and skin of females, this value continued to rise up to 2 years of agr. In the aorta, tendon, and skin of the males it reached a plateau at 8 months of age. An increase in elastin with age was found in the tail tendon of both sexes, and more elastin was found in male than in female rats at 8 months of age. The results of this work are givrri in a valuable series of tables and graphs. The influence of sex differences and hormones on elastin and collagen in the aorta of chickens was studied by Cembrano et al. ( I 960). In onr series of experiments collagen and elastin were estimated in the aortas of chickens of both sexes maintained on a standard diet. I n another series the samr estimations weir made with cockerels that had been gonadectomized or treated with estradiol or with hens treated with testosterone. The results show that the collagen and elastin contents are significantly higher in males than in females. Gonadectomy in males decreased significantly the content of collagen and elastin, so that the values became similar to those observed in females. The treatment of males with estradiol lowered the
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collagen and elastin content of the thoracic and abdominal aortas and the treatment of females with testost>eroneincreased the two proteins to levels similar to those observed in males. 4. Variations in Amino Acid Content of Elastin Preparations from Aorta
Changes in the amino acid composition of apparently pure samples of elastin, as an effect of age, have been reported by Lansing (19.54). In preparing his samples of elastin for analysis Lansing treated the dried defatted tissue by digesting a t 98°C with 0.1 N NaOH for 45 min. This reduced the material to constant weight and on microscopic examination the elastin preparation was apparently free from collagen, muscle, and other contaminating structures. Material so purified was hydrozyed and the amino acids estimated by microbiological assay (Lansing et al., 1951). With elastin from aorta highly significant increases in the contents of aspartic and glutamic acid were noted in the samples from senile subjects while the mean values for valine, proline, and glycine were somewhat lower. I n contrast to the findings for aorta, pulmonary elastin did not show increases in aspartic and glutamic acid with age, but these amino acids were usually present in greater amount in pulmonary elastin than they were in typical preparations from the aorta of young subjects. As pointed out by Lansing it is doubtful that the shift in amino acid distribution could represent a change with time in the amino acid composition of a pure protein. The elastin studied may be a mixture of two or more proteins and the changes observed may be related to differences in the ratios of the separate constituents. Hall (19.51, 1955) showed that treatment with boiling urea solution was capable of separating from a preparation of aortic elastin a fraction rich in hydroxyproline, aspartic acid, glutamic acid, arginine, lysine, and histidine and leaving behind a substance which had the classic amino acid analysis of young aortic elastin or ox ligamentum nuchae elastin. These observations suggest that the preparation of “old elastin” described by Lansing et al. contained a second component high in the acidic amino acids which is closely associated with true elastin. 5 . Age Calcification of the Aorta In an attempt to separate the normal effects of age from those due to visible atheromatosis, Lansing (19.54) collected human aortas which possessed an over-all minimum of atheromatous plaques. Little difficulty was experienced in selecting suitable tissue from young subjects but considerable selection was necessary for the older specimens. From this material atheroma-free sections were cut out and the adventitia and intima were stripped off. Elastin was isolated from the separated tissue and its
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calcium content mrasiired. The results showed that in plaque-frer areas there is a progressive increase with age in the calcium content of thr elastin, the lrvel reaching about 4 70 of the protein dry weight in subjects 60 years old. This led Lansing to the conclusioii that the change in composition of human arterial elastic tissur, which is almost invariably associated with overlying at,hrromatosis, is age-conditioned. He pointed out, howevrr that agr is riot alone effective since thr pulmonary artery is normally resistant to elastic tissue change at, all ages. However, the pulmonary artery does develop typical elastic tissue changes as well as athrromatosis when it is subjevted to stress as in pulmonary hyprrtension. The site of deposition of calcium in the aging human aorta has heen studied morr closely by Weissman and Weissman (1960). The gross calcification in the athrrosclrrotic plaques of the intima of the aging aorta is immediately obvious (Duff and McMillan, 1951) and appears to havr little connection with thr rlastic structurrs. On the other hand, apparrntly independently of the developmrnt of plaques, progressive drpositiori of calcium takes place in the media with advancing age. It is important to know if this calcification of the media, whieh evidently leads to loss of elasticity, is dirrctly associated with elastin rathrr than collagen or some othrr components of the elastic tissue. Calrified deposits from thc atherosc*lrrotic plaqur have heen studied by Carlstrijm rt al. (19.52) who tentativrly identifird the crystallites as hydroxylapatitr and showed that the X-ray diffraction pattern of material from thr plaques is similar to that givrn by bone. Weissman arid Weibsman (1960) prepared purified elastin from thr media of aortas from individuals aged 8-86, Only those sprrimrns frre from moderate to rnarkrd intimal plaque formation were employrd. Sections of the stripprd-out media from the specimrns were disintegrated in a homogenizer in et harioI solution. Purified elastin was prepared from the resulting defatted powdrr by extraction with water followed by autoclaving to remove rollagen. Thr purified elastic fibers preparrd in this way showed an increase in calcium contrnt and a decrease of nitrogen with inrreasing agr in agreemcwt with thr results of Lansing. Thr fibers gave the characteristic X-ray diff rztction pattern of hydroxylapatite while neithrr the collagen frartion (prrpared as gelatin) nor the polysawharide fraction separated during thr isolation of the elastin gave any crystalline pattern. This indicated clearly that hydroxylapatitr crystallites are assoriatrd intimately with elastin fibers. In the hope of tracing this assoriation to the molecular lrvel thr fibers were dissolved by the artion of rlastasr. Brief treatmrrit of rlastic fibers with this rrizymr yields a soluble, nondialyzable protein (Partridge c.t al., 19,5.5) and it was found that aftrr elastasr trratment followed by centrifugation to remove particulate. matter the solublr protein producaed still clontainrd calcium and still displayed
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hydroxylapatite structure on X-ray diffraction analysis. These results do not provide rigorous proof that deposition of hydroxylapatite crystallites has taken place in spaces between the polypeptide chains of the elastin molecule. This would imply some degree of regular orientation and would require further detailed analysis by X-ray methods. It does however indicate that calcification occurs within the fine strncture of the elastic fiber and suggests that the increasing rigidity of the elastic membranes of the aorta associated with advancing age may be attributed to this cause.
F. Relationship between Collagen and Elastin The formation of collagen fibers in vivo is now generally agreed to result from the activities of the fibroblasts, which are universally found in association with growing connective tissue. Although it was suggested by Doljanski and Romlet (1933) that in tissue cultures the formation of collagen fibers could occur in situations far removed from the cells, the work of Fitton-Jackson (1936) seems to offer conclusive evidence that fibrogenesis is indeed initiated by the fibroblasts and that the process of fiber formation commences a t the surface of the cells. Fitton-Jackson (18.54,1956) showed that characteristic granular particles, observed within the cytoplasm of the fibroblast, contain both protein and mucopolysaccharide and she suggested that the fibroblasts evolve both collagen precursors and also the polysaccharides from which the ground substance is derived. In a region near the surface of the cell the initial phase of fiber formation takes place with the production of a primitive collagen fibril with a band periodicity of about 210 A (Fitton-Jackson, 1956). The formation of soluble precursors has also been detected in chemical studies; thus Harkness et al. (19.54) have shown that a hydroxyproline-containing protein, extracted from t issurs by neutral salts, incorporates labeled glycine at a rate suggesting that it is the true precursor of collagen fibers. The chemical composition of the soluble precursor is closely similar to that of collagen, and under suitable conditions of pH and salt concentration it forms fibrils in vitro which havc the characteristic banded structure of collagen, but differ from mature collagen in a number of properties such as thermal stability and resistance to hydrogen bond-breaking solvents. No similar causal relationship has even been established between cells, soluble precursors, and mature fibers in the study of elastin. Indeed the “elastoblast” (Loisel, 1897; Krompecher, 1927) although postulated, has never been recognized as a morphological and functional entity. This has led a number of authors to suggest that collagen and elastin may he formed from the same mixture of soluble peptide or protein precursors, or even that preformed collagen may afterwards be converted into elastin, or elastin to collagen. The early interest in a possible relationship between collagen and elastin
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was renewed when a group of workers (Burton et al., 1953) showed that preparations of rollagen, after treatment with alkaline buffers or pancreatic enzymes, could give rise to material which had certain staining reactions and other histological characters similar to those of elastin. This interest led to a great deal of work in many laboratories and had valuable results in displaying properties of collagen that were previously not well known ; it was also valuable in displaying the nonspecific proteolytic activity of pancreatic elastase with many denatured protein substrates. D. A. Hall (19,59) has reviewed much of the data concerning the possible relationship between collagen and elastin and further extensive quotation from the literature seems unnecessary. Since reliable amino acid analyses have now become available for collagen from various tissues and animal species (Bowcs and Kenten, 1918; Eastoe, 19.55; Bowes et al., 1955; Eastoe, 1957; Leach, 1957, Jackson et al., 1958; Williams, 1960) and for elastin from a more limited number of sources (Newman, 1949; Newman and Logan, 19,50; Lansing et al., 1951; Partridge and Davis, 19.55; Fitzpatrick and Hospelhorn, 1960) a direct comparison of the composition of the two proteins is available. It turns out that both proteins have unusual and highly characteristic amino acid compositions, but except for a preponderance of glycine and proline in both, the two proteins have little in common. Collagen has a high content of hydroxyproline (about 14 94) and is relatively rich in aspartic acid (7 %), glutamic acid (11.6 %), lysine (4..5 %), and arginine (9%). Tyrosine is absent or very low (about 0.2%). Elastin on the other hand contains little hydroxyproline (1.4-1.6 %) but contains about 1.5 % of tyrosine. It has a very low content of the acidic and basic amino acids and is correspondingly rich in the monocarboxylic-monoamino acids, particularly valine (17.5%). As pointed out by Harkness et al. (1957) conversion of preformed collagen into elastin would imply the removal and loss of a very large proportion of the hydroxyproline and polar amino acids. Indeed the valine content of elastin is about seven times that of collagen and thus conversion of collagen fibers to elastin would involve a minimum loss of six-sevenths of the dry weight of the protein; the loss would be greater if some of the valine were not reincorporated. In view of what is now known about the energy requirements for protein synthesis it would appear not to be possible that a process involving such radical breakdown and resynthesis could occur in vitro under the conditions described by Burton and his colleagues. I t is therefore necessary to look for other explanations for their observations. Various phenomena may be suggested as giving rise to confusion as to the nature of fibers observed in connective tissue from pathological sources, or isolated tissue that has been treated chemically or with enzymes. The dramatic shrinkage that takes place when collagen fibers are denatured is
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a phenomenon that has been known for many years and was carefully described by Lloyd and Garrod (1946). The collagen fiber in its natural state has an elastic stretch of about 7 % of its unstretched length, is strongly birefringent, has a typical X-ray fiber diagram, and shows a characteristic banded structure in the electron microscope. A variety of conditions cause the partial or complete denaturation of the fibrous protein and this is accompanied by marked changes in all these properties. Denaturation changes usually begin to occur quite sharply at some specific temperature, but this temperature or small temperature range is very dependent upon the properties of the medium in which the fiber is immersed. Thus the temperature at which rat-tail tendon begins to denature in water is about 55-60°C; in 10% calcium chloride solution it is about 45°C; in acetic acid it is about 25°C; and in formic acid or thioglycolic acid it is less than 0°C. When denaturation occurs the fiber may shrink to about one-quarter of its original length in a few minutes, and there is a corresponding increase in thickness of four or five diameters. It then acquires rubberlike elasticity very similar to that of elastin and the fiber resembles elastin in appearance. The fiber also loses birefringence and this is accompanied by a drastic change in the X-ray fiber diagram and loss of the characteristic banded appearance in the electron microscope. After partial or complete denaturation collagen fibers acquire rather different staining characters; they still take up acidic or basic stains, but in addition they begin to show a new affinity for orcein and other stains that are usually regarded as specific for elastin (Gillmanet al., 1954; Bangaet al., 19.56;Lorincz, 1960). Thischange in staining character may be due to the exposure of new hydrogen-bonding sites arising from the rupture of the organized system of interchain hydrogen bonds in native collagen (Ramachandran, 19.55; Rich and Crick 1955). Although native collagen is resistant to the action of elastase, denatured collagen is dissolved by elastase about ten times faster than elastin itself (Banga, 1953); indeed, elastase is a proteolytic enzyme with a very wide peptide-bond specificity and catalyzes the hydrolysis of most denatured proteins (Naughton et al., 1960; Thomas and Partridge, 1960). Many reagents including hydrogen bond-breaking salts and salicylates (Courts, 1958), acidic and alkaline solutions (Courts, 1960), ionizing radiations (Bailey et al., 1962), and proteolytic enzymes have the effect of rupturing some of the bonds that contribute to the stability of native collagen and this results in lowering the temperature at which thermal contraction takes place. In extreme cases of such damage the denaturation temperature may be reduced below body temperature or even below room temperature, in which case contraction takes place without further heating. As suggested by Tunbridge et al. (1952) it seems probable that much of the increased deposition of elastica-staining material observed in the ex-
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posed parts of the skin of elderly subjects (“senile elastosis”) may be derived from collagen. I n electron-microscope studies Tunbridge and his colleagues showed that in these conditions there is little increase in true elastic fibers in senile skin, but that the exposed areas contain large quantities of bent and broken collagen fibers coated loosely with amorphous particulate material. They also showed that short periods of treatment of native collagen with pepsin produce material having similar staining properties and similar appearance under the electron microscope. It has been observed that these fibers are rapidly dissolved by elastase (Findlay, 19.54) and this had led to the assumption that they are related to elastin, but as mentioned above, elastase attacks denatured rollagen with extremr rapidity. Similarly Thomas and Rook (1949) and Hannay (1951) have stated that the elastic-staining material (“elacin”) obsrrved in the skin of patients suffering from pseudoxanthoma elasticum is present too abundantly to have originated from the elastic fibers originally present and suggrsted that, the material is degenerate collagen. A similar conclusion was reached by Tunhridge et al. (1932). Just :is collagen fibers may suffer changes in staining properties so may elastin if it is damaged by the action of enzymes or by too severe isolation procedures. Rupture of a peptide bond results in the appearance of a new 0-amino and a new a-carboxyl group and these added to the very few acidic and basic* groups in elastin result in a much increased affinity for acidic arid basic dyes. Concurrently the affinity for phenolic dyes may be reduced, particularly in acid or alkaline solutions, due to the repulsive forces set up by the increased charge. In view of the better appreciation both of the properties of collagen and elastin and of the changes which may take place in collagen fibers as a result of pathological or experimental conditions, there is now little reason for amhigiiity in the identification of the two main classes of fibers in connectivr tissue. Indeed there is slender justification for the impressive series of composite names which have found their way into the literature; these do no more than to describe stages in the progressive breakdown of the ordered structure of collagen which ultimately culminates in the produrtion of gelatin. Of course, chemical isolation of elastin structures, where this is possible , will always result in unambiguous identification since the two protein families, collagen arid elastin, share very few chemical or physival properties in common. However, when isolation is not possible, accurate identification should still be practicable through improved histological trrhniques. Thus, unlike the various elastolytic enzymes, t h r collagenase from Clostridium histolyticum is highly specific ; it digests native collagen fibers a i d collagen in all stages of denaturation; on the other hand elastiii and most other proteins are quite resistant to purified preparations of this enzyme (Mandl et al., 1953; Maiidl and Zipper, 19GO). Again, de-
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251
natured collagen, but not native collagen, is dissolved by crystalline trypsin, while native elastic fibers are quite rcsistant to purified trypsin. For many purposes histological staining reactions may be sufficient to differentiate clearly between elastin and structures derived from collagen, although it is probable that no single staining technique can be regarded as reliable in all ck-cumstances. The use of a series of stains together with a caomparative series of control sections from healthy tissues (cf Gillman el al., 1934) is to be recommended wherever partial denaturation or damage by enzymes is suspected. Dyes with acidic and basic functions, used under varying conditions of pH and salt concentrations may be of special value in detecting areas of damage in structures such as skin or arterial walls. The final solution of the problems raised by the concepts of Burton and his colleagues must await the unambiguous establishment of the route of elastin hiosynthesis. This work has progressed little in recent years, probably for the lack of a suitable biological system in which the formation of elastic fibers could c8onveniently be observed. Recently however, Sheldon and Robinsori (1960) have examined elastic cartilage from the ears of rabbits following administration of papain. Observations with the light and electron microscopes showed that some 8 hr after the intravenous injection of the enzyme both the elastic components of the matrix and the amorphous metachromatic material disappear, leaving a matrix which consists of delicate fibrils of collagen. After some days the cartilage regenerates and both the metachromatic material and the elastic fibers are reconstituted. Only one type of cell was identified in this tissue, the chondrocytes, and it was shown that during regeneration of the cartilage a concurrent series of changes were observable in the cytoplasm of the cells. I t is of course possible that cells may have differing functions of protein synthesis without showing any corresponding morphological differences in the electron microscope, but the results of this work appear to suggest that the same type of cell is responsible for the synthesis of all the components of connective tissue including the protein complexes of the acid mucopolysaccharides, collagen, and elastin. Should this prove to be the case, it would in no way suggest that collagen and elastin are interconvertible either by biological or by chemical means; it would indicate only that collagen and elastin synthesis draws upon the same amino acid pool and that possibly some intermediates in the synthesis are shared in common.
111. PHYSICAL PROPERTIES OF ELASTIK
A . Elastic Yield qf the Wet Fiber The protein fibers of connective tissue can exist in either of two forms in the presence of water: (1) a form with high tensile strength but little elastic extensibility and (2) a form with rubberlike extensibility and low modulus
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of elasticity. Collagen and reticulin normally exist in form (1) but can be made to pass into form ( 2 ) by thermal contraction in the presence of water or by the use of various hydrogen bond-breaking reagents. Elastin normally exists in form ( 2 ) and, at temperatures above the freezing point of water, cannot be converted into form (1). The condition of rubberlike elasticity is an unusual property, and only a few polymeric substances show it a t normal temperatures. These substances have been called “e1ast)omers” and have the following typical properties (Baumann, 1959): (a) They must stretch rapidly and considerably under tension, reaching high elongations from a t least twice to several times their original length, with little loss of energy as heat. (b) They must show high t,ensile strength and high modulus (stiffness) when fully stxetched. ( c ) They must retract rapidly, exhibiting the phenomenon of snap or rebound. (d) After release of stress, they must recover their original dimensions fully. All typical elastomers contain long polymer chains. In order to exhibit rubberlike properties quite long sections of these chains must have rotatable links and must be freely mobile with respect to one another. This gives the ability to stretch and to retract rapidly. On the other hand the gross mobility of the chains must be restricted by the provision of a certain degree of firm cross bonding; this restricts the slipping of one chain past another and allows the material to regain its original unstretched shape when the stress is released. It is important that the cross-links should be relatively few and widely spaced so that stretching to large extension can occur without rupture of strong bonds. In rubber, the introduction of a network of cross-links is achieved by vulcanization and this converts the thermoplastic raw rubber into a true elastomer. Long polymeric molecules, when free from strain, do not lie in a position of full extension, but in a randomly crumpled position giving maximum entropy. If such a system is pulled by a mechanical stress it will extend, with straightening of the chains, and on release it will revert to its original dimensions. An increase in temperature increases the kinetic motion of the free portions of the chains and causes greater crumpling; this results in further retraction of the system. Thus with vulcanized rubber an increase in temperature results in a decrease in extension for a given load or an increase in the load required for a fixed extension. However rubber differs from protein fibers in that it is formed from long hydrocarbon chains wit,h few potentialities for cross bonding by secondary valence. It thus forms a self-lubricating system and no solvent or plasticizer is required to reduce interchain adhesion. Protein molecules on the other hand are
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abundantly supplied with dipolar and hydrogen bond-forming radicals which are capable of taking part in the weak interchain linkages that promote crystallinity. In order for a protein to exhibit rubberlike elasticity these active groups must be satisfied by a polar solvent such as water, formamide, acetic acid, or phenol; the dry protein is always inextensible and brittle. Preparations of purified elastin fibers, or narrow strips teased from ligamenturn nuchae, behave as typical rubberlike solids when suspended in water or dilute salt solutions. The Young's modulus is much smaller than for collagen and the extension a t break is 20 to 30 times as great (Krafka, 1937). Meyer and Ferri (1936) and Wohlisch et al. (1943) showed that whole ligament behaved as a rubberlike solid up to 100% extension and that extensibility was independent of time up to 50% extension. Lloyd and Garrod (1946) studied strips of elastin from ox aorta after removal of collagen with 90 % formic acid a t 45°C for 72 hr. Load extension curves were determined for the strips in equilibrium with water at a range of temperatures. For a given extension the load was 60 % higher a t 75°C than a t 15°C and the shape of the family of curves was similar to those obtained with rubberlike polymers. Hoeve and Flory (1958) made a carefur reinvestigation of the stressstrain curves for fiber bundles obtained from unpurified ox ligamentum nuchae, using sensitive strain gauges. The experiments were carried out a t equilibrium in a 30 % glycol-water mixture a t a range of temperatures. The choice of this solvent mixture was determined by the desirability of using a system in which the volume of the swollen polymer is independent of temperature, thus greatly simplifying the mathematical treatment. It was found that the curves conformed to the condition for ideal rubberlike elasticity as defined by thermodynamic theory (0 th et al., 1957, 1958). Contrary to the conclusions of Meyer and Ferri (1936) no evidence could be found for interchain crystallization a t high extension, and this is consistent with the finding of an amorphous X-ray diffraction pattern for stretched elastin (Astbury, 1940). As observed in the microscope the samples of ligament used by Hoeve and Flory consisted of many separate fibers of elastin with irregularly curled thinner fibers of collagen between them. Treatment with elastase removed the elastin component leaving collagen fibers which, though coherent, accounted for only about 20 % of the initial (dry) specimen. The lengths of these residual fibers, when straightened out under tension, were about twice those of the relaxed native sample. The collagen fibers displayed low elongation and high modulus; they underwent shrinkage (in water) a t 65" to about one-fourth their length. These observations leave little doubt that the residual fibers were in fact collagen, and the presence of a separate fabric of collagen
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filaments appcars to have a marked effcct on the strcss-strain curvcs abovr about 70 70 cxtension. A t extensions of this order the collagen fibers evidently become taut and the stress rises abruptly with further extcnsion. In contrast with rubber, the steep rise in stress at clongations IWW the maximum attaiiiable length is not precipitated 1)y carystallization, but hy a permanently carystallinc romponent (collagen) iiitcrwovcn with thr drformable (elastic) component. The information obtained from the strcss-strain curves of purified clastiii fibers or whole ligament, together with thc X-ray diffrac.tion data, appcars to providr firm ground for the belief that elastin is an amorphous system of pcptidc chains cross-linked a t intcrvals hy firm bonds of a typc. a h yet nnknown. I n t,his its structure is vrry differcnt from that of (bollageti, whirh bchaves as a highly crystalliiie fibcr. €Iowcver when collugcii is denatured by heating in the presence of water, or when the strength of some of the secondary interchain linkages is rcdwed hy thc prcsciiw of hydrogen bond-brcaking reagents, the ordered sy m of hclically roilcd backbonc chains retracts, and the shortcried fiber develops rubbcrlikc elasticity. Thc stress-strain behavior of thermally coiitractcd collagen has been studicd by Wiedcrhorn arid Rearden (1952). Thew authors showed that when contracted collagen is swollen in appropriatr media its load-extension curves conform to t,hc ritbbcr elasticity theory. From the shapc of thc w w e s it was possible to establish that the material behaves as if it wcrc a iictwork polymer cross-linked at long intrrvals by covalciit, bonds. The molecular weight of the chain brtmem points of cross-linking was approximately 53,000. This is nrar the niolec.ular weight cdculatcd from X-ray data (60,600) assuming the c-axis of the unit ccll to he thc characteristic 625 A spacing.
B. Hydration, Xwelling, and Solubilit!j From the foregoing it appears that elastin can bc regarded as a mosslinked polymer gel and its swelling and solubility properties are consistent with this view. Elastin appears to be entirely insoluble in any reagent except those that attack the primary chain structurc by hydrolysis and the breakiw of covalent bonds. Little recent work has been reported on the water relations and swclling properties either of native elastin or elastin that has heeii isolated by various procedures or treated with enzymes or hydrolytic reagents. This is to be deplored sirice the study of the hydration of a protein fiber is often a sensitive means of revealing the pattern of lateral bonds which lends s t r w tiiral stability. A comparison of thc hydration of elastins derived from different animals, or different tissues of the same animal, would be particiilarly valuable since the methods are rclativcly insrnsitive to thc presencv
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of small quantities of foreign protein; uncertainties due to the absence of criteria of absolute purity could he avoided to some extent by these means. Techniques for the study of the hydration of protein fibers are of two main types: measurements of swelling and solubility and measurement of the water content of fibers when in equilibrium with moist air at various temperatures and various relative humidities. There is an abundant literature of this kind for keratin, collagen, gelatin, and nylon, but references to elastin are scarce. Green (1948) allowed fibers of elastin, collagen, thermally contracted collagen, and tanned collagen to equilibrate in closed vessels with water vapor at known partial pressures. The isotherm curves relating the water content) of the sample to the equilibrium relative humidity showed some hysteresis, and water uptake was related to some extent with the past history of the sample. Thus samples of elastin that had been dried at 100°C took up about 30 % less water than those that had never been dried below 5.2 % water. Elastin was prepared from bovine ligamentum nuchae by repeated boiling with water, and standard hide powder was used as the source of collagen; all samples were dried at room temperature by a standard procedure. At 80% relative humidity 100 gm of dry elastin took up 17.8 gm of water and the same weight of collagen, 30.9 gm. After thermal shrinkage collagen took up rather less water and tanning also reduced the water uptake. The experimental data were analyzed by means of the equations of Brunauer et al. (1938) and showed that both the number of hydration centers on the absorbent and the energy of absorption were influenced by the previous history of the sample. The value V , was derived representing the amount of water required to fill up a first molecular layer or cover the whole of the active centers on the protein with one layer of absorbed molecules. V,,, could be related to the total number of polar groups which can absorb water; thus the absorption of water by native collagen ( V , , 561 moles of HzO per lo6 gm) was roughly equivalent to the total number of polar groups on the protein side chains, but with elastin the absorption of water (V, , 328 moles of HzO per lo5 gm) was much greater than could be accounted for by the relatively few side chain polar groups (168 moles/106 gm) and the peptide backbone must also be available for combination with water by hydrogen bonding. Lloyd et al. (1946) compared the solubility and swelling of collagen and elastic fibers (and a number of other proteins) in a range of solvents including the short-chain primary alcohols, the lower carboxylic acids, formamide, thioglycolic acid, lactic acid, and phenol. Collagen dissolved in formic acid, thioglycolic acid, and lactic acid at 20°C; but although elastin suffered considerable swelling it did not dissolve in these or any solvents tested. Lloyd and her colleagues interpreted their results on the basis
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that swelling reaches equilibrium when the internal excess osmotic pressure is balanced against cohesive forces due to cross-bonds of various types. The protein fibers swell in varying degrees according to the temperature, the nature of the solvent, and the number and t,ype of cross-bonds which can be opened under the influence of the solvent. If the nature of the solvent, is such that8 some, but not all, of the normally existing lateral bonds are released, the protein suffers marked swelling, but if all are freed the protein dissolves. Gelatin, rat-tail-tendon collagen, and nylon could all be induced to dissolve in strong aqueous solutions of active hydrogenbond breakers, but hair keratin, which contains covalent disulfide crossbonds, swelled markedly but resisted solution. In similar experiments with wool keratin Griffiths and Alexander (1933) found that in 8 M lithium bromide the fibers supercontract irreversibly to 60 O/o of their original length without any apparent splitting of S-S bridges. I n the presence of small concentrations of bromine however (loys M Brz in 8 M LiBr) the covalent disulfide bridges are oxidized and the wool almost completely dissolves. In spite of the great interest in the effects on proteins of hydrogen bondbreaking reagents, work in this field has usually been confined to the use of a few well-known compounds such as urea or guanidine salts, which are by no means the most active in reaction with fibrous proteins. As many as seventy-eight reagents were examined by Courts (1958) for the property of liquefying swollen gelatin granules under standard conditions a t 18°C. The most active group included sodium benzene sulfonate, sodium p-aminosalicylate, urea nitrate, lithium bromide, potassium iodide, and potassium thiocyanate. Also highly active were: sodium benzoate, thiolacetic acid, sodium salicylate, thiourea, and zinc chloride. In recent unpublished work carried out in the present authors' laboratory a preparation of bovine Zigamentum nuchae elastin purified by autoclaving (Partridge et al., 1955) was tested for solubility in 2 M , 1.5 M , or 1 M concentrations of each of these reagents a t 18", 37", 45", and 95°C. The fibers swelled extensively, but in each case the protein remained insoluble and could be recovered quantitatively by centrifuging. Solvents of the phenolic type (phenol and m-cresol) did not dissolve elastin at 95°C either when in the anhydrous form or when containing 5 or 10 % of water. Hall (19.51) reported that when elastin is heated to 95°C in 100 to 200 times its volume of 40 % aqueous urea it goes into solution after about 5 days. However, concentrated urea solution becomes markedly alkaline under these conditions of heating, and there is little doubt that the eventual solution of the protein is due to the hydrolysis of peptide bonds. Heating in neutral urea or thiourea solutions results in the extraction of no more than a few percent of protein either from Zigamentum nuchae elastin or preparations from bovine aorta.
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257
IV. ISOLATION AND ANALYTICAL CHARACTERIZATION OF ELASTIN A . PuriJication Procedures The methods at present in use for the isolation and purification of elastin depend upon the extreme inertness of this protein fiber, upon its insolubility in protein solvents, or its resistance to hydrolysis during treatment under mild conditions by dilute acid or alkalies. Methods which use acidic or alkaline reagents, although extremely rapid and effective, have been criticized on the ground that the resistance of elastin to hydrolysis is only relative, and even mild treatment with hydrolytic reagents probably results in some changes in the structure and composition of the protein; thus mild alkaline treatment might be expected to introduce new carboxyl groups by release of amide ammonia from asparagine and glutamine side chains, and rather more vigorous treatment may result in the rupture of sensitive peptide links such as those adjacent to serine or threonine (Desnuelle and Bonjour, 1952). 1. Extraction at Neutrality
Collagen is usually the most difficult major constituent to remove from elastic tissue. This protein is fairly stable to the action of cold acids and alkalies but is converted to soluble gelatin by boiling with water or heating in an autoclave; most processes for the isolation of elastin depend upon extraction at 95°C or above. Thus Schneider and Hajek (1928) heated pieces of ox ligamentum nuchae under pressure in water at 120°C for 2 hr to remove the collagen fibers and then boiled with several changes of distilled water until the washings no longer gave a biuret reaction. The material was obtained in the form of small blocks or clumps of fibers and was almost certainly contaminated to some extent by other tissue substances. However, methods based on autoclaving with water at neutrality have been used by many workers and it is clear that from ligamentum nuchae a product of fair purity can be obtained by this simple means. The method was st,udied in some detail by Partridge et al. (195.5))who found that a product of constant composition could be obtained by exhaustive autoclaving. These authors extracted the finely minced fresh ligament with 1 % sodium chloride solution to remove soluble serous proteins and then autoclaved at 1 atmosphere pressure for periods of 45 min until the extract gave a negative biuret test. Usually three or four extractions were necessary. After dehydration with ethanol and extraction with 50 % ethanol-ether mixture the product was dried and powdered finely in a hammer mill. This product was found to contain traces of collagen and mucopolysaccharides, and to remove these it was again exhaustively extracted by autoclaving, washed with a large quantity of hot distilled water, and redried with ethanol-ether.
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Prepared in this way the material consisted almost entirely of separate, smooth rodlike fibers of about 6.5 p diameter (Fig. 1). The fibers gave the staining reactions typical for elastin ; elementary analysis showed N, 16.8 % (Kjeldahl); S, 0.2 %; P, less than 0.02 %. The carbohydrate content of the preparation was very small; the color given by the anthrone reagent (Morris, 1948) being equivalent to 0.17 % carbohydrate (as glucose) and with the orcinol reagent (Sorensen and Haugaard, 1933) 0.30 CTO (as glucose). Analysis for hexosamines showed the presence of small amounts of both glucosamine and galactosamine but the amount of either was not more than 0.05 % of the protein. Partridge and his colleagues thought that elastin prepared as above could be regarded as a chemically homogeneous protein in a reasonably high state of purity. Their reasons for believing so can be stated as follows: ( a ) The fiber preparation appeared to be homogeneous under the microscope and gave the staining reactions typical for native elastin fibers. (b) Very little carbohydrate, lipid, or phosphorus could be detected. (c) The amount of material that could be extracted from the fiber preparation by cold dilute alkali, formic acid, or other protein solvents was negligibly small. ( d ) The amino acid analysis, followed during preparat,ion, reached coristancy of composition (Table I, Section VI) and agreed with other preparations prepared by different procedures in other laboratories. The amino acid spectrum for elastin is very unusual, being remarkably high in valine, alanine, glycine, and proline and remarkably low in aspartic and glutamic acid. I t also contains no detectable tryptophan, A foreign protein as an impurity depresses the content of the abundant amino acids, particularly valine, and markedly increases the content of tryptophan and the acidic and basic amino acids. Thus quite small quantities of serum proteins or collagen can be detected in preparations of elastin. (e) Soluble elastin (see Section VI,A) prepared by partial hydrolysis of elastin fibers was fract,ionated and the head and tail fractions analyzed quantitatively for amino acids. The composition of the two fractions was suhstaiitially the same and not vcry different from that of the parent protein. 2. Use of Acids and Alkalies Products similar in amino acid composition may be prepared from ligamenturn nuchae by a number of other methods which involve the use of acidic or alkaline reagents. Thus Lansing et al. (1952) following Lowry et al. (1941) digested the dried, defatted tissue from the ligamenturn nuchae of horses and the tunica media of fresh human aortas with 0.1 N NaOH at 95°C and took small samples at 5-min time intervals for histological and
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259
chemical analysis. Material from five aortas was digested over a 60-min period. There was a progressive reduction in the amount of insoluble residue up to 4 0 4 5 min of digestion after which time the residual dry weight was constant to 60 min. These data indicate that digestion of aorta tissue in hot dilute alkali for less than 45 min is not adequate to remove the tissue components other than elastin. Microscopic examination of samples digested in NaOH for time periods less than 45 min revealed variable amounts of collagen and muscle after staining with Mallory’s procedure. At and after 45 min the elastin was optically homogeneous, and apparently free from contaminating structures. Elastin prepared in the same way from the ligamenturn nuchae of horses was similarly free from collagen insofar as could be determined by light microscopy and electron microscopy. The material had a refractive index of 1.534, was resistant to digestion by crystalline trypsin, and stained effectively with orcein, resorcin-fuchsin, or the Verhoeff procedure. Inorganic constituents, including calcium and phosphorus were removed from the purified elastin by extraction overnight a t room temperature in 0.1 N formic acid. After alkaline hydrolysis the product was free from tryptophan as judged by paper chromatography. The purified elastin from human aorta gave a positive anthrone test for carbohydrate but no carbohydrate could he detected in elastin from ligamenturn nuchae. Lloyd and Gnrrod (1946) isolated elastin from bovine aorta by heating with 90% formic acid a t 45°C for 72 hr. They provided evidence that collagen is readily soluble under these conditions but gave no detailed analysis of the elastin produced. The use of concentrated formic acid was again proposed by Hass (1942) and was studied in some detail by Ayer et al. (1958). These authors cut annular slices from the aortas of dogs and treated them with 88% formic acid in sealed tubes at 45°C for periods of from 3-216 hr. The treated slices were then sectioned and stained. All evidence of collagen, muscle, and cells vanished between 18-24 hr unveiling with great clarity the structure of the elastic tissue which retained its special staining characteristics. The treatment produced marked swelling but the lamellae appeared to retain their structure unchanged except perhaps for a tendency for the elastic fibers to split longitudinally. In the published micrographs the lamellae can be seen to be composed of loose aggregates of long elastic fibers of various thickness, which anastomose about fenestrations of variable size. Prolonged exposure of the tissue to formic acid (216 hr) appeared to cause no change in staining characteristics but caused progressive subdivisions into progressively finer fibers. As with dilute sodium hydroxide, treatment with concentrated formic acid results in slow hydrolytic cleavage of peptide bonds. Ayers and his
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colleagues followed the loss in weight of aorta tissue on treatment with 88 96 formic acid a t 45OC. The weight,loss was rapid up to 36 hr, rcprescntirig thr solubilizat~io~i of collagen and removal of ground substance, muscle, and (.ells. The residual elastin represented 37% of the dry weight of the aorta. After 36 hr there was very little weight lost until 84 hr when the elastin itself began to dissolve. By back-extrapolation of the curve it was estimated that the amount of elastin lost by hydrolysis in the initial 36 hr was about 3.1 %. Slices cut from Achilles tendon almost completely dissolved after 18 hr. The small residue of elastin occurred only in the sheat,hs surrounding b u d Irs of collagenous fibers.
3. Variation in Properties Due to Isolation Procedure Experience in the author’s laboratory indicates that elastin of high purity can be prepared readily from ligamentum nuchae of the large ruminants either by autoclaving with water or by treating with alkali under the conditions given by Lansing and his colleagues. The same is not true when the starting material is the tissue from large arteries or the elastic cartilage from the external ear. With these materials simple autoclaving with water is not sufficient to remove all carbohydrate or tryptophan-containing protein and further extraction with cold alkali is necessary in order to obtain a product of constant composition. Although the analytical composition of ligamentum nuchae elastin prepared by careful alkali treatment is the same as that prepared by exhaustive autoclaving, there are some differences to be observed in the properties and behavior of preparations made by the two procedures. There is some reason to suppose that the excessive heating and repeated drying involved in the autoclaving procedure may result in some degree of hardening or cornification of the outside layers of the purified fibers. Effects of this sort are well known in protein chemistry. Thus Evans and Butts (1949) observed degradative changes in soybean proteins when they were autoclaved for long periods in the presence of carbohydrates. These changes resulted in greater resistance to proteolytic enzymes, but no similar changes were observed when purified soybean protein was heated alone. Liesegang (1942) found that heating moist gelatin above 100°C greatly reduces its solubility in hot water and destroys its power to be hydrolyzed by trypsin. Most proteins display a measurable loss of solubility if the water content is too far reduced and it is thought that fibers of autoclaved elastin may become “case-hardened” to some extent by similar mechanisms. This hardening effect is manifested in a number of different ways. Histologists and electron microscopists have frequently observed a surface layer of greater density which appears to surround some of the fibers of autoclaved elastin. A dense surface layer is also suggested by the mode of action of pancreatic elastase; the early stages of attack by this enzyme result in the
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261
production of pitlike corrosions on the surface of the fibers, suggesting that the immediate surface-layer is more resistant than the interior of the fiber. Gotte and Serafini-Fracassini (1962) found that whereas elastin from ligamentum nuchae, prepared by treatment with hot alkali, was readily reduced to elementary microfibrils by ultrasonic treatment, the fibers produced by the autoclaving procedure resisted disintegration by this method; this in spite of the fact that the preparations had almost identical analysis. A resistant surface layer is also suggested by observed differences in the adsorptive affinity of the fiber preparations for elastolytic enzymes. Variations in the rate of hydrolysis by enzymes due to differences in preparation procedure have been reported by several authors. Grant and Robbins (1957) examined three samples of elastin prepared in different ways: A, prepared from beef aorta by boiling with 1 N NaOH for 1 hr. (This sample was inert to trypsin and chymotryspn but was exceedingly sensitive to elastase.) B , prepared from beef aorta by boiling with 0.1 N NaOH for 1 hr. C, prepared from beef ligamentum nuchae by boiling with 1 N NaOH for 1 hr. The rates of elastolysis of the three samples, as measured with a standard preparation of elastase, were found to be in the ratio A:B:C = 100:17:15. The adsorptive capacities of A and B for elastase were assessed by measuring the loss of soluble enzyme by adsorption after stirring with a fixed weight of elastin at pH 4.7 for 30 min a t room temperature. Contact with elastin preparation A resulted in the loss of 89 % of the enzyme activity while contact with B resulted in only 63 % loss. In both cases the enzyme could be eluted from the fiber preparations by treatment with 0.2 N acetic acid Similar experiments were performed in this laboratory with preparations of ligamentum nuchae elastin prepared by autoclaving and by boiling with 0.1 N NaOH and with 1 N NaOH. The autoclaved elastin was found to be less adsorptive than either of the alkali-treated preparations; but its adsorptive capacity could be progressively raised by subsequent treatment with alkali, and very highly active preparations could be made by severe treatment with boiling 1 N NaOH. FitzPatrick and Hospelhorn (1960) prepared elastin from different connective tissues by alkaline extraction and by a method based on alternate digestion with crystalline trypsin and collagenase until no further protein was liberated. This second method was presumed to leave the elastic fibers in a state closely approaching their native condition. This product “enzyme elastin” was isolated from lung and aorta of man and dog. The alkali-processed elastin contained rather less serine, threonine, niethionine, and arginine than t,he enzyme-elastin. In addition there was a striking difference in the rate of attack by pancreatic elastase and also by papain, the enzyme-elastin being considerably more resistant. From the foregoing it appears that autoclaved elastin may in fact be rather more inert than is normal to the native fiber, but on the other hand,
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alkali-treated elastin acquires new properties and becomes more reactive if the treatment is prolonged. Bendall (1955) determined the titration curve of autoclaved bovine ligamenturn nuchae elastin and found that the preparation showed the presence (in lo6 gm of protein) of about 17 moles of acidic groups and 15-16 moles of basic groups. These values are in substantial agreement with those expected from the amino acid analysis. The soluble a- and P-elastins of Partridge et al. (1955) were also titrated. The soluble proteins were prepared by mild acid hydrolysis of ligamenturn nuchae elastin and the titration results showed during this process up to 31 moles of a-amino residues (in los gm of protein) were released by hydrolysis of peptide bonds with the simultaneous release of an equivalent number of a-carboxyl residues. Milder treatment with acid or alkali is insufficient to result in the dissolution of the protein, but the production of smaller numbers of new a-amino and a-carboxyl groups can still be detected by titrating the fibrous preparations. It appears that the variable properties of alkalior acid-treated preparations of elastin may be understood on the basis of a variable content of charged groups artificially produced. These charged groups correspond with points of chain rupture and may affect swelling in water, ease of hydrolysis by acids, alkalies, or elastolytic enzymes. They also affect staining characteristics and the adsorptive capacity of the preparations towards elastases or other proteolytic enzymes. Since it seems to be virtually impossible to remove all extraneous substances from the elastic fibers of aorta or ear cartilage without the use of acids or alkalies, some degree of hydrolytic damage must be accepted, and the most generally useful procedure appears to be extraction with hot alkali under controlled conditions. From titration data Gotte et al. (1962) showed that the increase in a-amino and a-carboxyl groups that resulted from extraction of ligamenturn nzlchae or aortic elastin with 0.1 N NaOH for 45 min at 100°C was very small (within the experimental error of the measurement,). Similar results were obtained with material that had been extracted by heating with water at 110°C when this was followcd by extracting the fibers with 0.5 N NaOH at 25OC for four periods each of 1 hr. Material prepared by either of these methods was regarded as a reasonably satisfactory representation of the protein in its native condition. Nevertheless, the importance of adequate definition of sources and preparation procedure is obvious; it might be added that in much kinetic work with enzymes, the data has been vitiated by failure to appreciate the importance of the previous history of the starting material.
B . Composition of Elastins from Difercmt Tissues No syslematic survey of the amino acid composition of mammalian elastins has yet been reported, but from the information available it ap-
ELASTIN
2G3
pears that only small differences in composition are to be expected when comparing elastins from the same tissue in different mammals. Thus Neuman (1949) compared elastin preparations from aorta of sheep, cattle, and pig. The elastins were prepared by boiling in water followed by treating with 40% urea for 40 hr at 100°C. Amino acid analysis was carried out after hydrolysis by microbiological assay with Clostridium perfringens and Leuconostoc mesenteroides. The results showed little variation from one species to another; pig aorta elastin however, differed in isoleucine, glutamic acid, and tyrosine content. It is possible that these differences were due to impurities. Two preparations from cattle ligamentum nuchae and a preparation from sheep ligament were also assayed by an identical procedure. No significant difference was found in the amino acid composition of the ligament elastin from that of the aorta of the same species. Microbiological assay was also used by Lansing et al. (1951) who prepared elastin from human aorta by boiling with 0.1 N NaOH for 45 min. During this hydrolytic procedure tryptophan was found to decrease progressively and only a trace remained in the purified material. The amino acid composition reported differed somewhat from that given by Neuman for the aortas of the large ruminants, but the distribution had the same characteristic pattern. Apparently significant differences were found in the composition of aortas from young and old subjects. The senile elastins contained more aspartic acid, glutamic acid, serine, lysine, and arginine than elastin from young aortas. This result was criticized by Hall (1959) who thought that the difference in amino acid composition may be due to the presence of a contaminating protein which is not entirely extracted by sodium hydroxide solution during the purification treatment. Lansing and his colleagues themselves recognized this possibility and thought it unlikely that a homogeneous protein should change in character with advancing age. No recent work appears to have been reported on this subject and the problem awaits further investigation. Paper chromatography was used by Bowes and Kenten (1949) to examine the amino acid composition of elastin preparations from ligamentum nuchae and skin. The results indicated either that the elastic fibers of skin differ from those of ligamentum nuchae in amino acid composition or that they are less resistant to hot water. This interesting suggestion does not appear to have been pursued. The modern ion-exchange methods for amino acid analysis are possibly more accurate, and certainly more reliable than microbiological procedures; with careful use the experimental variations are small and the methods are sufficiently reliable to show up small differences in amino acid composition where comparative surveys are carried out in the same laboratory under identicalconditions. Gotte et al. (1961) used the method of Moore and Stein
264
S. M. PARTRIDGE
(1951) to assay the amino acid composition of elastins prepared from various tissues of the ox. Purified elastic fibers were prepared from ligamentum nuchae, aorta, and the elastic cartilage of the external ear by heating under pressure with water following the procedure of Partridge et al. (195.5). Some of the preparations were then further purified either by ( a ) furt,her autoclaving with water a t neutrality or ( b ) extraction with 0.5 N NaOH at 25°C for four periods each of 1 hr. Extraction of the ligamentum nuchae preparation with alkali resulted in the removal of rather less than 2 % of the total protein; with all three preparations the first alkali extract removed a significant amount) of protein but the amount extracted fell to zero with the fourth extraction. The total soluble protein removed from the aorta preparation was 3 % of the dry weight and nearly 10 % was removed from the material from ear cartilage. The amino acid compositions of the preparations are given in Table I and are compared with samples prepared by extracting the dried, defatted tissue powder with 0.1 N NaOH a t 98°C for 45 min. The results illustrate the course of the successive stages in purification and show that while elastin from ligamentum nuchae is readily purified either by simple autoclaving or alkali extraction, aorta contains substances of protein character which are much more difficultj to remove by alkali extraction. The elastica of ear cartilage is even more resistant to purification by the use of alkalies. With all three tissues, digestion with hot 0.1 N NaOH offers the readiest means of obtaining elastin preparations approaching constant composition. When prepared by hot alkali digestion the preparations from aorta had almost identical composition with those from ligamerdum nuchae; nevertheless t8he values for lysine in the preparations from aorta were always rather higher than in those from ligamentum nuchae and the difference is greater than the expected experimental error. The product from ear cartilage, after cold alkali extraction or digestion with hot, dilut,e sodium hydroxide solution, had the pattern of amino acid distribution characteristics of elastin, but was rather higher in aspartic and glutamic acids and rather lower in proline, glycine, alanine, and valine. Further purification by alkali digestion was not, possible because the elastin fibers tended to dissolve. The final analyses of the three elastin preparations are so similar that it is tempting to regard the proteins as identical and to ascribe the small differences as due to the presence of resistant impurities. That t,here are real differences between the elastins of different tissues can be shown by an examination of the physical properties of the different products. Slight differences in staining reactions have already been noted, but recent work in this laboratory has shown that there are marked differences in the rate of alkaline hydrolysis between the different samples. Figure 3 shows the
265
ELASTIN
effect of continued treatment with 0.5 N NaOH a t 25°C. The samples of elastin used were all prepared by autoclaving with water by a standard procedure. After 72-hr treatment with dilute alkali the whole of the ear TABLEI Amino Acid Composition of Bovine Elastins" , b Tissue Ligamcnlum nuchae
I
Aorta Method of isolation
Amino acid
Autoclaved
Zxtractec ith 0.1 h NaOH at
Autoclaved
9a0c
Autolaved am extracted rith 0.5 N NaOH a t 25°C
Extractet rith 0.1 1 NaOH a1 98°C
--
Aspartic acic 1.1 Serine 0.9 1.1 Threonine 2.4 Glutamic acid 13.5 Proline 26.7 Glycine 21.3 Alanine 17.7 Valine Methionine Trace 3.8 Isoleucine Leucine 9.0 1.5 Tyrosine Phenylala6.2 nine 0.5 Lysine 0.1 Histidine 1.3 Arginine 1.6 Hydroxyproline Tryptophan <0.1 16.8 Nitrogen
16.9
*
25°C
--
Auto,laved and extracted vith 0.1 N NaOH a t 98°C
3.5 1.6 1.6 6.4
3.4 1.4 1.3 6.3
2.1 0.9 1.o 4.6
23.9 19.9 16.0 0.3 4.0 8.8 2.3 6.0
12.1 25.2 20.9 16.7 Trace 3.6 8.3 2.1 5.3
14.0 26.8 22.2
-
13.7 22.9 16.8 13.4 Trace 3.3 9.9 3.3 5.9
12.8 22.9 17.2 13.7 0.6 3.0 9.9 3.3 5.8
14.3 25.8 20.3 16.0 Trace 3.0 9.4 2.6 5.6
1.2 0.3 1.7 1.5
1.o 0.2 1.0 1.5
1.1 0.1 0.8 1.8
1.8 0.5 2.5 1.9
1.6 0.2 2.4 1.8
0.5 0.1 1.3
0.5 16.9
0.4
-
-
-
-
16.6
16.8
16.4
16.1
16.7
15.2 26.8 22.8 18.2 Trace 3.5 8.8 1.6 5.5
0.8 1.5
Autoclaved a n extractec with 0.5 1 NaOH at
1.o 1.0 1.0 2.4
2.0 1.1 1.6 3.4
0.6 Trace
Autoclaved
1.4 1.4 1.5 3.4
0.7 0.9 1.o 2.4
--
5
Ear cartilage
-
-
Trace 3.8 9.0 1.8
-
Taken from Gotte et al. ( 1 2). Values given as grams of E ino acid per 100 gm of dry ash-free protein.
cartilage elastin had dissolved, but in this time only 60 % of the aorta and 30% of the ligamentum nuchae elastin had gone into solution. The cross section of the fibers of elastin from ligamentum nuchae is much greater than those from aorta, and these in turn are much thicker than the fibers from ear cartilage. That the differences in rate of hydrolysis were not due to differences in fiber diameter was shown by wet-grinding some of the liga-
266
S. M. PARTRIDGE
mentum nuchae fibers. This preparation showed a large proportion of crushed fibers, but its rate of hydrolysis was identical with that of the unground material. Differences in the rate of hydrolysis of the order observed strongly suggest differences in the degree of cross-linking of the
24
4a
72
Time of extraction with 0.5 N NaOH,hours
FIG.3. Time-course of solubilization of elastin from three bovine tissues ((iotte el al., 1962). The elastin sample8 (prepared b y autoclaving) were suspended in 0.5
N NaOH and maintained a t 25°C with gentle rocking. The amino acid composition of the samples is given in Table I. Key: 0 = elastin from bovine ear cartilage; A = elastin from bovine aorta; X = elastin from bovine ligamenturn nuchae.
proteins concerned. More peptide bonds must be hydrolyzed in a highly cross-linked protein-to release soluble fragments-than is necessary in a protein which is less highly cross-linked. This sit,uation occurs among the keratins, when the highly cross-linked (high sulfur-con taining) hard keratins of horn or hair are much more resistant to hydrolysis than the soft, low-sulfur keratins of the epidermis.
ELASTIN
267
Two views are possible about, the small differences in composition found for ear cartilage elastin: elastin of classic composition may be physically mixed with a second protein which is difficult to extract by dilute alkali; or the elastic fibrous protein of ear cartilage is homogeneous and different in composition from the elastin of aorta or ligamentum nuchae. A possibility of wider significance is that the cells that are responsible for the synthesis of “elastin” in ear cartilage produce two soluble proteins which are later integrated together by new covalent cross-links a t the site of fiber format,ion. A process of this sort could obviously give rise to as many different types of elastin as there are different types of tissue, and a similar mechanism is t.hought to account for the chemical and physical difference between keratins found in different) tissues of the same animal (Ward and Lundgren , 1954).
C . A ssociation with Mucopol ysaccharides Most authors who have studied the chemical nature of elastica have been impressed with the difficulty of freeing the elastic fibers from mucopolysaccharides and as a result the idea has gained currency that certain polysaccharides may be combined with t,he fibrous protein by chemical bonds. Hall et al. (1952) found up t,o 2 % of polysaccharide in elastin preparations purified by boiling with di1ut)e acetic acid, and Banga and Schuler ( 1 9 3 ) reported even higher contents of carbohydrate in similar preparations. These authors believed t,hat substantial quantities of mucopolysaccharide formed an integral part of the fiber substance, and accordingly suggested that elastin should be regarded as a glycoprotein. HOWever it has now become clear that most of the polysaccharide in the tissues is interfibrillar or lies at the surface of the fibers; by reasonably mild purification procedures a protein with a very low carbohydrate content can be isolated both from ligamentum nuchae and from aorta. This point was carefully investigated by Loeven (1960a) who prepared a large number of samples of elastin from bovine ligamentum nuchae both by autoclaving with dilute (2%) acetic acid and by boiling for two periods of 45 min with 0.1 N NaOH. Extraction with 2 % acetic acid at 1 atmosphere pressure under the conditions used was sufficient to remove all collagen (as judged by hydroxyproline determinations) but did not reduce the hexosamine content beloweabout 0.35%. It should be observed that the presence of acet,ic acid appears to inhibit the extraction of some of the mucoproteins and mucopolysaccharides of the ground substance since autoclaving in water according to the procedure of Partridge et al. (1955) reduces the hexosamine content of elastin from Zigamentum nuchae to below 0.1 %. In Loeven’s experiments boiling with alkali reduced hexosamine to 0.05 % indicating a residual mucopolysaccharide content of not more than about 0.1-0.2 %.
268
S. M. PARTRIDGE
This residue of polysaccharide was very small, but was quite const'ant over a large niimher of samples; it was regarded t>hereforeas being chemically hound. Looven showed that the firmly bound residue could be released from the protein by treatment with a preparation of pancreatic elastase that cont,ained elastomucase. In view of the small amount of firmly bound curbohydrat,e in purified elastin it is doubtful if there could be any useful purpose in regarding the fiber substance as a mucoprotein; many albumins and globulins contain similar small residues of carbohydrate and to describe them all as mucoproteins would only serve to negate the value of an accepted and useful system of classification. Buddecke (1960) attempted the complete analysis of the interst,itial mucopolysaccharides from the human aorta. It was found that t,he dried, defatted arterial tissue could be dissolved almost completely by treatment with papain a t a temperature high enough to denature collagen. Accordingly the powdered tissue was incubated for a total of 18 hr a t 65OC in buffer solution with the addition of papain. The solution was then dialyzed and high molecular weight peptides were removed by precipitating several times with ethanol at pH 2-3, 7, and 10. The mixture containing the mucopolysaacharides was then lyophilized and extracted with 90 % phenol. This dissolved neutral mucoproteins and other material of protein character and the residue contained the acid mucopolysaccharides. These were fractionated by column chromatography on powdered cellulose and yielded 5 fractions: chondroitin sulfate A and B (.27%), hyaluronic acid (14 %), chondroitiri (14 %), heparin (2 %), and keratosulfate (22 T I ) . In normal aorta the mucopolysaccharides represent about 1.3 % of the dry weight of the tissue but this increases to 2.5% in the sclerotic artery. In arteriosclerosis there is an increase of 50 % in chondroitin sulfate and generally a slight increase in keratosulfate, while hyaluronic acid and chondroitin are reduced to 40 % and 15 % of their original values. The neutral mucopolysaccharides of aorta occur as glycoproteins, low in uronic acid. Histochemical methods have been used to follow changes in the acid mucopolysaccharides of human aortic tissue. Bertelsen and Jensen (1960) examined aortas from subjects up to 90 years old including some fetuses. The aortic tissue was found to contain both hyaluronic acid and sulfated mucopolysaccharides. The aortas of fetus and newborn contained hyaluronic acid as the major component. In aging the amount of sulfated polysaccharides increases up to about 50 years, especially in the media and intima, and after this time it is nearly constant. After 20 years of age, a periodic acid-Schiff (PAS) positive material is deposited in increasing amounts in the media and later in the intima. It was thought likely that the acid mucopolysaccharides are elaborated by the fibroblasts and the
ELASTIN
269
endothelial cells. Young elastic fibers do not stain with toluidine blue but older fibers are stained orthochromatically with this dye. Age changes in the mucopolysaccharide content of the human aorta have recently attracted a great deal of attention because of a possible association with the onset of sclerosis. Kaplan and Meyer (1960) compared the mucopolysaccharide patterns of isolated fractions from whole human aortas at various ages. The acetone-dried powder from whole aorta was homogenized in 0.1 N HC1 and digested with pepsin. The peptic digest was then neutralized and digested with trypsin and protein degradation products were removed by the Sevag (1934) procedure and several alcohol precipitations. The product was finally fractionated as the calcium salt at 18 %, 23 %, 28 %, 33 %, 43 %, and 52 % alcohol. The fractions were analyzed for uronic acid, glucosamine, and galactosamine and characterized by their reactions with pneumococcal hyaluronidase, testicular hyaluronidase, and A-adapted2 and heparitin-adapted enzymes. Optical rotation and infrared spectrophotometry were also used to characterize the mucopolysaccharides. The total yield of mucopolysaccharide showed no correlation with age, nor did the total hexosamine content of individual aortas; total mucopolysaccharides varied between 0.85 % and 1.2 % of the dry weight of the whole aorta tissue. Hyaluronic acid and chondroitin sulfate C were found to decrease with age while chondroitin sulfate B and heparitin sulfate increased. These results show a pattern of aging which was quite different from that of costal cartilage and it was thought that degenerative and reparative processes were superimposed on the normal age changes. Gore and Larkey (1960) studied the ground substance of human aorta and found it to be largely mucopolysaccharide in nature. In subjects older than 50 years the mucopolysaccharides were found to constitute about 0.5% by weight of the aortic tissue. The mucopolysaccharides show anticoagulant properties which were thought to be due to the chondroitin sulfate B fraction bhich represented about 4 0 4 0 % of the total mucopolysaccharides. Age changes in the skin were investigated by Shibaeva and Zherebtsov (1960) who showed a gradual development of hyperelastosis with age. Up to 25 years of age the normal pattern of histochemical reactions was found, but after this age increased metachromatic reactions gradually developed and thick elastic fibers appeared in the hyperelastotic areas. After 30 years neutral mucopolysaccharides began to accumulate and at, 70 years dense amorphous masses of modified elast>in appeared along with acidic polysaccharides. In view of the complex nature of the system of mucopolysaccharides and mucoproteins in the ground substance of aortic tissue it, is not surprisCell-free extracts of FEavobacterium digest chondroitin sulfate B when the organism has been adapted to either chondroitin sulfate A or B.
270
S. M. PARTRIDGE
ing that quantitative isolations carried out in different laboratories by different methods should sometimes lead to conflicting results. Although the methods at present in use frequently give very satisfactory separations with artificial mixtures of purified mucopolysaccharides they often fail when a natural tissue is analyzed. Very little appears to be known about the effects of variations in molecular weight on chromatographic and precipitation procedures with mucopolysaccharides, and it seems quite possible that variations in molecular size are sometimes the cause of failure to isolate clean fractions. In the analysis of the interstitial matrix of elastic tissue the emphasis so far has been towards the acid mucopolysaccharide fraction, but it has recently become apparent that proteins containing neutral carbohydrate are often present in greater quantity than the acid mucopolysaccharides and their protein complexes. These substances, of predominantly protein character, often contain sialic acid; because of their high concentration in the elastic tissue of the large arteries they may be of special importance in maintaining the integrity of these vulnerable structures. An attempt was made by Bertelsen (1960) to isolate the glycoproteins from human aorta and to investigate age changes in this fraction. The tissue was freed from adventitia, disintegrated, and defatted. It was then extracted with 0.5 N NaOH for 4 days at, 0°C. The filtrate was neutralized and the acidic mucopolysaccharidesprecipitated with 2 volumes of ethanol. The filtrate contained the mucoproteins. These were precipitated by increasing the alcohol content to 84%, giving a fraction that was free from uronic acid. It was found that both the acidic mucopolysaccharides and the glycoproteins increased with age but the percentage increase was much greater with the former. There was also an increase in the hexosamine contents of both fractions with age. The periodic acid-Schiff reaction was faint with the acid mucopolysaccharide fraction but was very strong with the glycoprotein fraction. These findings were in agreement with the histological picture showing t,hat the deposit, of periodic acid-Schiff-positive substance is not distinct in adult aorta until after the second decade, thereafter it increases gradually with age.
D. Association with Lipids and Lipoproteins The state of combination of the lipids of the human aortic wall has attracted greet, iiit,erest in the past, few years because of it,s significance in theories of the origin of the lipid material in the atherosclerotic plaque. Much work has also been reported on changes in the composition of the extractable lipids of the media, but these compounds, although important in the study of the development of atherosclerosis do not directly affect the elastic fiber and must be regarded as outside the scope of this review.
ELASTIN
271
Nevertheless the suggestion has been made from time to time that elastin is so closely associated with certain substances of lipid character that it should be regarded as a lipoprotein. Lansing et al. (1952) pointed out that elastic fibers exhibit a weak but positive osmiophilia and are colored a light blue-black by Sudan black. Both these tinctorial reactions suggest the presence of lipid material. These authors also observed that elastase digestion of elastin is accompanied by the release of small fatlike droplets capable of concentrating Nile blue sulfate. F a t analysis of hydrolyzates indicated the presence of a small but consistent lipid moiety in acetonemethyl alcohol-refluxed, sodium hydroxide-extracted elastin. These results suggest the presence of a small tightly bound lipid fraction which resists conventional fat extractants. La Bella (1957) found that when defatted elastin was hydrolyzed by pancreatic elastase about 0.5% of the dry weight of the product could be extracted by fat solvents. On the basis of a positive reaction with Schiff’s reagent this substance was thought to be an unsaturated fatty acid. La Bella suggested that the protein-bound unsaturated substance may be related to the unknown grouping in elastin, with an absorbance peak at 2750 A, which was reported by Partridge and Davis (1955). Gero e l al. (1960) observed that in the morphogenesis of atherosclerosis the deposition of lipid substances in the human aorta is preceded by an increase of metachromatically stainable mucoid substance. By means of immuno-electrophoresis it could be shown tjhat one part of the lipids in the aortic intima is identical with the @-lipoprotein of plasma (Ott et al., 1958). This substance has been shown to form specific complexes with polyanions of high molecular weight and, based on this, Gero and his colleagues believe that part of the lipoprotein of aorta exists as a complex with mucopolysaccharide. This supposition is supported by the histological finding that the deposition of lipids occurs mainly in those areas of the aorta that are rich in metachromatically stainable substances. The mucopolysaccharides of sclerotic human aortic intima were isolated and separated by paper electrophoresis into two fractions: one fraction represented the sulfated mucopolysaccharides (periodic acid-Schiff-negative) and the other fraction (of lower mobility) the nonsulfated mucopolysaccharides. The mixture of mucopolysaccharides obtained by extraction was mixed with purified human @-lipoprotein and the system was examined by paper electrophoresis. The @-lipoprotein (stained by Sudan black) was found to migrate with the nonsulfated mucopolysaccharide fraction. The association of lipoprotein with periodic acid-Schiff-positive mucopolysaccharides or mucoproteins has been observed by many histologists both in the intima and in t,he mcdia. The complexes appear to be laid down as thin layers following the contours of the elastic lamellae. There appears to be little
272
S. M. PARTRIDGE
doubt that a large part of the lipoprotein is deposited in the interfibrillar spaces and as such can be regarded as a structure quite distinct from the elastic element. There remains the possibility that the protein of the elastic fibers may itself be associated with a small amount of bound lipid or other fat-soluble substances in the native condition, and this has recently been investigated in some detail by Loomeijer (1961). This author examined elastin from bovine ligamentum nuchae and bovine aorta purified b y autoclaving with water and extraction with fat solvents. The protein was partially hydrolyzed with N HC1 at 150°C for 4 hr. This resulted in a mixture of peptides, some of which retained the yellowish-white fluorescence of the original protein. The fluorescent peptides were adsorbed on charcoal and recovered by elution with pyridine. After removal of the solvent a small portion of the residue was found to be soluble in ether. This material was further hydrolyzed by refluxing with N HC1 in ethanol-water and the residue was extracted with ether. The water-soluble fraction contained amino acids but the ether-soluble fraction was free from nitrogen and contained a substance which gave a brilliantly fluorescent band on paper chromatograms. This substance was purified chromatographically. On the basis of physical properties and the infrared spectrum it was suggested that this substance is a carboxylic acid (possibly ketonic) wit,h a chain length of about 12 atoms. The acid appears to be bound to peptide by primary valency and Loomeijer suggests that the fluorescence of elastin may be due to the presence of this material. It was estimated that the content of the solvent-soluble fluorescent acid in purified elastin may be of the order of 0.1-1 %. Further discussion of the fluorescent prosthetic group in elastin is given in Section VI. In the work of Loomeijer and work on similar lines carried out in the author’s laboratory no evidence was found for the presence in important amounts of the common fatty esters, phospholipids, or steroids in purified preparations of elastin; in these circumstances there seems to be little reason to classify elastin with the lipoproteins. The uptake of lipophilic dyes, which has been mentioned by several authors as indicating a lipoprotein character, can as well be explained by the presence of massive nonpolar radicals in the fluorescent prosthetic group, as described by Loomeijer, or to interactions with lipophilic sections of the peptide chain due to the large concentrations of nonpolar amino acids such as valine, the leucines, and phenylalanine.
E. Determination in Tissues Methods at present in use for the determination of elastin in tissues are based on the current methods for the purification of elastin (Section IV,A)
ELASTIN
273
and depend upon the relative insolubility of elastic fibers in hot water or arids and alkalies. Ideally the aim is to choose conditions such that all other tissue components arc extracted without, solubilization of any part of the elastin; this may then be determined gravimetrically, or by estimating nitrogen, or by other means. Lowry et al. (1941) described a procedure based on the extraction of muscle protein and other noncollagenous substances with 0.1 N NaOH a t room temperature; collagen was then converted to gelatin by autoclaving with water. The collagen and elastin contents were then determined gravimetrically, elastin by weighing the final dried residue and collagen from the difference in the dry weight of the residues. Neuman and Logan (1950) modified this procedure by using hydroxyproline determinations for estimating the collagen in the autoclaved extract and the elastin in the final residue. For this purpose the hydroxyproline content of collagen was taken to be 13.4 gm amino acid per 100 gm dry protein. Neuman and Logan believed that the hydroxyproline content of elastin differs for different species and, for estimating elastin, used the values: 1.5% for pig; 1.91 % for ox; and 2.3 % for rat. The method proved suitable for small amounts of tissue and an accuracy of better than f 5 % was claimed for the determination of collagen; the results for elastin were rather less reproducible. A partial survey of the tissues of a number of mammals was attempted using this method; the elastin content of the aorta of ox, pig, and rat was given as 39.8 %, 57.1 %, and 47.7 % dry weight, respectively; 4.9 % and 3.9 % of elastin was found in the chordae tendineae of the ox and the pig and 4.6 %, 1.2,5%, and 0.55 % in the spleens of ox, pig, and rat respectively. Collagen is very resistant to cold alkali, but is readily soluble in hot dilute sodium hydroxide. This reagent also dissolves muscle proteins, cellular constituents, and components of the ground substance. If elastin is the only fibrous protein to be determined gravimetrically, digestion of the tissue in a single step with hot alkali should be adequate for effective isolation. Such a procedure was investigated by Lansing and his colleagues (1952) who showed that elastin could be prepared in a reproducible form by digesting whole defatted tissue with 0.1 N NaOH at 98°C for not less than 45 min. After this time the weight of the residue was constant and remained so up to 60 min with the tissues analyzed. The method has proved to be a rapid and convenient procedure for routine estimations of elastin in tissues with a relatively high content of elastica, but is less satisfactory for tissues which contain much muscle or other cells. Human aorta tissue usually has a high calcium content, much of which appears in the residue after alkali digestion. If the residue of elastin is determined gravimetrically it is therefore essential to correct for ash as well as for moisture.
274
S. M. PARTRIDGE
Harkness et al. (1957) examined the collagen and elastin content of the walls of the larger arteries of dogs. Sect,ions of the arteries were aut,oclaved 6 hr a t 2.2 kg/cm2 pressure and then washed twice with hoiliiig distilled water to extract the collagen degradation products. The collagen content was estimated by determining hydroxyproline in the extract after complete hydrolysis. The residue was heated for 45 min a t 100°C in 0.1 N NaOH to dissolve material other than collagen. The residual elastin was fat-extracted and weighed. As a check the residue was estimated for hydroxyproline, but it was found that the values varied from 1.0% to 2.05 % in different dogs. The thoracic aorta of dogs contained 29-32 % of t,he dry weight as elastin and the abdominal aorta only 11-16 70; the pulmonary arteries contained 16-20%. The whole system of arteries was found to fall into two distinct types. The first type is virtually confined t'o the intrathoracic aorta, from the heart to within a few centimeters of the diaphragm, and in this vessel about 60% of the total fibrous protein is elastin. The second type comprises all the other arteries of the body. In these about' 30 % of the fibrous protein is elastin and 70 % collagcn. The transition from one type to the other is relatively abrupt. Similar extraction methods have been adapt'ed to a variety of tissues hy other workers. Wilson et al. (1954) examined t~hceffect of age and grade on t,he collagen and elastin content, of t m f and veal. They concluded that the elastin and collagen content of the longissirnus dorsi of h e d animals is not, a critical measure of the tenderness of meat. There was no difference in collagen and elastin content of samples from cows and steers. Samples from older animals contained less collagen and elastin than veal. On the other hand, Lorinz and Szeredy (1959) concluded that chemical det,ermination of connective tissue could be applied to the grading of meat. These authors determined collagen and elastin together by extracting tJhe muscle protein with 0.05 N alkali a t room temperature for 24 hr; t,he result>swere calculated by subtracting the nitrogen extracted from the total nitrogen of the sample. Higher total connective t,issue and elastin values were found with meat from young animals and low-grade animals. Histological examination of the treated t.issues agreed with the resuks of collagen and elastin determination. Ornes and Roddy (1960) investigated the elastin content of animal skin before and after treatment for leather manufacture. Two methods were used for the determination of elastic tissue. In the first, squares of limed calfskin, after deliming, were treated with a commercial pancreatic preparation containing elastase to dissolve the elastin. The squares were then autoclaved for 4 hr at 15 Ib steam pressure, and the residue was treated alternately wit8hhot 0.Ti N HC1 and hot water to remove lime and acid. Finally the residue was extracted with acetone to remove fat, and was
ELASTIN
275
dried. Comparison of the residues from the treated squares with control squares gave a measure of the weight of elastin and collagen removed. In the second method specimen squares were weighed and autoclaved in distilled water without prior enzyme treatment. After washing they were dried and reweighed, the residue being taken as elastin. The residual material was examined histologically. Stained preparations showed the elastic tissue as a basketlike network in the epidermal area and also demonstrated the elastic tissue formations of the arteries. It was possible by these methods to determine the influence of leather-processing operations on the elastic tissue; in normal calfskin processing there is a reduction in the amount of elastic tissue compared with that found in salt-cured calfskin, and by use of sufficient pancreatic enzyme it was possible to remove up to 86 % of the elastic tissue in the bating operation. In the medical field there has been a growing interest in comparing the elastin content of a variety of human tissues as a function of age. Bertolin and Scapinelli (1958) determined the elastin content of the yellow ligaments from the human lumbar tract by extracting the dried and defatted tissue with hot 0.1 N NaOH. The elastin content averaged 70.26% in subjects 25-37 years old, and 66.20% in subjects 45-68 years old. Scarselli and Repetto (1959) found that the elastin content of human lung increases progressively with age: a t 1 0 3 0 years the content (grams/100 gm of dry fat-free tissue) was 3.9 %; a t 43-58 years, 7.0 %; 64-70 years, 9.8 %; 78-94 years, 13.5 %. The collagen and elastin content of the lung in emphysema was investigated by Pierce et al. (1961). The fresh lung was first extracted with 0.1 N NaOH for 7-14 days a t room temperature, the NaOH being changed each day. The resulting “lung skeletons” were sectioned for histological examination. Blocks of tissue were then analyzed for collagen and elastin by the method of Lowry et al. The mean dry weight of the lung skeleton was 17 gm in the control group and 20 gm in the emphysematous group; of this collagen averaged 46.3 % and 47.4 % respectively, and elastin 18.9o/o and 17.9 % respectively. Thc amino acid composition of elastin of emphysematous lungs was not found to differ significantly from that of normal humari lung tissue or of ox ligamenturn nuchae. It was concludcd that pulmonary emphysema does not result from any primary dissolution or loss of lung collagen or elastin. To summarize the present position concerning the determination of elastin in tissues it appears that procedures based on the successive removal of all other tissue components, by the use of suitable extractants, are satisfactory provided conditions are carefully chosen to secure adequate purification of the elastic residue without any loss of elastin by partial hydrolysis and dissolution. When muscle or other cells are abundant in the tissue or the content of mucopolysaccharide and mucoprotein
276
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is high, an initial treatment by extraction of the fresh tissue at low temperatures with dilute (0.054.1 N ) sodium hydroxide as proposed by Lowry is advisable. Removal of collagen from aortic tissue or yellow ligaments can readily be effected by digestion with 0.1 N NaOH for 45-60 min at 98°C; but as indicated in Section IV,R, elastin from different tissues varies in the ease with which it is dissolved by hot or cold alkali, and in the analysis of tissues such as the elastic cartilage of the external ear there is risk of loss of elastin by partial hydrolysis and dissolution. Gravimetric procedures for the final determination of the residual elastin appear to be satisfactory when the extraction is carefully performed but the results should be corrected by determining the ash and water content of the residue ; alternatively the residual elastin may be determined by estimating the total nitrogen of the residue after acid hydrolysis. The procedure studied by Ayer et al. (1958) based on the removal of collagen and other tissue components by treatment with 88% formic acid at 45°C for 36-72 hr has not been developed for routine analysis, but appears promising. However, precautions against loss of elastin by hydrolysis and dissolution are equally necessary with formic acid as with dilute alkalies. V. ELASTOLYTIC ENZYMES The literature relating to collagenases and elastases has recently been the subject of a comprehensive review by Ines Mandl (1961) and no purpose would be served by attempting a similar description here. I t is proposed instead to summarize briefly some of the salient features arising from the work of the last decade and to draw attention to a number of recent researches that have led to advances in the study of elastolysis by enzymes.
A . Occurrence of Elastases The term “elastase” is used to describe any enzyme which will dissolve fibers of the insoluble protein elastin, and it is implicit, in this definition that the elastin fibers used as substrate in the assay procedure should be undamaged and free from contamination by other substances. As indicated in Section IV this ideal is not easy to achieve. Isolation of elastin by means of hot alkali or acid may give rise to partial hydrolysis and the appearance of new a-amino and a-carboxyl groups if the treatment is overprolonged. The elastin fibers so prepared carry a greater surface charge; they show a higher adsorption affinity for proteolytic enzymes, and are more susceptible to enzyme attack. If degradation has proceeded far enough there is danger that the preparation may be digested by enzymes which would not attack the native fibers of the tissues and which would not normally be regarded as elastolytic. On the other hand, insufficiently purified material may be protected by a surface coating of mucopolysac-
ELASTIN
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charide or other components of the ground substance. Such preparations may be only slowly attacked by pure elastases but are rapidly attacked when the enzyme system contains a mucolytic component (Loeven, 1960b). Elastin preparations which are unduly slow in their reaction with elastases may also result from overheating or overdrying. It is therefore essential that in claiming elastolytic activity for an enzyme preparation, the source and previous history of the substrate should be specified. No universally accepted specification for the substrate, either as to source or method of preparation, has yet been agreed upon, but in assay procedures most workers use elastin from ligamentum nuchae because of the relative ease with which it can be prepared. Purification procedures have varied from the st>rictavoidance of acid or alkali (Thomas and Partridge, 1960) to the use of hot dilute sodium hydroxide (Lansing, 1954). In the opinion of the present author (based on the considerations given in Section IV) the use of material from ligamentum nuchae prepared by autoclaving in water until free from collagen-followed by a short extraction with dilute alkali at room temperature-would provide a reproducible substrate of acceptable purity. 1. Elastase Activity of Proteolytic Enzymes
With elastin preparations as described above, the classic proteolytic enzymes of the pancreas-trypsin and the chymotrypsins-have little activity and do not produce dissolution of the fibers. In the early literature there are many reports of the dissolution of elastin fibers by pancreatin or by impure preparations of trypsin, but it was not until the work of Balo and Banga (1949) that the existence of a separate elastolytic enzyme in the pancreas was recognized. Soon after this Banga (1952) obtained a purified crystalline enzyme which was thought to be substantially free from nonelastolytic components of the pancreatic system of enzymes. The pancreas of all mammals so far investigated contain an elastase with similar enzymatic reactions (Lewis et al., 1956; Marrama et al., 1959), but immunological differences have been observed between pancreatic elastases from different species (Moon and McIvor, 1960). Elastase is secreted in the pancreatic juice as an inactive zymogen, LLproelastase” (Grant and Robbins, 1955; Lamy and Lansing, 1961) which, like other pancreatic enzymes, is activated by trypsin or enterokinase. Although it is now generally agreed that pure preparations of trypsin and chymotrypsin do not digest elastin there have been frequent reports in the early literature that pepsin is capable of dissolving elastic fibers. This was repeated recently by Fisher et al. (1960) who observed moderate elastolytic activity by three different crystalline pepsin preparations at pH 1.2. The action of pepsin was estimated to be approximately one-eighth
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S. M. PARTRIDGE
that, of the elastase preparations tested. Lewis el al. (1956) and Thomas and Partridge (1980) found no measurable elastolytic activity wit'h their assay procedures and it is assumed that, the contradiction arises because of differences in the method of preparation of t,he substrate. In view of what is known about the st,ructure and composition of elastin and its apparent lack of crystallinity and organized tertiary structure it would not be expected that proteinases capable of digesting elastin would show a high degree of specificity for this substrate. This seems to be the case, and with one recent exception all elastolytic enzymes so far reported are active general proteinases, most with a wide peptide bond specificity. Purified preparations of pancreatic elastase readily attack most denatured proteins and are resisted only by certain native proteins such as collagen, which are characterized by structural organization of a high order. Highly active proteinases, with wide peptide bond specificity, are found among the latex-bearing plants, and three of them, papain, ficin, and bromelin, are commercially available. These enzymes, which are SH-activated, readily attack elastin yielding free amino acids and peptides of low molecular weight (Miyada and Tappel, 1956; Wang et d., 1958; Thomas and Partridge, 1960). Elastase activity is not a universal property of proteolytic sulfhydrylactivated enzymes. There are abundant reports in the literature describing the disappearance of elastic fibers in vivo preceding the repair of damaged tissues, but there is no evidence as to how this is brought about. The tissue cathepsins, most of which are SH-activated, have received little systematic study, but Thomas and Partridge (1960) reported that cathepsins extracted from kidney and spleen by the method of De La Haba et al. (1955) did not digest elastin either in the presence or the absence of cysteine. Microorganisms have recently proved to be a rich source of elastolytic enzymes. Among the molds several organisms have been found which rapidly digest elastin incorporated in the growth medium (Sbarra el al., 1960); but the low concentration of enzyme protein in solution in the medium makes the isolation of many such enzymes difficult. Elastolytic bacteria have frequently been described in the early literature and also more recently, but the enzymes responsible were not isolated or characterized until the work of Mandl and Cohen (19Fi9, 1960). These authors found elastolytic activity in Pseudomonas aeruginosa isolated from a patient suffering from periodontitis, and other elastolytic Pseudomonas were also obtained from various sources. Many bacterial proteinases have since been tested for elastolytic activity in various laboratories and some have been found which are capable of dissolving elastin at a rate comparable with purified preparations of pancreatic elastase. The only microbial
ELASTIN
279
elastase that has yet been brought to a state of substantial purity is the enzyme from Flavobacterium elastolyticum isolated by Mandl and Cohen (1960). All the elastase preparations isolated from microorganisms so far reported are active proteolytic enzymes and digest a variety of denatured proteins. However, it is claimed by Mandl and Cohen that treatment of partly purified preparations of Flavobacterium elastase with alumina Cr removes associated nonspecific proteolytic enzymes and leaves in solution a low concentration of an enzyme which digests elastin but has no action on other proteins. The yields obtained were very small but the results are of considerable theoretical interest as the first demonstration of true substrate specificity among elastolytic enzymes. 2. Methods of Assay
All the methods of assay in use at present are based on measuring the amount of the substrate that is solubilized by the action of the enzyme in a given time. This may be carried out either by measuring the loss in weight of the undigested elastin (Banga, 1952) or by estimating the soluble protein and peptide products formed. For this purpose optical density (Grant and Robbins, 1957), the Folin reaction (Banga et al., 1959), or refractive index (Hall and Czerkawski, 1959) can be used. The color released from dyed elastin is also used as an index of solubilization: Naughton and Sanger (1961) used elastin dyed with congo red; azoelastin was used by Robert and Samuel (19rj7); and orcein was used as the dying agent by Sachar et al. (1955). In most of these methods some 10-20 mg of elastin is taken for each tube of the assay; the elastase unit is rather large and the assay procedures are expensive in enzyme when they are used to control purification procedures. A much more sensitive method, which measures quantities of elastase of the order of one-hundredth the Banga (1952) unit, was devised by Sbarra et al. (1960). In this assay elastin-agar plates arc used and the method is based on measuring the diameter of the clear zone which is produced by elastase deposited in circular holes cut in the gel. This method of assay is also of particular value in screening microorganisms for the production of elastase. The kinetics of the enzymatic dissolution of elastin are very complex and the reaction of elastin fibers with elastolytic enzymes is commonly characterized by a lag phase or a markedly sigmoid time course (cf. Hall and Czerkawski, 1959; Naughton et al., 1960). The length of the slow initial phase is influenced by the enzyme-substrate ratio and the source and method of preparation of the elastin used in the assay; the course of the reaction with different enzymes is also influenced by the presence or absence of salts, reducing agents, and many other substances. As a result
280
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of these factors, it is difficult to compare the activity of enzyme preparations in terms of the various kinds of unit and different assay conditions adopted by different laboratories. Variations in the length of the lag phase are particularly troublesome as a source of discrepancy between apparently similar assay procedures used in different laboratories. The lag phase is thought to arise from the cross-linked nature of elastin; when elastin is attacked by a proteolytic enzyme a finite number of peptide bonds must be broken, and a finite time must elapse, before the release of a soluble fragment of the network (cf. Hall and Czerkawski, 1961). The lag phase is very marked during the hydrolysis of elastin with acids or alkalies (Partridge et al., 1955; Gotte et al., 1961). This reaction is independent of the particle size of the elastin preparation, presumably due to the rapid diffusion of hydrogen and hydroxyl ions within the particle. With enzyme reactions, hydrolysis appears to proceed at the surface of the fibers or the microfibrils and the reaction rate is particle size-dependent (Loeven, 1960; Scarselli, 1960). Here the lag phase is less evident, but the reaction commonly starts slowly, and later accelerates, giving a more or less sigmoid time curve instead of the zero-order kinetics that would be expected for the initial phase of a surface reaction. Time curves of an entirely different character are obtained if the same enzymatic reaction is carried out a t constant pH with an automatic titrator and the number of peptide bonds broken in unit time is measured. Here there is usually no lag phase, and the release of a-carboxyl and a-amino groups follows a logarithmic time course. As yet, assay procedures based on the liberation of a-amino and a-carboxyl groups have not been used extensively for routine purposes, and indeed such assays yield information which is different in kind from that given by measurement of elastin solubilization. Nevertheless, in recent work Lamy et a2. (1961) were able to find a restricted set of conditions under which the initial phase of the reaction with pancreatic elastase exhibited zero-order kinetics both when measured by estimating the elastin solubilized or by estimating the a-amino groups released. Using these conditions empirical units of elastolytic activity can be defined which have a simple arithmetic relationship when determined by a variety of methods.
B. Properties and Mode of Action of Elastases It has already been remarked that most enzymes with elastolytic activity have proved to be proteinases with a wide peptide-bond specificity. Thus papain, bromelin, and ficin have a similarly broad hydrolytic action and are all active elastolytic enzymes. Sanger et a2. (1955) found that the oxidized A chain of insulin was hydrolyzed in a variety of positions by either crude papain or activated mercuripapain. There were five major
ELASTIN
28 1
sites at which papain hydrolyzed the insulin A chain, pepsin (which has a much smaller elastase activity) rvhibits three major sites of action, and chymotrypsin (which does not digest elastin) only one. Naughton and Sanger (1961) in a study of the peptide-bond specificity of the pancreatic enzyme used as starting material crystalline pancreatic elastase prepared by the method of Lewis et al. (1 956). This preparation was fractionated by chromatography on carboxymethylcellulose and showed the presenre of several protein peaks, but elastolytic activity was associated with only one of them. Material from the isolated peak had insignifirant trypsin and rhymotrypsin activity as measured by assay against synthetic substrates, and the peak was regarded as enzymatically pure. The enzyme prepared in this way was completely inhibited by diisopropyl phosphoroM DFP, showing that elastase, fluoridate (DFP) a t a concentration of like trypsin and chymotrypsin, is a DFP-sensitive enzyme. Using P32DFP, radioactive diisopropoxy-P32-phosphinyl-elastase was preparcd and was found to give a single radioactive peak in the same position as elastase when chromatographed on carboxymethylcellulose. From the specific activity of the P32-DFP-elastase it was calculated that the molecular weight of elastase was 28,500 assuming that 1 mole of DFP reacts with 1 mole of elastase. This figure compares with the molecular weight 25,000 given by Lewis et al. (1956) from ultracentrifuge and diffusion measurements. The reaction of the purified preparation of elastase with the A and B chains of insulin was then studied, and the peptide-split products were identified. The results showed that, unlike trypsin and chymotrypsin, elastase can attack a wide variety of peptide bonds involving neutral amino acids having aliphatic side chains. Naughton and Sanger suggested that the digestion of elastin by pancreatic elastase can be explained in terms of a general specificity for bonds involving the carboxyl groupsof neutral amino acids such as leucine and valine, which are present in high concentration in elastin. In work from the same laboratory, (Naughton et al., 1960) the same preparation of radioactive DFP-elastase was used to study the amino acid sequence at the active center of the enzyme. It was found that elastase, like trypsin and chymotrypsin, contains the sequence Asp.Ser.Gly around the reactive serine residue. The results given above suggest that pancreatic elastase is a single and highly characteristic component of the pancreatic complex of enzymes, but other workers have concluded that the active principle is itself a system of more than one component. Thus Lamy et al. (1961) isolated two elastases from the euglobulin fraction of pancreatin by chromatography on Amberlite IRC-50. One of these components had a high proteolytic activity towards casein, but the other was much more highly specific towards elastin and digested casein only slowly. Dvonch and Alburn
282
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(19.59) also isolated two elastolytic components using starch electrophoresis. It is not] yet clear whether these multiple elastolytic fractions represent split products arising from the same zymogen or if they are independent enzymes of the pancreatic system. However the discovery of a pancreatic elastase with little nonspecific proteolytic activity is of considerable significance. The complete separation of elastase from other enzymes of the pancreas has proved a difficult matter and the crystalline enzyme prepared by Banga (1952) is now known to contain several other factors. Mucolytic activity was first reported by Hall et al. (1952) and Hall (1957) showed the presence of a separate mucolytic factor which enhanced the activity of the proteolytic enzyme. Loeven (1960a,b) confirmed the separate nature of the two enzymes; he isolated elastoprot~einaseunmixed with elastomucase and confirmed that the latter has a marked synergistic effect with elastin preparations prepared by autoclaving with 2 % acetic acid. These preparations still contained about 1% of polysaccharide, and during the reaction with the mixture of enzymes, hexosamine con t,aining polysaccharide was released in the initial phase. 1. Action of Etastolytic Enzymes o n the Cells o j the Epidermis
Recently an interesting feature of elastolyt,ic activity has come to light concerning tjhe effect of proteolytic enzymes with elastase activity in releasing the epidermis from the collagen structures in the skin; the same enzyme preparations are effective in enzymatic unhairing and in bringing about the separation of cells in tissue cultures. Cordon et al. (1960, 1961) suggested that fibers of elastin may act as an anchoring material for the epidermis of the skin and examined the relationship between the hairloosening ability and elastase activity of depilatory enzymes. Fifteen commercial enzymes including papain, bromelin, trypsin (not purified), and a keratinase were used. The results obtained with three different, methods of determining elastase activity did not rank the enzyme preparations in the same order and no exact correlation was found between the hair-loosening effect and the elastase activity of the enzyme preparations. It was found that a “keratinase” from Streptomyces fradiae had the greatest elastase activity of any of the enzyme preparations tested. A similar line of thought recently led Fiizi et al. (1960) to examine the use of elastase for the isolation of cells during tissue culture. The effect of pancreatic elastase in separating the cells for the preparation of cell suspensions was examined. It was found that pancreatic elastase solutions in phosphate buffer were effective within 3-10 min for rabbit. epithelial cell cultures and human amnion epithelial cell cultures. The toxicity of the elastase preparations towards the cells did not appear to exceed that of trypsin.
ELASTIN
283
2. Biological Inhibitors
Like other pancreatic proteinases the elastase from pancreas is inhibited by substances in the serum of mammalian blood. A nondialyzable inhibitor of pancreatic elastase was observed in human, cattle, and rabbit serum by Balo and Banga (1949) and in 1959 Tolnay and Bagdy reported 50-90 % inhibition with 1:100 dilutions of human, cattle, rabbit, and guinea pig serum. Wu and Laskowski (1960) found that a crystalline trypsin inhibitor from bovine blood also inhibited elastase. The inhibitor was similar in composition to ovomucoid. Soybean trypsin inhibitor also inhibits pancreatic elastase (Graham, 1960). On the other hand, soybean trypsin inhibitor or mammalian blood serum does not inhibit Fluvobucterium elastase at equivalent concentrations (Mandl and Cohen, 1960). According to Walford and Schneider (1959) the serum elastase inhibitor migrates electrophoretically with the a,-globulin or albumin fraction of the serum and the highest concentrations of inhibitor were found in sera with the highest a-globulin content. The substance involved seems to be very similar to the serum trypsin inhibitor, but according to Balo and Banga (1949) the two are not identical. However inhibition of pancreatic elastase by serum can be completely reversed by adding a stoichiometric amount of trypsin (Graham, 1960). Walford and his colleagues (1959) reported that human and chicken serum inhibitors inhibited only the elastolytic action of pancreatic elastase and had no effect on casein hydrolysis, but Robert and Samuel (1957) found that both hemoglobin digestion and elastolysis were inhibited to the same degree. 3. The Specificity of Elastases As indicated earlier the mechanism by which some proteolytic enzymes are enabled to attack certain proteins, but not others, is little understood. It is well known that proteolytic enzymes are markedly specific for individual peptide bonds, both in proteins and in synthetic peptide substrates; but since proteolytic enzymes usually attack denatured proteins much more readily than the same proteins in their native condition, it is obvious that more is involved than the nature of the amino acid sequences in the substrate. I t is commonly assumed that the high specificity of native proteins in many biological reactions is due in part to the helical arrangement of portions of their peptide chains and in part to other arrangements of the interhelical and nonhelical regions to give a specific tertiary structure. As a result of the unique arrangement of the chain in the macromolecular particle, the particle must present a unique two-dimensional pattern of atoms at its surface. This pattern may react with a similarly unique pattern on the surface of the enzyme and an enzymecatalyzed reaction with very high specificity may result. The highly specific nature of the attack of bacterial collagenase on colla-
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gen can readily be understood on this basis, since collagen is known to have a highly ordered structure, both in the arrangement of the peptide chains in helices and in the apposition of one helix with another. However the same considerations do not apply to the reaction of elastin with proteolytic enzymes. There is much evidence (Section VI) to show that in elastin the peptide chains are arranged in a highly random fashion, and are prevented from forming crystallites by the presence of cross-links a t fairly wide intervals. Nevertheless, it has been shown recently that Fluvobacterium elastase (Mandl and Cohen, 1960) digests elastin but does not attack other proteins. Similarly there have been recent indications that one of the proteolytic enzymes of the pancreas may be a specific elastase (Lamy et al., 1961) although the classic crystalline pancreatic elastase of Banga certainly is not. There are two types of mechanism that could account for an elastolytic reaction of high specificity. First, the enzyme could attack the crosslinks in elastin without rupture of peptide bonds. This kind of mechanism has been suggested by several authors, and raises the possibility of elastolysis by a nonproteolytic enzyme. Alternatively the specificity of the reaction may be due to the presence in elastin of an amino acid sequence of some length which is not present in other proteins. I n this case the enzyme would differ from most other proteolytic enzymes which seem to display specificity for short sequences or a single type of peptide bond. The work of Mandl and Cohen appears to show that the specificity of the Fluvobucterium elastase is of this second type. Evidence was presented which suggests that the purified elastase is a true proteolytic enzyme. Crude preparations digest proteins such as casein, hemoglobin, and gelatin, but in its purified form it attacks only elastin and possibly synthetic substrates with a definite amino acid sequence (Mandl and Cohen, 1960). The complete specificity requirement has not yet been ascertained but i t is thought that the proline-glycine link might be susceptible. A random copolymer of proline and glycine, with all possible sequences of these two amino acids, was split by the enzyme and yielded proline only a t the C-terminals and glycine only a t the N-terminals. However various smaller peptides tested, even though they contained a prolyl-glycyl sequence, resisted attack by purified preparations (Mandl, 1961).
VI. CHEMICAL STRUCTURE OF ELASTIN From the foregoing sections of this review we may conclude that the elastic fibers of yellow connective tissue are composed in the main of a protein which is characteristic of this type of tissue and which is quite distinct, from collagen or other fibrous proteins. Elastic fibers havc characteristic staining properties, but they vary in thickness and morphological
ELASTIN
285
habit in different tissues. The thicker fibers of ligamentum nuchae may be up to 10 p in diameter but electron microscope studies show that each fiber is a ropelike structure composed of numerous regular microfibrils each about 10 mp in diameter. There is much evidence to show that the elastic fiber may be penetrated by other components of the connective tissue, including collagen and mucopolysaccharides, but it seems unlikely that the microfibrils are so penetrated. The microfibrils appear to be the elementary unit of the fiber structure and may be regarded as homogeneous filaments of the protein “elastin.” Due perhaps to differences in the degree to which the fiber structures are penetrated by collagen and the ground substance, there are considerahle differences in the ease wit,h which pure elastin may he prepared from tissues of different character. Thus a protein of constant composition may be prepared from ligamentum nuchae of cattle by autoclaving with water alone, but the elastin of aorta requires additional treatment with alkali to reach a constant composition. When aortic elastin is so prepared its amino acid analysis is almost identical with that of elastin from the ligamentum nuchae of the same animal. Nevertheless it is possible that there may be small differences affecting one or two amino acids. From some other tissues, particularly the elastic cartilage of the external ear, it is even more difficult to isolate a protein of constant composition and the fibers appear to be penetrated by some protein-containing material which is not readily extracted by alkali. It has not proved possible so far to isolate from ear cartilage an elastin which is identical in composition to that of ligamentum nuchae and it is not yet known whether the small differences in amino acid composition observed in samples of ear cartilage elastin arise from some unextracted impurity or whether the two proteins are different in constitution. Should the latter be true, it would be expected that the two proteins would show corresponding differences in chemical and physical properties and one such difference has been noted. The data in Fig. 3 shows that ear cartilage elastin is much more readily hydrolyzed by dilute sodium hydroxide than aortic elastin and this in turn is more easily dissolved than elastin from ligamentum nuchae. Recent work in this laboratory has shown that there are equivalent differences in the rate of hydrolysis by acids; that the effect is not due to differences in the diameter of the fibers was shown by finely grinding and crushing the fibers of ligamentum nuchae elastin, when it was found that such crushing had no effect on the rate of hydrolysis. It seems therefore that the protein of the microfibrils is probably homogeneous, and closely similar in amino acid composition for all the tissues of a single mammalian species. No general survey of the elastic tissues of the mammals has yet been undertaken but there is enough evidence to
286
R. M. PARTRIDGE
suggest that large differences in amino acid composition do not occur and that elastin, like collagen, may he regarded as a single protein or as a closo family of proteins with a wide distribution over the animal kingdom.
A . Soluble Proteins Derived from Elastin Partridge et al. (1955) observed that when elastin from ligamentum nuchae of cattle was repeatedly extracted with 0.25 M oxalic acid a t 100°C the fibers completely dissolved after about 5 hr total extraction. On dialysis in cellophane only about>5 % of the total nitrogen of the reaction mixture diffused through the membrane. The bulk of the product was rz protein which was soluble in distilled water or buffer solutions a t temperatures below 25”C, but on raising the temperature of the solution in dilute buffer at pH 4-6 a “coacervate phase” consisting of liquid droplets separated. The soluble material was thus similar in properties to the “hemielastin” of Horbaczewski (1882). The soluble protein showed a single boundary peak in the Tiselius apparatus in buffers of ionic strength 0.02 at all pH values in the range p H 2-9, but its isoelectric point was markedly dependent upon the salt concentrat,ion. At ionic strength 0.2, in the presence of sodium chloride, the isoelectric point both from electrophoretic mobility measurements and membrane potential determinations was pH 3.94.0. At lower ionic strength (0.02) the protein was isoelectric a t pH 4.8 in the electrophoresis experiments and pH 4.7 in membrane potential determinations. In spite of the apparent electrophoretic homogeneity it was found that the protein could be separated into two fractions with different physical properties. When the temperature of a solution of the protein in dilute buffer was raised, the bulk of the protein formed the reversible coacervate phase, but the remainder was completely soluble a t any temperature up to 98°C even when in concentrated solution. The coacervate-forming material was called the a-protein and the soluble substance the P-protein. Osmotic pressure determinations showed that the a-protein had a mean molecular weight of 60,000-84,000 in different preparations while the ,!?-protein had a mean molecular weight of ,5500. Ultracentrifuge and fractionation experiments carried out on the a-protein suggested that this substance was polydisperse over a fairly wide range, but little material was found with a mean molecular weight in the range between the a- and ,!?-proteins. The amino acid analysis of the a- and ,!?-proteins was compared with that of the purified elastin fibers from which they were prepared. It was found that the composition of the three proteins was closely similar thus showing that the original fibers must be homogeneous as regards amino acid composition even though they show a fibrillar structure. There were
ELASTIN
287
small differences in the content of certain amino acids between the two soluble proteins and the fibers, but these were thought to arise from the loss of small peptide sequences during the partial hydrolysis and subsequent dialysis (Partridge and Davis, 1955). The N-terminal groups in elastin fibers and the two derived proteins were estimated by the fluorodinitrobenzene technique. The elastin of the fibers contained 0.29 moles of N-terminal residues in 100,000 gm and the a-and /?-proteins contained 24.4 and 32.5 moles of N-terminals respectively in the same weight of protein. Ry comparison with the molecular weight of these samples it was deduced that P-protein consists of molecules composed on the average of two chains, each containing 27 amino acid residues; the a-prot?ein has, as a mean value, 17 such chains containing 35 residues each. Partridge and Davis (1955) were of the opinion that these results are to be understood on the basis that elastin is a cross-linked protein. The production of soluble fragments by partial hydrolysis with organic acids is due to the splitting of peptide chains at certain points where susceptible linkages are to be found (Partridge and Davis, 1950). This gives rise to fragments which still retain the cross-links that were originally present in the fibrous elastin. From the results found for n-elastin the average length of chain associated with one cross-link must be about 35 amino acid residues. The origin of &elastin was puzzling and in order to throw some light on this Partridge et al. (1955) followed the course of the partial hydrolysis reaction, estimating the proportion of a-and /?-elastin produced at each stage. Figure 4 shows the results. It will be seen that the release of soluble proteins shows a marked lag phase. The release of P-protein shows less lag than the release of a-protein. After about 2 hr of heating the release of /?-protein reaches a relatively steady rate and continues so until the whole of the fibrous protein is solubilized. The release of a-protein on the other hand is negligible until after 2 hr when it rapidly accelerates until all the protein is dissolved. Curves of similar shape were found for the hydrolysis of elastin by pancreatic elastase (La Bella, 1961). This author fractionated the soluble protein resulting from elastolysis by addition of trichloroacetic acid (TCA) . The TCA-insoluble fraction, which resembled a-elastin, was produced in small amount early in the incubation but its release rapidly accelerated towards the end of the process. On the other hand the TCA-soluble material (which resembles ,&elastin) was produced in greater amount in the initial stages but towards the end of the reaction it represented only 20 % of the soluble protein released. Partridge et al. explained their results by assuming that the production of the double-chain P-protein was the result of the splitting of four bonds around a single cross-link. This process
288
5. M. PARTRIDGE
1.8
1.6
B-
1.4
Y
2 3
1.2
4
C
.A
:. 2a
1.0
0
w 0
5
0.8
i
1
2
3
4
5
Serial number of extract
FIG.4. Extraction of 5 gm of elastin powder by heating with successive 50-ml quantities of 0.25 M oxalic acid a t 100°C for 1 hr. Key: A = grams of total protein dissolved in each extract; X = grams of a-elastin released; 0 = grams of @elastin released. (Partridge et al., 1955.)
would involve a short time lag, but would then proceed at a steady rate provided the supply of susceptible bonds was in excess. The production of the multichain molecules of the a-protein was visualized as a more complicated process. Here an average of about 34 bonds must be broken before the release of the multichain fragment and the time lag for the process would be correspondingly long. The process of release of the large
ELASTIN
289
fragments would be expected to accelerate rapidly following destruction of the network by release of the smaller @-proteinfragments. a-Elastin may be converted into a substance resembling the @-proteineither by further hydrolysis with acids or by the action of proteolytic enzymes. Other workers however have suggested that the @-protein may arise from a more easily soluble fraction pre-existing in the elastic fiber. The strong coherence of the microfibrils in the elastic fibers has impressed many workers and several have suggested that the fibrils are cemented together by a matrix substance that may be either mucopolysaccharide in character or may consist of a protein of similar chemical and physical properties to the elastin of the microfibrils (Lansing et al., 1952). Partridge and Davis rejected the suggestion of a polysaccharide-cementing substance on the ground that their preparation of elastin contained no more than 0.3% of polysaccharide and showed no tendency to break up into any fibers smaller than about 4 p , but the suggestion put forward by Lansing and his colleagues has never been closely investigated. Partial hydrolysis of elastin by reagents other than organic acids also gives rise to a mixture of soluble proteins similar to a-and @-elastin. Thus Wood (quoted by Hall et aE., 1952) first demonstrated that partial hydrolysis with dilute sodium hydroxide yields a protein which forms a reversible coacervate on raising the temperature of its solutions. Later Wood (1958) showed that on prolonged heating in aqueous solution a-elastin is converted into an insoluble gellike form. Reconstituted fibers of heat-treated a-elastin resembled fibrous elastin in their elastic behavior and X-ray-diffraction pattern but unlike purified elastin they were dissolved by 1% acetic acid at 100°C and by crystalline trypsin. Ioffe and Sorokin (1954) investigated a novel procedure for the hydrolysis of elastin using copper sulfate and 0.4 N barium hydroxide at 37°C for 60 hr. The first product of hydrolysis was a protein which resembled a-elastin in that it showed reversible coacervation on raising the temperature. This substance was subsequently degraded further to yield a soluble fraction and a fraction containing peptides. Alkaline hydrolysis was much more rapid in the presence than in the absence of copper ion. Hall and Czerkawski (1961a) reported several methods for preparing a-elastin: alkaline hydrolysis with 4 N NaOH at 37°C for about 20 hr; hydrolysis with 80% (w/v) sulfuric acid at 95°C for 3 4 min; refluxing with 0.5 N ethanol-HC1 for 3 hr; incubation at 3°C with pepsin a t pH 2; and finally partial hydrolysis with pancreatic elastase. Hall and Czerkawski (1961b,c) distinguished two main phases in the reaction of pancreatic clastase with elastin. In the first a-elastin is produced and later this is broken down to material of lower molecular weight. The second half of t,he reaction could be inhibited by sodium dodecyl sulfate,
290
6. M. PARTRIDGE
and when this was present the reaction gave a high proportion of a-elastin. Controlled elastolysis, with highly purified pancreatic elastase in the presence of borate buffer containing about 0.1 % sodium dodecyl sulfate, yielded an a-elastin that was little degraded by further hydrolysis. In the ultracentrifuge it gave a sharp symmetrical peak indicating a high degree of homogeneity. The product had 32 moles of amino groups in lo6 gm of protein. La Bella (1961) investigated the soluble proteins produced by the action of pancreatic elastase on elastin from ligamentum nuchae. Early in proteolysis approximately 50% of the total solubilized protein was precipitated by 5 % trichloroacetic acid. The proportion of trichloroacetic acid-insoluble protein then progressively increased so that, when solubilization was essentially complete, this fraction comprised 80 % of the total. The course of the reaction was thus similar to that observed during hydrolysis with organic acids (Fig. 4).
B. Ultraviolet Absorption and Fluorescence The preparation of a soluble form of elastin rendered possible the examination of the ultraviolet absorption spectrum of the protein by the methods normally in use for soluble proteins. Partridge and Davis (1955) measured the spectrum of the mixed soluble proteins obtained by partial hydrolysis of elastin with oxalic acid. The spectrum was examined both in alkaline (0.1 N NaOH) and in acid (0.1 N HCl) solution and in both solutions there was fairly strong absorption in the region 2700-3150 A. It is usually assumed that absorption shown by proteins in this region is due to tyrosine and tryptophan only. The absorption of tryptophan at 2800 A is not much affected by change in pH but the peak absorption of tyrosine in acid solution at 2750 A is shifted to 2940 A when the solvent is changed to 0.1 N NaOH, the effect being due to ionizationof the phenolic hydroxyl. As shown by Holiday (1936) the acid and alkaline curves may be used as a basis for estimating the tyrosine and tryptophan of a protein and the curves for soluble elastin were analyzed using the equations of Goodwin and Morton (1946). The calculated tyrosine content was 1.1 $4 and the value for “tryptophan” was 3.2 %. The value for tyrosine was in good agreement with that found by column chromatography of the products of hydrolysis, but no tryptophan could be found in elastin by any other method. I t was therefore assumed that an unknown substance with an absorption peak near 2750 A both in acid and alkali was present in elastin. The soluble proteins derived from elastin retain the yellow color of the fibrous protein, and also its strong blue-white fluorescence, and the pigment cannot be removed by further mild hydrolysis and exhaustive dialysis or repeated precipitation with alcohol. It thus appears that a fluores-
ELASTIN
29 1
cent chromophore is firmly combined to the peptide chains in elastin. Partridge and Davis also observed that when elastin was analyzed by the method of Moore and Stein (1951), about 5 % of the weight and 5 % of the nitrogen could not be accounted for as amino acids or amide nitrogen, and it was suggested that elastin contains about one-twentieth of its weight as a nitrogenous substance which is not an amino acid. A number of workers have attempted to isolate the compound responsible for the yellow color and fluorescence of elastin. Loomeijer (1958) hydrolyzed elastin to a mixture of peptides with activated papain and showed that the yellow fluorescent pigment did not appear in the free state but remained attached to certain of the peptides produced by the hydrolysis. Milligram amounts of a pigment were isolated after complete acid hydrolysis of elastin. The yellow pigments were adsorbed on charcoal and eluted with hot pyridine. The pigments were brought into ether and acidic substances were extracted with 2 % sodium hydroxide solution. The pigment was then purified by large-scale paper chromatography. The pigment was insoluble in water and light petroleum but soluble in methanol, ether, and chloroform. The absorption spectrum showed two distinct maxima between 200 and 300 mp which were independent of the pH of the solvent; and the compound had no distinct acidic or basic properties and it was suggested that its structure was related to the pyrimidines, purines, or pteridines. The early work of With (1947) had already suggested that the yellow color of native elastin may be associated with porphyrin pigments. In later work Loomeijer (1959) hydrolyzed bovine elastin with pancreatic elastase and isolated fluorescent peptides. The compounds were thought to consist of fluorescent substances of lipid character attached to peptides of various composition. Three different fluorescent compounds were isolated and purified by chromatography on silica-gel columns using benzene-ether as eluent. Preliminary analysis of one of the compounds suggested an aliphatic structure possibly containing carbonyl groups and having a weakly acidic character. A similar investigation was carried out by Karkela and Kulonen (1959) who partially hydrolyzed elastin by acids, alkalies, and enzymes under varying conditions and fractionated the products by adsorption on alumina and elution with ammonia. They obtained fractions in which the yellow color was enriched and determined the infrared and ultraviolet absorption spectra. They concluded that the yellow pigment was tightly bound to the peptide chain and that it did not appear to be a bile pigment. La Bella (1961) studied the soluble products released from elastin by pancreatic elastase. Elastin was prepared from beef Eigamentum nwhae by repeated extraction with hot 0.1 N NaOH in order to remove extraneous material as completely as possible. The purified product was then treated
292
S . M . PARTRIDGE
with purified pancreatic elastase and the soluble products werc separated into two fractions by adding trichloroacetic acid (TCA) to a conccntrstion of 5 %. Most of the yellow pigment was associated with the trichloroacetic acid-insoluble protein fraction, which was Schiff -positive (cf. La Bella, 1957, 1958). This product could be degraded further by pancreatic elastase, which splitnpeptides and amino acids from it; but most of the fluorescence, yellow pigmentation, and Schiff -positive material remained nondialyzable throughout. Both the TCA-soluble and TCA-insoluble fractions exhibited identical fluorescence maxima at 340,405,440, and 460 mp but the fluorescence emission of the former fraction was twice that of the latter. The component fluorescing a t 340 mp appeared to be localized at the surface of the fiber, but the material responsible for the other fluorescence peaks appeared to be distributed throughout. La Bella suggested the possibility that the TCA-soluble protein (which seems to correspond with the P-elastin of Partridge and Davis, 1955) may be derived from an amorphous cementing substance in the elastin fiber and the TCA-insoluble protein (similar to &-elastin) may be the substance of the microfibrils. He also pointed out that the fluorescent substance may be concerned with cross-linking in elastin. The fluorescence remains nondialyzable even following prolonged enzyme hydrolysis which results in a large proportion of the original protein being degraded to a dialyzable form. This suggests that the fluorescent moiety stabilizes the peptide chains in such a manner that these regions are protected from enzyme attack.
C. Composition of Peptides from Enzymatic Hydrolysis Recent work in this laboratory (Thomas and Partridge, 1962) has been based on the view that elastin is to be regarded as a cross-linked gel; randomly coiled peptide chains are envisaged as running roughly parallel to the fiber length and these are provided at intervals with stable lateral linkages of a type which a t present is entirely unknown. In order to provide chemical evidence for the presence of such a linkage it is necessary to isolate from the protein degradation products an H-shaped peptide with the link st.ill intact. This has been attempted by several workers, but the difficulty lies in the very large number of peptides of the required order of size that result from the attack of elastolytic enzymes. However it was thought that peptides containing the cross-link would in general be of larger molecular size than the rest of the population for the reason that they must contain double chains. Another factor, possibly operating in the same direction, concerns the probability of steric hindrance of enzyme action in the immediate vicinity of the cross-linkage. It would therefore be expected that a process based on the systematic ‘Lsieving’’of the pep-
ELASTIN
293
tides resulting from exhaustive enzyme treatment would result in the selective separation of peptides containing the cross-linkage. Such a process was attempted by treating the elastin from ligamentum nuchae first with several successive additions of pancreatic elastase and then further degrading the product with papain until all but a few per cent of the product was dialyzable through cellophane. The diffusate was then treated, in series, with a range of ion-exchange resins with varying internal pore diameter. The resins were of the sulfonated polystyrene type, in bead form, and had nominal divinylbenzene (DVB) contents of 2 %, 435 %, 8%, and 20% (cf. Partridge et al., 1950). It was found that the material which was adsorbed on the 20%, 8%, and 436% DVB resins consisted mainly of amino acids and small, unpigmented peptides which appeared to be linear; the material which passed through short columns of these resins but was adsorbed on the 2 % DVB resin was deep yellow in color and displayed a strong blue-white fluorescence. The ultraviolet absorption spectrum of this peptide fraction was found to be of the same shape as that of a-elastin and it was concluded that the sieving process had resulted in the enrichment of the mixture with peptides containing a chromophoric nucleus originally present in elastin. This result was regarded as indicating an association of the chromophoric grouping with the cross-links assumed to be present in the protein. Attempts to develop the sieving process as a tool for the isolation of a pure peptide containing the chromophore were not successful because it was found that the resins did not act as pure “ionic sieves” (Partridge, 1952) ; the colored peptides displayed strong adsorptive effects which appeared to be nonionic in character. However it was found that the strong adsorptive affinity of the colored peptides could itself be utilized for further enrichment of the fractions. Partially enriched material was further degraded by retreatment with pancreatic elastase followed by exhaustive treatment with carboxypeptidase. It was then fractionated on a 100-cm column of 2 % DVR sulfonated polystyrene resin in buffer using an increasing pH gradient. Colorless peptides were eluted as a series of peaks in the early part of the chromatogram, but the colored fraction, which remained on the column, was displaced as a single sharp front with dilute alkali. The colored peptide so purified was shown by paper chromatography to be free from simple peptides and the material migrated as a single band during high voltage paper electrophoresis a t all pH values in the range p H 3-9; it was therefore regarded as a pure substance or a mixture of closely similar substances. The peptide was soluble in water but insoluble in all but, aqueous solvents. It was readily adsorbed by cellulose and by most other common adsorbents from aqueous alcohols, but could be eluted by water or dilute
294
S. M. PARTRIDGE
alkali. Because of these properties the substance was difficult to purify further. Since the fraction was the limit product of enzyme action it was thought. to represent a peptide sequence consisting of a small number of amino acid residues lying on either side of the chromophoric nucleus; this being so, its molecular size and composition would be fixed by the position of susceptible bonds at each side of the chromophoric residue. Nevertheless, owing to the slowness of the final stages of the enzymatic reaction it was TABLEI1 Stages i n the Degradation of Elastin to Yield a Limit Peptide Containing the Chromophoric Nucleusa 1 9
Moles a-carboxyl groups Moles a-amino groups Moles FDNB reacting Moles N-terminal residues recovered (as DNP-alanine) Weight recovered as amino acids (gm) Weight of non-amino acid residue (gm) N-content (calc) of residue
a-Elastin
Fraction
Fraction D
Fraction
0.27 0.25 0.24 0.24
1.57 1.87
2.02 1.93 1.81 0.74
2.04 2.0 2.2 0.76
C
-
LP
Fraction
DC
2.19 2.22 1.86 0.75
930
-
638
548
407
70
-
362
452
593
10.1
10.2
3.4
3.1
(%I Extinction (X275) given by 1 mg/mlc a c
0.51
2.7
3.0
Taken from Thomas and Partridge (1962). Values expressed per 1000 gm dry ash-free protein or peptide. Not corrected for tyrosine.
expected that the product might be coiitamiriated with a proportion of similar molecules bearing additional amino acid residues. A series of colored peptide fractions resulting from the successive stagcs in the stepwise degradation with enzymes were analyzed with the results given in Table 11. All the products, including a-elastin, titrated with equal numbers of a-amino and a-carboxyl groups and, in each, the number of moles of fluorodinitrobenzene (FDNB) taken up (corrected for the lysine content) was equivalent to the a-amino groups titrated. The results are expressed as moles per 1000 gm of dry peptide and it will be seen that the series of peptides approach a limiting molecular weight such that 1000 gm dry weight contains about 2.2 moles of a-carboxyl or a-amino groups. The product of the reaction with FDNB was studied by hydrolyzing the
EIASTIN
295
dinitrophenyl (DNP) peptide and separating the products by quantitative paper chromatography. It was found that about half the DNP-labeled residues was DNP-amino acid and of this fraction 94 % was DNP-alanine. The remaining 50 % was combined with the fluorescent, chromophoric nucleus and remained on the baseline in paper chromatograms. As will be seen from Table 11, in the final fraction (DC), 0.75 mole of DNP-amino acid was recovered by acid hydrolysis and paper chromatography and almost the whole of this (94%) was DNP-alanine. If it is assumed that the limit peptide is a single homogeneous substance, then the molecule must contain two terminal a-carboxyl groups and two terminal a-amino groups one of which is the a-amino group of alanine; from the titration figures, the minimum molecular weight must be near 910. The fractions were analyzed for amino acids and the final fraction (DC) was found to contain (in 910 gm) 0.87 mole proline, 0.84 mole glycine, 2.15 moles alanine, and 0.28 mole valine, together with traces of other amino acids. These figures approach integral proportions and suggest that the bulk of the enzymatic end product is a peptide containing proline (1 mole), glycine (1 mole), and alanine (2 moles); this peptide however is mixed with about 30 % of a second larger peptide containing an additional valine residue. Subtraction of the weight of material recovered as amino acids from the weight of peptide taken leads to an estimate of the amount of non-amino acid material, and hence an indirect estimate of the molecular size of the chromophore. If a molecular weight of 910 is assumed for the peptide this leads to a value of 530 for the chromophore; by a similar argument its nitrogen content is about 10%. The last line in Table I1 shows the rise in extinction at X 273 as the peptides are enriched with the chromophore. The values are roughly consistent with the presence in a-elastin of about 7 % of the chromophore, a figure which agrees with the estimate of nonamino acid nitrogen previously given (Partridge and Davis, 1955). The information now available about the chromophoric peptide is summarized in Fig. 5 . The nature of the chromophoric nucleus (represented by the circle) is unknown; the peptide molecule cannot be represented by a single chain because it contains two amino groups which titrate as a-amino, but only one of them can be liberated as a DNP-amino acid (DNP-alanine) by acid hydrolysis. The data given do not provide formal proof for the structure in Fig. Yj since no direct evidence has been given that the sequence around each chromophoric nucleus is indeed unique ; nevertheless the results suggest, with high probability, that the chromophoric nucleus in elastin is part of a bridge structure serving as a chemical link between adjacent peptide chains. The view that the structure and properties of elastin can be understood on the basis of the presence of chemical cross bonds at fairly wide intervals
296
S . M. PARTRIDGE
along the peptide chains has long been held, and originally rested upon analysis of the stress-strain curve given by the wet protein (Section II1,A) and upon its swelling and solubility behavior (Section II1,B). Recently other evidence (Section VI,B), bearing in the same direction, has come to light, and this includes information derived from kinetic analysis of the hydrolytic breakdown of the structure by enzymes or by acids or alkalies. Hall and Czerkawski (1961~)noticed the swelling and loosening of the structure of elastic fibers which precedes solubilization by the action of enzymes, and investigated the extension of the fibers under constant load COOH
I
I
NH
t=0
HC-CH, I LH
I
DNP
FIG.5. Structure of elastin around the chromophoric nucleus (indicated by the circle). The nucleus links two short peptide chains together. The structure is represented as the DNP-derivative of the peptide isolated, which contains glycine (1 mole), proline (1 mole), and alanine (2 moles). One mole of alanine is N-terminal but the position of the other three amino acid residues is not known (Thomas and Partridge, 1962).
during the process of elastolysis. There was initially no change in extension, but this began to increase rapidly when about 5 76 of the protein had dissolved and eventually became very great (about, tenfold increase in length). The stress-strain curves were analyzed using the equations of Treloar (1938) which relate the molecular weight ( M ) of a free chain fixed a t both ends to the modulus of rigidity of the elastic substance. The calculation led to a value of 2600 for thc molecular weight of the free portion of the chain in the untreated elastic fiber. This figure compares with the value 3900 found for the length of the free chain associated with one crosslink in a-elastin by Partridge and Davis (1955). Hall and Czerkawski observed that their value for M increased rapidly during elastolysis and was doubled when only a small fraction of the protein had been dissolved.
ELASTIN
297
This is the expected behavior for a cross-linked polymer and may arise either by rupture of the cross bonds or by rupture of some of the peptide chains between the cross bonds. VII. CONCLUSIONS In view of the many apparently conflicting theories of the nature of elastin-some of them amounting to a denial of the chemical individuality of the substance of the fibers-it is obvious that further study of a t least the salient features of structure of this protein is urgently required. However, in spite of early confusion, more recent papers have shown that a certain measure of common outlook is emerging which, it is to be hoped, will crystallize into a more generally acceptable view. In the present state of the subject it is difficult for the present author to draw final conclusions in any terms other than his own interpretation of the evidence. With this stipulation, it may be concluded that there is now abundant evidence in favor of the view that elastic fibers are composed of bundles of thin homogeneous microfibrils, and that the substance of these microfibrils is a unique protein. This protein contains in its structure a chromophoric residue which gives elastin its characteristic yellow color and fluorescence. The microfibrils of all elastic fibers in the different tissues of the same animal species have a similar analytical composition, although they may differ to some extent in details of molecular structure. The elastic properties of the wet fiber may best be understood on the basis that the component microfibrils are composed of a protein with inherent elastic properties brought about about b y a cross-linked structure. These cross-links are not the familiar S S bridges since elastin contains little, if any cystine, but if the linkages are indeed of covalent character they must be due to bridges of a hitherto unknown type. Finally, evidence has been presented leading to the view that the radical comprising the chromophore in elastin may also be involved in bridging adjacent peptide chains. The recent discovery of a large number of different proteolytic enzymes c.apable of degrading elastin has provided a very valuable tool for the study of the structure of the protein, and by means of controlled degradation-coupled with modern techniques for the examination of the split products-it should be well within the present compass to obtain an unambiguous representation of this unique material. Such a project. if successfully concluded, would form a firm basis for many urgent investigations in the study of connective tissue diseases, in leather chemistry, and food science.
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ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
. .
D B WETLAUFER' Department of Biochemistry. Indiana University School of Medicine. Indianapolis. Indiana
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304 I1. Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 A . Radiation Absorption Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305 B . Interpretation of Absorption Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 I11. Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 A . Instrument Performance; Stray Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 B . Absorptivity Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 C . Turbidity and Light Scattering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 D . Fluorescence Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 E . High-Resolution Techniques . . . . . . . . . . . . . . . . . .................... 310 F . Differential Spectrophotometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 G . The Far-Ultraviolet Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 H . Spectral Studies on Solid-state Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 IV . The Absorbing Components of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314 A . The Aromatic Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314 R . The Sulfur-Containing Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 C . Histidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 D . Other Common Amino Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 E . The Peptide Bond . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326 V . Spectrophotometric Titrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 A . General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334 B . Protein Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335 C . Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 VI . Difference Spectra and Chromophore Environment . . . . . . . . . . . . . . . . . . . . . 346 A . Introductory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 B . Difference Spectral Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 C . Interpretation of Spectral Perturbations . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354 D . Phenolic Hydrogen-Bonding Model Studies . . . . . . . . . . . . . . . . . . . E . Analysis of Difference Spectra in Quantitative Terms . . . . . . . . . . . . . . . 362 F . Thermal Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366 G . The Solvent-Perturbation Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370 H . Difference Spectra in the 2200-2400 A Range . . . . . . . . . . . . . . . . . . . . . . . . 373 I . Difference Rpectr a. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374 VII . Analytical Applic.tt.ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 A . Accounting for Protein Absorptivity as t.he Sum of Its Part,s., . . . . . . 375 B . Tyrosine and Tryptophan Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378 1 Present address : Department of Physiological Chemistry. Medical School. University of Minnesota. Minneapolis 14. Minnesota
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C. Determination of Protein Concentration by Peptide-Bond Absorptivity, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Miscellaneous Problems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . Spectr:tl Transformations in Renin., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Effects of Metal Ions on the Ultraviolet Spectra of Proteins. . . . . . . . . C. Spectral Intensification a t High p H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Summary., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ............................................................
380 380 380 381 381 382 383
I. INTRODUCTION The ultraviolet spectra of proteins were reviewed by Beaven and Holiday (1952), and by Doty and Geiduschek (1953). The review of Dannenberg (1951) should also be noted. I n this present chapter, we shall largely accept these earlier works as the bases on which to build, and concentrate most of our attention on developments in the past8 decade. The recent important review by Beaven (1961) was not available to the author until this chapter was substantially completed. The ultraviolet spectra of proteins can for present purposes be divided into three regions: above 2500 A, between 2500 and 2100 A, and below 2100 A. We can paraphrase this division by calling the first region simple -only a few absorbers and easily sorted out; calling the second region complex because of the multiplicity of the contributions to absorptivity; and calling the third complex but exciting, because of the peptide-bond absorption and its conformation-dependence. Because the first region is simple and most easily accessible to experimentation, the majority of reported protein spectral studies concern it. As a consequence, we have our most extensive information on the main contributors to this region, the aromatic amino acids. While the second region is less promising because of its greater complexity, we attempt to survey its components in some detail, to permit the reader to judge for himself what hidden delights may dwell therein. Two factors have led to recent activity in studying the shorter-wave region. First, the means : several commercial spectrophotometers of high quality, capable of operating to appreciably below 2000 A, have become available in the past few years. Second, the objective: the attraction of studying the absorptivity of that most characteristic component of protein structure, the peptide bond itself. Because of its fundamental importance for all protein studies, this aspect of protein spectra will be emphasized in the present discussion. The small perturbations that are seen in spectra of amino acid side chains when they are incorporated into proteins are even more a matter of concern today than a t the time of Beaven and Holiday’s (1952) review,
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in which they were a major subject. The mode of presentation of these effects has changed-they are now presented as difference spectra-but the basis for interest in them remains the same: to achieve understanding sufficient to exploit the effects for protein structural deductions. The section here on difference spectra is concerned with these matters. The technique of “spectrophotometric titration” of tyrosyl groups in proteins has been applied extensively in the past decade, but the extent of its discussion here has been somewhat limited in deference to the chapter by Tanford in this volume. Some of the spectral effects of specific covalent modifications of proteins, i.e., iodination, oxidation, tyrosinase action, etc., will be discussed briefly. Heme proteins, flavoproteins, and other conjugated proteins will not be discussed except as regards studies involving their amino acid components. Study of the fluorescence and fluorescence polarization of amino acids and proteins appears to constitute one of the most sensitive new approaches to protein study. While results from these studies will be cited in the general discussion, a full discussion is beyond the scope of this review. It is our understanding, however, that a review (monograph) is in preparation on this subject by G. Weber, who has contributed so importantly to the application of fluorescence to biochemical studies. Both Weber (1961), and Beaven (1961) give useful discussions of fluorescence studies related to proteins. The question of energy transfer through proteins by various mechanisms has been considered in a recent symposium (Augenstine, 1960) and will not be discussed here, nor shall we discuss the studies of protein and amino acid luminescence from the solid state (phosphorescence): Debye and Edwards (1952, 1956). The photoinactivation of proteins will also not be considered. The recent paper by McLaren and Luse (1961) is suggested as a leading reference.
11. TERMINOLOGY
A . Radiation Absorption Measurements Although some progress toward standardization of terminology has been achieved, a multiplicity of terms is still found in the current literature. For this reason it is necessary to comment briefly on the terms employed here. Absorbance, A (also absorbancy, A ; optical density, O.D.; extinction, E ) is defined: A = log,, (lo/l) where l ois the intensity of light incident on the sample, and I is the intensity of light emergent from the sample. Absorptivity (absorbancy index, a ) a = A / b c , where b is the optical path length, and c is the concentration of the absorbing
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species. When absorptivity is expressed in concentration units of moles per liter and the path length is given in centimeters, we have defined the molar absorptivity (molar extinction coefficient, e ; molar absorbancy index, am);when the molecular weight of the absorber is unknown or uncertain, absorptivity is conveniently expressed as A k (or E k , ) , which is the absorbance of a 1% solution with a 1 cm optical path, calculated on the assumptions of Beer’s law. The terms of first choice above are those recommended by the Subcommittee on Applied Spectroscopy of the Society of Analytical Spectroscopists and the American Society for Testing Materials (Hughes et al., 1952; Editorial, Anal. Chem., 1961). Most American manufacturers of spectrophotometers seem to have adopted the suggestions of this report, judging by the generality of “absorbance” scales on their current products. Gibson (1949) and committees of the Optical Society of America have suggested that absorbance be reserved for homogeneous isotropic media (such as glasses), and that absorbancy be employed for substances confined between the windows of a cell, after correction for reflection and absorption (if any) of radiation by these windows. With a few exceptions, chemists generally have not noted or accepted this distinction, probably because practically all measurements of concern to chemists are made on fluid media in cells, with the corrections indicated above. It is the author’s opinion that the greatest good is served by retaining the least number of special terms consistent with clarity and general usage; for this reason we employ here the term absorbance.
B. Interpretation of Absorption Processes Certain terms from the field of molecular spectroscopy are seen occasionally in the literature surveyed in this review. A simple glossary of such terms is presented below. N + V transitions involve excitation of an electron from a bonding situation to a higher energy level which does not involve removal of the electron from the molecular domain. I n a molecular-orbital (MO) picture, such a transition involves excitation of an electron from one molecular orbital to another of higher energy. An example is the absorption of benzene around 2500 A. When the transition occurs between two ?r-orbitals (MO language), it is designated A + ?r*. (The asterisk may be employed to signify an excited state. Many authors and/or journals omit use of an asterisk, which results in a generally ambiguous situation.) N + Q transitions involve excitation of an electron from a nonbonding situation to a higher energy level still in the molecular domain; or in molecular-orbital terms, excitation from a nonbonding atomic orbital to a higher energy molecular orbital. An example is the weak absorption of acetone around 2800 A, which can be more specifically designated n + T * [transition from localized, nonbonding (n) orbital, to antibonding, delo-
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calized (T*)orbital]. Another example is the vapor-phase absorption of water around 1700 A, which can be designated n + CT*[nonbonding, localized orbital (n)to a localized antibonding orbital (.*)I. N -+ R transitions involve excitation of an electron to a higher energy state with removal of the electron from the molecular domain, analogous to a Rydberg transition in an atom. In the limit, the highest energy excitation produces a positive molecular ion and a free electron. An example is the ultraviolet absorption of the alkyl halides. The spacing of the absorption bands corresponding to N --+ R transitions can be described by the Rydberg formula. For a more detailed discussion of state designations and orbital promotions, see the papers of Platt (1953), Matsen (1956), Robinson (1961), Kasha (1961). Criteria for distinguishing between n + r * and P 4 P* transitions are summarized in Sidman’s (1958) review. It may be mentioned that an analysis of solvent effects on spectra is one of the most generally employed tools for preliminary diagnosis of the character of an electronic transition, a point which will be discussed further in connection with model studies for difference spectra of proteins (Section VT,D). Charge-transfer spectra arise from excitation of an electron in one molecule to an excited level in another molecule. I n the general case, the two molecules are involved in a loose electron donor-acceptor complex which is stabilized by resonance between a no-bond and a dative electronic structure. There is also the class of contact charge-transfer spectra, examples of which are provided by Iz and O2in various solvents (Evans, 1955; Tsubomura and Mulliken, 1960). In this situation the acceptor does not appear to form a complex in the ground state even with the strongest electron donor, but with all donors, whether strong or weak, gives rise to an absorption spectrum interpretable as resulting from donoracceptor interaction in the excited state. The interested reader may consult the papers of Mulliken (1952), Tsubomura and Mulliken (1960), and McGlynn (1960). E x d o n theory deals with the theory of electronic excitation processes in ordered arrays of absorbers (Kasha, 1959). Applications of exciton theory to protein problems have not been numerous, but some important results, especially on the conformation-dependence of the peptide absorption around 2000 A, will be discussed in the section on peptide-bond absorption. 111. EXPERIMENTAL
A . Instrument Performance; Stray Light Most ultraviolet absorption studies in the past 10 years have been done with commercial spectrophotometers. Performance of these is discussed
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in standard references (Hiskey, 1955; Scott, 1955; West, 1960) and in certain critical discussions (Goldring et al., 1953; Beaven and Johnson, 1962). All such instruments may give false readings due to stray light at high sample absorbance. Such errors are discussed perennially (Hogness et al., 1937; Saidel et al., 1951; Mehler, 1954; Fridovitch et al., 1957; Mitz and Schleuter, 1957); it will be enough to say here that an experimental proof that Beer’s law holds is usually sufficient proof of the absence of significant stray light (at a given wavelength and monochromator slit width, absorbance is proportional to the concentration of absorber).
B. Absorptivity Errors While wavelength calibrations of spectrophotometers are generally reliable and easily tested, we have less confidence in some of the other factors that go into a reported value of a molar absorptivity at a specified wavelength, e x . To quote a compiler of a recent (1958-1959) review of spectral data (Phillips, 1962), “For molar absorbance [absorptivity] data, reproducibility is poor. Thus, only two out of five maxima agreed within 0.02 of a unit of log 23, only three out of five within 0.05, and fully 20% of the maxima differed by 0.10 or more. When it is considered that a log E difference of 0.10 corresponds to a 1.26 to 1.00 ratio of E’s, large variations in molar absorbances are clearly to be expected in published data.” It is probable that some improvement could be expected if absorbance measurements were always carried out near the minimum-error absorbance (due to scale-reading uncertainties) : 0.43 for a single beam, 0.87 for a double-beam spectrophotometer (Hiskey, 1949; West, 1960). Not only instrumental errors are likely to be involved-on the other hand, these cannot be excluded without testing. The relatively simple, but much neglected, test of measuring the apparent absorbance of a standard absorber is recommended here. Alkaline chromate solutions are probably the most reliable standards, and an exhaustive and critical compilation (Haupt, 1952 a,b) of absorptivity values is available throughout the spectral range 4500 to 2200 A. Working standards may conveniently be glass filters or perforated-plate attenuators. Sample impurities and errors in concentration determination contribute to variations in reported absorptivities, as does lack of scrupulous cleanliness in preparing sample solutions and sample cells; these factors probably underlie an appreciable fraction of the reported cases of variance. We will note here the disturbing report by Rosenberg and Stolkowski (1953) that the ultraviolet absorptivity of ratalase, peroxidase, and carbonic anhydrase varies with dilution. Indeed, even the method of dilution was reported to influence the observed absorptivity. This is a surprising and disturbing claim. We do not know whether it has been verified. Nonetheless, it should
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
309
serve to remind us that it is worthwhile occasionally to test even our fundamental operating assumptions, especially when the means for testing are simple.
C . Turbidity and Light Scattering Light scattering can be appreciable in protein systems; in precise work one may try to correct for this by measuring the apparent absorbance out of the region of probable true absorption, and extrapolating this correction into absorbing regions. A plot of the logarithm of the apparent absorbance (turbidity) versus the logarithm of X (the wavelength), has proved to be useful. The method has been recommended by Schauenstein and Bayzer (1955), and has also been used by Reddi (1957) and by Leach and Scheraga (1960b) in their studies on scattering protein solutions. A note of caution: it has been observed in suspensions of algae that anomalous “selective” light scattering (Latimer and Rabinowitch, 1959) occurs in the regions of certain absorption bands. This selective scattering appears to be related to the anomalous dispersion of refractive index sometimes observed in absorption bands. To the writer’s knowledge, no similar effects have been observed in protein solutions, but there appears to be only one published investigation that bears on this question. This is the study of Schauenstein and Bayzer (1955), who showed that for the 2800 A band of y-globulin, the same absorptivity was obtained from molecularly disperse solutions as was obtained from a turbid solution (very low salt concentration) after applying turbidity corrections as described above. This was taken as evidence of the additivity of scattering and absorption in this system, and implies that there was no significant selective scattering through the absorption band. Methods for measurement of true absorbance in strongly scattering systems (such as suspensions of cellular or subcellular particles) have been developed by Shibata and co-workers (Shibata et al., 1954; Shibata, 1959) and by Keilin and Hartree (1958).
D. Fluorescence Errors In strongly absorbing systems, luminescence (here implying fluorescence or phosphorescence or both) can contribute to deviations from Beer’s law. Errors attributed to luminescence have been noted for simple organic compounds by Braude et al. (1950), by Ungnade et al. (1951), and for enzyme solutioiis by Mehler et al. (1957). If we assume that a fluorescing sample solution can be approximated as a point source, luminescent emission reaching the detector should vary inversely as the square of the distance between detector and sample. In spectrophotometers where it is possible to vary the distance from sample to detector, this provides a
3 10
D. B. WETLAUFER
simple way to test for luminescence. Absorbance is independent of this distance in the absence of luminescence (and scattering). Another way, less generally convenient, would he to test the apparent absorbance with and without a narrow-band pass filter (transparent a t the incident wavelength) between sample and detector. 1
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2300 2400 2600 2800 3000 3200 W A V E L E N G T H I N A FIG.1. Absorption spectra of the aromatic amino acids at pH 6 (S. Malik, 1961). 2000
E . High-Resolution Techniques The coupling of electronic and vibrational modes of excitation gives rise to discrete narrow absorption bands, which are exemplified in the spectrum of phenylalanine (Fig. l), where at least six saw-toothed projections appear to emerge from the otherwise smooth contour of the absorption envelope. Such a system of maxima within an absorption band is collectively called the fine structure of the band. Tryptophan
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
311
and tyrosine show (Fig. 1) much more diffuse fine-structure bands; indeed in their spectra as shown, the fine structure is so blurred that often it would be better defined by inflection points than by maxima. The exact position of the fine-structure bands of particular chromophores varies from protein to protein, and therefore provides a means for characterizing proteins (Beaven and Holiday, 1952). The moving-plate spectrograph of Holiday (1937) still appears to offer the most precise way of locating the fine-structure bands of proteins and amino acids in solution a t ordinary temperatures. This method is reviewed by Beaven and Holiday (1952). An alternative means of locating fine-structure bands has been advocated by Annau (1958). By using a solution of xanthine (which has a smooth absorption envelope with a peak a t 2800 A) in the reference path of his spectrophotometer, and measuring optically dense (A,,, 1.5) protein solutions, Annau achieves an exaggeration of the fine structure of several proteins. This method unfortunately offers the distinct likelihood of displacing fine-structure bands on the wavelength coordinate. While Annau’s results were in rough agreement with those obtained by the moving-plate method, the latter is certainly both more sensitive in resolving weak bands and more accurate in locating band positions. Decreasing the temperature of an absorber generally has the effect of sharpening the fine-structure details of its spectrum. The aromatic amino acid residues of proteins are no exception, as was shown in the pioneering investigations of Lavin and Northrop (1935) on pepsin. These authors showed that band sharpening occurs to a considerable extent when spectra are taken in glycerol at -100°C. Control spectra of the aromatic amino acids taken under the same conditions were also presented. Low-temperature spectra of sublimed thin films of aromatic amino acids were reported by Sinsheimer et al. (1950), who describe their experimental arrangements in detail. Their principal objects of study were, however, not proteins and amino acids, but purine and pyrimidine bases. Brown and Randall (1949) briefly reported similar observations on films of aromatic amino acids, employing a moving-plate spectrograph to measure fine-structure shifts. Although it is relatively convenient to make observations on sublimed films of crystalline materials, the crystalline state is not generally satisfactory for spectral comparison with dilute solution because of the likelihood of resonance coupling of an electronic transition in the crystal lattice, or, a t best, an undefined “environmental effect” on the chromophore. On the other hand, the range of solvents for low-temperature spectroscopy is severely limited. Lavin and Northrop (1935) proposed that a suitable solvent for a protein “should neither inactivate nor denature the material,” which would certainly be ideal, but from a current point of view, this is
3 12
D. B. WETLAUFER
almost certainly impossible. (We cannot conceive it likely that any protein will not undergo some kind of conformation change when it is transferred from water to a glass-forming solvent and subjected to a temperature decrease of a hundred degrees or more.) Practically, the recurrent problem in attempts to measure low-temperature spectra of proteins is that of finding adequate solvents which will form supercooled glasses rather than crystalline solids at the convenient low temperature of liquid nitrogen (77°K). Glycerol remains vitreous to about - 100°C but cracks at lower temperatures. Various nonaqueous mixed-solvent systems which remain vitreous at 80°K were described by Freed et al. (1958), who were primarily interested in the possibility of fine structure in the low-temperature fluorescence spectra (they found no such fine structure), and made no report of the absorption spectra of the proteins they studied. As a practical alternative to obtaining clear glasses, Keilin and Hartree (1949) suggest the use of aqueous glycerol solvent in a turbid, microcrystalline state, obtained by partial thawing followed by re-cooling. Estabrook (1956), and Lindemeyer and Estabrook (1958) have employed this solvent system in the study of the visible spectra of different cytochromes. These workers observed band sharpening to an extent which permitted quantitative resolution of two very closely related cytochromes in mixtures. Further low-temperature studies of protein spectra have been carried out by Beaven et al. (1961), who used thin layers of glassy solvent to minimize scattering. Their findings are reported to include appearance at low temperatures of a new band near 2960 A in proteins and peptides containing tryptophan. Normal human hemoglobin (Hb A) and fetal hemoglobin (Hb F) earlier were shown (Beaven and Holiday, 1952) to display tryptophan fine-structure peaks at 2910 A and 2898 A respectively, when examined in aqueous solution. Beaven et al. (1961) show that, under their low-temperature conditions, the fine-structure band in Hb F sharpens and moves to 2886 A, but that Hb A shows no wavelength shift at low temperature. Thus, low-temperature conditions may increase substantially the resolving power for analysis of mixtures of closely related proteins. There obviously should be a close connection between fine-structure shifts and shifts of absorption bands as revealed by difference spectra. It remains to be shown whether the two methods are simply equivalent, or whether they may complement one another in some useful way.
F. Diferential Spectrophotometry Giese and French (1955) have shown how minor features of an absorption curve can be emphasized by differentiation; to date, however, the method has not been used in the study of protein or amino acid spectra. Various
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
313
experimental arrangements have been devised (Martin, 1957 ; Olson and Alway, 1960) for recording the first derivative of absorption spectra.
G. The Far- Ultraviolet Range The spectral range between 2100 and 1800 A is becoming accessible as commercial spectrophotometers are being provided with optical components suitable for these wavelengths and with provisions for flushing the optical path with nitrogen. This region has become of increasing interest following the demonstration by Imahori and Tanaka (1959) that the peptide absorption around 1900 A is conformation-dependent. In addition to oxygen absorption in the vapor phase of the optical path, dissolved oxygen makes absorbance contributions of varying magnitude in common solvents. This has been demonstrated in water at wavelengths between 2150 and 2000 A, with the significant feature that the molar absorptivity of dissolved oxygen is much higher than that in the vapor phase (Heidt and Johnson, 1957). This result has been interpreted as due to hydrogen-bonded structures between oxygen and water (Heidt and Johnson, 1957), and alternatively, as a contact charge-transfer process (no oxygen-water complex formation) by Tsubomura and Mulliken (1960). Irrespective of which interpretation is more nearly correct, the investigator of solution spectra should be aware of the generality of oxygeninduced absorbance not only in aqueous solutions, but much more strongly in organic solvents (alkanes, alcohols, ethers, aromatics, etc.). It is also useful to remember for aqueous spectral studies, that nearly all common inorganic ions, including chloride and hydroxyl ions, absorb appreciably around 2000 A. The work of Buck et al. (1954) on the ultraviolet absorption spectra of some inorganic ions is useful in this connection. Useful experimental details for obtaining spectra in this wavelength region with commercial instruments are given by Johnson (1960), and by Rosenheck and Doty (1961).
H . Spectral Studies on Solid-State Samples Solid-state spectral studies relating to proteins have not been numerous, but those associated with the peptide-bond absorption are of special interest. I n particular, we note Peterson and Simpson’s (1957) singlecrystal spectroscopy of myristamide, and the demonstration of dichroism in oriented films of helical polypeptides by Gratzer et al. (1961). The results of these studies are discussed in Section IV,E. Since great care and exacting technique is required for solid-state studies, we may do no better than to refer interested readers to the original papers for critical discussions of experimental aspects. Low-temperature spectra of materials in solution in glassy solvents are discussed in Section II1,E.
D. B. WETLAUFER
314
IV. THEABSORBING COMPONENTS OF PROTEINS
A . The Aromatic Amino Acids
.
i Spectral Characterization An examination of the spectra of phenylalanine, tyrosine, and tryptophan in Fig. 1 shows a great qualitative similarity. At wavelengths > 2000 A, all three amino acids show spectra composed of two major absorption bands, a strong band at 2100-2200 A, and a weaker band at 2600-2800 A. The separation of these two bands is approximately the same for all three aromatic chromophores. It is true that there are large quantitative differences between these three spectra, particularly in the weaker (long-wave) bands. The logarithmic spectral presentation of Fig. 1has the advantage of emphasizing similarities rather than differences, which is desirable here, since the similarities do not seem to be widely recognized. Spectra of the aromatic amino acids are seen with a linear absortivity coordinate in Fig. 2. It seems likely that both absorption bands K* transitions in all three chromophores [see Matsen represent similar K (1956) for a discussion of transition assignments in benzene]. The spectrum of the phenolic-ionized tyrosyl group (Fig. 9) is similar to that of un-ionized tyrosine, except that both its peaks are somewhat intensified and shifted about 200 A to longer wavelengths. The spectral change of tyrosine on ionization is discussed in many texts and references [for example, Edsall and Wyman (1958), and Tanford (1961), and in detail by Beaven and Holiday (1952)], and is assumed to be generally familiar. --f
2. Tyrosine a. General. The absorption spectrum of tyrosine is shown in Fig. 1. The shoulder a t 2800 A is the only remnant of fine structure apparent in aqueous solution. It seems highly probable (Bovey and Yanari, 1960, refs. 32-34; Ito, 1!360a) that the 2750 A absorption band corresponds to a K 4 T * transition. The increase in absorptivity and the long-wave shift of the spectrum of tyrosine with ionization of the phenolic hydrogen is well known, and indeed provides the basis for studying the hydrogen ion equilibria of tyrosyl groups in proteins, as discussed in Section V. In another connection, displacement of the long-wave absorption peak from 2750 to 2950 A on titration with base, improves the resolution of tyrosyl from tryptophyl contributions in the simult,aneous spectrophotometric determination of these two residues. Analysis of proteins for these two components is discussed in Section VI1,B. I n addition to their major employment in spectrophotometric titrations and compositional analyses,
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
315
tyrosyl residues in proteins have been extensively studied by the difference spectral technique (Section VI). Model compound and difference spectral studies have been carried out on tyrosine and related compounds by Wetlaufer (1956), Laskowski (1957), Edelhoch (1958), Chervenka (1959), Bigelow and Geschwind (1960), Yanari and Bovey (1960), and Foss (1961) (see also Section V1,D). Studies of the fluorescence of tyrosine-activation spectrum, fluorescence emission spectrum, and quantum yield-have been reported by Teale and Weber (1957). The pH-dependence of the fluorescence of tyrosine and related compounds was studied by White (1959) ; fluorescence-polarization studies from the same laboratory were reported by Weber (1960a) for simple compounds, and for proteins (Weber, 1960b). Teale (1960) has carried out extensive studies on the fluorescence characteristics of a score of proteins. b. Covalent Tyrosyl Derivatives. Certain modifications or derivatives of tyrosine are encountered frequently enough in protein studies to warrant some discussion here. Gemmill (1955) has reported the ultraviolet spectra. and apparent phenolic ionization constants of thyroxine and a number of related iodinated amino acids. The spectra of iodinated phenolic compounds can be briefly described by saying that both the phenolic and the phenolate ion peaks are appreciably intensified and shifted to longer wavelengths. Gemmill (1956 a,b) also studied the iodination of tyrosine and thyronine as well as the proteins thyroglobulin, casein, and insulin, Roughly speaking, for all these materials, increased iodination (in alkaline solution) progressively increases absorptivity and induces a long-wave shift of the phenolate peak from 2950 A to about 3200 A. The demonstration by Thompson (1954) that chlorination of the aromatic ring of tyrosine can result from performic acid oxidation of proteins in the presence of chloride is also worth noting. If this occurs, it is certain to alter the spectrum of the oxidation product. Spectral alterations of the tyrosyl spectrum resulting from the formation of an ether linkage with the phenolic oxygen may be quite small, as with 0-methyltyrosine (Wetlaufer et al. 1958), or substantial, as with the aromatic ether thyronine (Gemmill, 1955). The ester linkage is also of some interest in this connection, since tyrosine-0-sulfate has been shown by Bettelheim (1954) to occur in bovine fibrinogen. The spectrum of tyrosine sulfate ". . . differs markedly from that of tyrosine, showing a much weaker absorption with a maximum near 2630 A." Another interesting modification of tyrosyl groups is that resultant from N-bromosuccinimide oxidation. From the studies of Schmir el al. (1959) and Corey and Haefele (1959) on tyrosine peptides, it is apparent that N-bromosuccinimide oxidation leads to breakage of the peptide bond
316
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linked to the tyrosyl carboxyl and formation of a dibromospirodienone lactone (A, 2600 A: €2600 = 104). Application of N-bromosuccinimide to proteins was originally concerned with oxidation of the tryptophyl residues, but Ramachandran and Witkop (1959) and Peters (1959) have demonstrated that tyrosyl and cystyl residues in proteins also react. These matters are discussed further in the section on tryptophan (IV,A, 4). c. Tyrosinase Action. Sizer’s (1947) finding that tyrosinase catalyzes oxidation of tyrosyl residues in certain proteins has been expanded by Ymunobu et a2. (1959). These workers studied the oxidation of a variety of N-terminal, C-terminal, and endotyrosyl peptides. On the basis of the sequence and nature of spectral changes in the ultraviolet and visible range, they propose three types of reaction routes: a “dopachrome sequence,” a “dopa quinone sequence,” and a ‘(protein sequence.’’ In both the dopachrome and dopa quinone sequences, the final products are nondialyzable polymers. The dopachrome sequence is characterized by the formation of an intermediate oxidation product with absorption peaks at 3050 and 4800 A, leading to a final product characterized by a single new peak at about 3250 A. The dopa quinone oxidation sequence is characterized by the formation of an intermediate with an absorption maximum at 3900 A. As the oxidation proceeds, this peak disappears and a slight shoulder appears a t about 4700 A. The protein sequence, while showing some variability (the oxidations of hypertensin I, 0-corticotropin, and a-lactalbumin were studied) in the intermediate stages of oxidation, all gave final reaction products showing absorption maxima at 3500 A. Oxidation occurred much more slowly than was observed in the dopachrome and dopa quinone sequences. Yasunobu and Dandliker (1957) showed that the sedimentation velocity of a-lactalbumin increased only slightly after tyrosinase action (oxygenuptake studies indicated that all five tyrosyl groups of lactalbumin were oxidized). They conclude that oxidized lactalbumin was not aggregated. While there is no evidence as to whether oxidized hypertensin and corticotropin were aggregated, the spectral similarity of these materials to oxidized lactalbumin suggests that they were not. We may speculate that the tyrosinase-oxidized phenolic groups in the protein oxidation sequence are somehow inhibited from undergoing intermolecular condensations, and that the 3500 A peak indeed reflects just this state of affairs. Further work is clearly needed on this question. Tyrosinase can clearly distinguish between C-terminal (dopa quinone sequence) and N-terminal tyrosyl (dopachrome sequence) residues in small peptides. I n larger proteins, notj only is the spectrally observed reaction sequence different, but there is a great range of reactivity of tyrosyl groups (both effects may be explained by assuming varying degrees
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
317
of steric hindrance). Peptide-chain cleavage apparently does not accompany tyrosinase activity. The great specificity of tyrosinase, the simplicity of monitoring its action spectrally, and in some cases, the discrimination shown among the several tyrosyl groups of a protein [it oxidizes only one of the six tyrosyl groups of ribonuclease (Yasunobu and Dandliker, 1957)] appear to recommend its serious consideration as a tool in protein structure studies. 3. PhenyEalanine The absorption spectrum of phenylalanine is shown in Fig. 1. The low intensity absorption peak centered slightly below 2600 A corresponds to a forbidden ?r + ?r* transition. Vibrational fine structure is quite evident in this region. Aside from increased blurring of the fine structure which is to be expected on passing into successively more polar media, change of solvent effects little change in the general size, shape, and locat,ion of the absorption envelope. That this “forbidden” transition does indeed occur is due to coupling of the electronic transition with various molecular vibrations in a way which removes, to a small extent, the symmetry barrier. Substitution on the benzene ring, particularly by polar groups, also has the effect of destroying symmetry and increasing the oscillator strength, i.e., the area under the peak. For example, the same transition in tyrosine (Fig. l ) , yields a peak whose maximum height (at 2750 A) is seven times greater than that for phenylalanine. Because the 2570 A band of phenylalanine is weak, it is often obscured in proteins by the much stronger tyrosine and tryptophan absorptions. It is occasionally visualized in protein spectra as ripples (fine structure) in the spectral region 2500-2700 A. These ripples can be amplified by the difference spectral technique, as is shown in Fig. 13. A typical phenylalanine difference spectrum, obtained in a comparison of the isoelectric amino acid with a solution of the same concentration at pH 1 is shown in Fig. 12. Difference spectra for phenylalanine in various solvents have been measured by Bigelow and Geschwind (1960), Yanari and Bovey (1960), and Donovan et al. (1961). Fluorescence activation and emission spectra for phenylalanine were measured by Teale and Weber (1957). It is worth pointing out that, although the absorptivity of phenylalanine is small around 2600 A, it is one of the strongest absorbers in the region of the N -+VI peptide absorption band, with €2060 = 9600, €1900 = 44,000 (Rosenheck and Doty, 1961).
4. Tryptophan a. General. The spectrum of tryptophan is shown in Fig. 1. This amino acid is the strongest absorber in the usual 2700-2900 A protein
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absorption band, its maximum absorptivity being about fourfold greater than un-ionized tyrosine and about twice as great as ionized tyrosine. Here, as with phenylalanine, some vibrational fine structure is seen. The assignment of electronic transitions corresponding to the 2600-2900 A absorption band of tryptophan should be assisted by Weber's (19604 fluorescence-polarization studies on indole and tryptophan. The results of these studies seem to indicate two independent electronic transitions in this spectral region. By analogy with phenylalanine and tyrosine, at least one of these transitions is certainly u -+ u*. Edwards (1949) observed that methyl substitution in the 1 or 3 position of indole leads to a small redshift of the 2800 A band. This result is consistent with a ?r -+ ?r* transition, but not with an n -+ U* transition (Sidman, 1958). Therefore, the evidence favors representation of this absorption band as a single u 4 U* transition, with a slight reservation. The low-intensity absorption at wavelengths > 2950 A is apparently more solvent-sensitive than the band envelope as a whole (Wetlaufer and Coffin, 1962), a result seemingly inconsistent with a single transition. The question is not yet fully resolved. Difference spectral studies on model compounds related to tryptophan have been published by Chervenka (1959), Bigelow and Geschwind (1960), Donovan et al. (1958, 196l), Yanari and Bovey (1960), and Foss (1961). The fluorescence characteristics of tryptophan and its derivatives have been studied by Teale and Weber (1957), by White (1959), by Weber (1960 a,b), and Teale (1960). b. Polytryplophan: Absorption and Fluorescence. The absorption spectrum of poly-DL-tryptophan was described briefly in a note by Patchornik and co-workers (1954). In butylamine-water (9: 1) solvent the spectrum was nearly the same as that of the amino acid itself in the same solvent. In a more recent note, Shifrin (1961) confirmed this result for poly-L-tryptophan in several solvents, and showed also a 35 % decrease in the fluorescence emission (per residue) compared to N-acetyltryptophan. These results re-emphasize a point already apparent from the studies of Weber and his students (Weber, 1961) : the fluorescence of protein chromophores is much more environment-sensitivethan is their absorptivity. Here, as in Section VI, neighboring parts of the molecule to which the chromophore is attached, as well as solvent molecules, constitute the environment of a chromophore. c. Oxidation of Tryptophyl Residues. Patchornik et al. (1958) reported that N-bromosuccinimide oxidizes tryptophan in peptide linkage with ensuing cleavage of the peptide bond. This was a finding of great interest to protein chemists, since it appeared to offer a highly specific method for peptide-chain cleavage in proteins. Studies on the mechanism of cleavage (Schmir et al., 1959; Corey and Haefele, 1959) quickly established that
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
319
tyrosyl peptides also undergo a similar course of reactions with N-bromosuccinimide. Close on the heels of these papers there appeared two separate studies on N-bromosuccinimide oxidation of proteins, one by Ramachandran and Witkop (1959), the other by Peters (1959). These reports agreed that for several proteins, not only were tryptophyl residues oxidized, but also tyrosyl, disulfide, and perhaps other groups as well. Both studies used ultraviolet spectral changes to monitor the extent of reaction of tyrosyl and tryptophyl groups. Ramachandran and Witkop found that the extent of chain breakage was much lower than would correspond to breakage at every oxidized tryptophyl residue, in the range: 20-40% of such expected stoichiometry. Peters studied the yield of new N-terminal amino acids in bovine serum albumin as a function of the mole ratio of oxidant to protein (Ox/Pr). The yield curve for both serine and glycine end groups rose from zero a t an Ox/Pr ratio of 5 or 6 to a maximum of 0.4 to 0.5 a t an Ox/Pr ratio of 15, and declined only slightly as the Ox/Pr ratio was increased to 60. Both Peters and Ramachandran and Witkop routinely used concentrated urea solutions as protein solvents, to promote complete accessibility of protein tryptophyl groups to the oxidant. N-Bromosuccinimide, under the conditions thus far employed, not only lacks specificity in oxidizing side chains, but also fails to effect peptidechain cleavage at about half of the sites of oxidative attack. Nonetheless, it shows useful possibilities even a t present, and certainly one may expect that ways of increasing its usefulness will be found. Further developments and possibilities in this direction are discussed in an interesting chapter by Witkop (1961) in the preceding volume of thk series. d. Reaction with Formaldehyde. The reaction of indole with aqueous formaldehyde results in the formation of N-methylol indole. The spectra of this product and compounds related to N-methylol tryptophan have been presented by Edwards (1949). There is surprisingly little difference between the spectra of indoles before and after reaction with formaldehyde: as an example, the 2800 A band of 3-Me indole shifts only about 30 A to longer wavelengths after conversion to the N-methylol compound, with a correspondingly slight increase in peak height.
B. The Sulfur-Containing Amino Acids I . Spectral Characterization The ultraviolet absorption spectra of cysteine, cystine, and methionine are shown in Figs. 2,3, and 11. The lowest energy (long-wave absorption band) transitions have been assigned (Mulliken, 1935; Walsh, 1953) as n + u* transitions. Transition assignments for disulfide spectra have been critically discussed in a recent paper by McGlynn et al. (1962). The
320
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FIQ.2. Far ultraviolet spectra of a-amino acids. All amino acids were in aqueous solution (2 mm quartz cells) at pH 5,except cystine (pH 3). The dibasic acids were measured as hydrochlorides and the absorbance corrected by subracting the absorbance contribution of chloride ion. Measurements were made in a Beokman DK2a extented-UV range ratio-recording spectrophotometer with continuous nitrogen flushing below 2OOO A (Sussman and Gratzer, 1962).
nonbonding electrons in the ground state might be expected (as is generally the case) t o be somewhat sensitive to environmental changes-but no studies clearly relating to this point have come to the writer's attention. The ionization of the thiol group of cysteine results in a strong increase in
ULTRAVIOLET SPECTRA OF PROTEINS A N D AMINO ACIDS
321
absorptivity (Fig. 11) with a new peak forming in the wavelength region 2300-2400 A (loglo €2380 = 3.65). Methionine is seen (Fig. 2) to have a spectrum somewhat like that of cysteine, but, of course, its sulfur exhibits no acid-base behavior in the usual pH range (pH 1 to pH 13). The mercaptide-ion spectrum of cysteine is shifted about 80 A to longer Wavelengths by deprotonation of the a-ammonium group and, similarly, the spectrum of the thioglycolate di-anion undergoes a short-wave shift of the same magnitude in the presence of polylysine (Kornguth et al., 1961) suggesting an association of some sort. If the --S--CH3, -SH, and in particular, the S-S- chromophores are especially sensitive to changes in environment, this may partly explain the frequent failure to obtain reproducible absorptivity values in protein spectra a t the (usual) spectral minimum near 2500 A. Studies of model systems pertinent to sulfur chromophores in protein spectra are clearly needed. 2. Cystine Peptides
The absorption spectra of several cystine-containing peptides as obtained by Otey and Greenstein (1954) are shown in Fig. 3. Some nonadditivity of absorptivities is evident; for example, the spectrum of Lcystinyl-L-cystine is obviously not simply the sum of 2 cystine spectra, even at wavelengths greater than 2500 A, where the disulfide group is the only significant chromophore. The question, “What is a suitable model for cystine absorptivity in a protein?” is apparently farther from a satisfactory answer than is the parallel question for aromatic amino acid residues. 3. Thionein and Metallothionein
The absorption due to cysteine residues in proteins is usually more or less obscured by the aromatic residues. With thionein, a cadmium- and zinc-binding protein from equine renal cortex, this is not the case. Its spectrum in both a metal-containing and metal-free form is shown in Fig. 4. According to Kagi and Vallee (1961), a t least 95% of all of the sulfur of this protein is present in the form of sulfhydryl groups of cysteine; further, the cysteine content of this protein is of the order of 30%. By chemical analysis, practically no aromatic amino acids are found in this protein, and this is clearly reflected in the spectrum. It is worth noting that Cd” bound to this protein effects a substantial and specific increase in absorptivity around 2500 A.
4. Oxidation of Cysteine, Cystine, and Methionine From a comparison of the spectra of cysteine and cystine it is immediately clear that oxidation of SH to disulfide in peptides or proteins can result
322
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in a substantial spectral change, particularly below 2500 A. Oxidation of cystine disulfide groups to cysteic acid also results in a decrease in absorptivity in this spectral region (Saidel et al., 1952). For reasons that are not generally clear (steric hindrance and thiazoline structures are two possibilities), oxidation of protein sulfhydryl to disulfide is often strongly inhibited. A classic method for oxidizing S H to 44,with IZ , was
210
230 250 270 WAVELENGTH ( m p1
290
1 4 ’ 1 ~ . 3. Al)sorplion spectra of cystine and cystine peptides (Otey and Greeiistein, 1954). Absorption curves in 0.01 N HCI of (I) I.-cystinyl-o-cystine and o-cystinyl1,-cystine; (11) L-cystinyldiglycine; (111) cyclo-L-cystinyl and L-cystinyl-L-cystine;
(IV) L-cystine; (V) glycylglycine; and (VI) glycine.
found (Fraenkel-Conrat, 1955) to yield a sulfenyl iodide in the case of the single -SH of tobacco mosaic virus subunit protein. Cunningham and Nuenke (1959) have recently shown that ovalbumin and B-lactoglobulin also form sulfenyl iodides under similar conditions, and call attention to the absorption of this grouping in the near ultraviolet (absorption maximum = 3500 A, no extinction coefficient given). Since triiodide ion unfortunately absorbs strongly a t the same wavelength, the utility of this absorption band is somewhat diminished.
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
323
There has been no report on the spectrum of methionine sulfone, which is found in hydrolyzates of performate-oxidized proteins. It will probably be not very different from cysteic acid at wavelengths > 2000 A, since simple sulfones are generally transparent to 1800 A (Koch, 1950). Methionine sulfoxide is a stable amino acid found in human urine, in a variety of plant tissues, and as an intermediate in the oxidation of methionine to the sulfone. Its absorption spectrum has not been recorded. The sulfoxide chromophore is usually a broad band, located around 2100 A and of about the same intensity a8 alkyl sulfides (Koch, 1950). An interesting 10
1;
05
z a
m
L Y O
o 05
fn m U
0 25 0 200
250 WAVELENGTH i m p )
200
250 WAVELENGTH i m p )
FIG.4. Absorption spectra of thionein (Kagi and Vallee, 1981). Absorption spectra of metallothionein (A), thionein (A, B), and cadmium thionein (B). Difference spectra of cadmium and zinc thionein (C), and the cadmium and zinc complexes of 2-mercaptoethanol (D). I n B, Cd++ was added to 8 X 1 0 P M thionein (0), the numbers ( 1 4 ) identifying the spectra also represent the multiples of the increments of Cd++ (5 X 10-6 M ) . I n D, 6.8 X 10-4 M 2-mercaptoethanol was added to 4 X 10-6 M ZnC1, or 2 X 10-6 M CdClz as shown. The ~ Z I of S the zinc complex was 8.3 X 108. The Q,LO of the cadmium complex was 1.46 X lo4. I n A, B, and C the buffer was 5 X 10-8 M phosphate and succinate, pH 5. In D, the buffer was 0.03 M Tris, p H 7.5.
feature is the great solvent-sensitivity of this band, which undergoes shortwave shifts as the polarity of the solvent is increased. 5. Thiazolines
Some 20 years ago Linderstr@m-Langand Jacobsen (1941) suggested that a thiaaoline structure, as illustrated by 2-methylthiazoline, may acCHZ-CHz
$\/ A \
CHa 2-Methylthiaeoline
324
D. B. WETLAUFER
count for some of the abnormally unreactive thiol groups in proteins. Although this grouping has not been established in any protein, it does exist in the peptide antibiotic bacitracin, as shown both by Craig's group (Weisiger et al., 1955) and by Newton and Abraham (1953) and in other naturally occurring peptides (Cecil and McPhee, 1959). Therefore, the possibility of thiazoline structures in proteins cannot be dismissed. One of the characteristic features of a thiazoline ring is an absorption band (loglo e = 3.4 at 2500 A for 2-methylthiazoline) around 2500 A (Weisiger et al., 1955; Martin and Parcell, 1961). That a protonated thiazoline structure might be formed in strongly acid solutions of glutathione was first suggested by Calvin (1954). This suggestion has been verified both by ultraviolet and by Raman spectral studies on glutathione and its S-methyl analog (Martin and Edsall, 1958). Further correlations between structure and thiazoline ring stability have recently been made by Martin and Parcel1 (1961). While the thiazoline structure is found only in strongly acidic solutions in glutathione, it is stable in bacitracin A up to about pH 7. The possible occurrence of thiazoline structures in proteins near neutrality cannot, therefore, be discounted on the basis of the behavior of glutathione. 6. Thiol esters
The possibility of a thiol ester linkage for an -SH group in papain has been suggested by Smith (1958). To the author's knowledge, there is no strong evidence for thiol ester groupings in proteins. Thiol esters have an absorption band (log,, €2300 N 3.6) very similar to that of ionized thiols (Noda et al., 1953). Although this band is located in a somewhat complex spectral region, it may possibly be of some use in testing Smith's hypothesis.
7. Serum Albumin and Cu" Finally, we should like to note another putative spectral expression of a sulfhydryl group. Klotz and co-workers (1952) found that a weak ( E = 200) absorption band is formed at 3750 A on the addition of one mole equivalent of cupric ion to bovine serum albumin. Evidence for the mercaptide nature of this particular protein-bound copper was adduced from demonstrations of competition with HgClz , Ag+, and organic mercurials. Subsequent work by Kolthoff and Willeford (1958) argues convincingly against the mercaptide nature of this combination. The matter is further discussed by Cecil and McPhee (1959). A recent note by Peters (1960) suggests that the N-terminal aspartyl residue of serum albumin may be implicated in binding the first mole of Cu'I. To the author's knowledge, this sort of spectral interaction with Cul' has not been shown
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
325
in other proteins, although Klotz has remarked that the copper-containing respiratory protein, oxyhemocyanin, has a similar absorption band in the near ultraviolet.
C. Histidine 1. General
As Fig. 2 shows, the imidazole group of histidine absorbs appreciably in the region between 1850 and 2200 A. The transition for this absorption has not been assigned. Because all three of the aromatic amino acids absorb strongly here, as well as cystine, methionine, and cysteine, it is easy to understand why there have been no extensive studies on absorption of histidyl residues in proteins. Unlike tyrosine or cysteine, ionization of the imidazole chromophore (ring nitrogen) perturbs the spectrum of histidine only very slightly-to about the same extent as ionization of the a-carboxyl or the a-amino group (Saidel et al., 1952). Donovan et al. (1961) have reported difference spectral data for the various ionic species of histidine. 2. Acetyl Imidazole and Chymotrypsin
The absorption spectrum of acetyl imidazole was reported by Stadtman (1954). Its absorption band is relatively broad, with A, ‘V 2440 A, and e2440 = 3.0 X lo3. An imidazolyl group was for a time considered (Dixon and Neurath, 1957) to be the site of acetyl group attachment in monoacetyl chymotrypsin. The recent studies of Wootton and Hess (1961) showed that the larger part of the apparent absorbance change observed at 2450 A was due to a general increase in Rayleigh scattering due to aggregation of acetyl chymotrypsin, which disaggregated concomitant with deacylation. Wootton and Hess showed further that a second form of acetyl chymotrypsin, which they identify as the “true intermediate” in ester hydrolysis, neither undergoes aggregation nor shows an absorbance change at 2450 A during deacylation.
D. Other Common Amino Acids Spectra of many of the common amino acids not yet discussed are shown in Fig. 2, kindly made available by Sussman and Gratzer (1962). The data of this figure are usefully supplemented by the earlier but less extensive (2000-2300 A) observations of Saidel et al. (1952). In addition to some duplication of the amino acids of Fig. 2, Saidel et al. showed spectra of cysteic acid, hydroxyproline, asparagine, valine, isoleucine, and glycine. The monoamino, monocarboxylic amino acids, including the hydroxyamino acids and the imino acids, are seen to have very similar spectra at wave-
326
D. B. WETLAUFER
lengths > 1850 A. The main absorber for this group of amino acids is the carboxylate group. The electronic transition corresponding to this carboxylate band has been given a A -+ A* assignment by Barnes and Simpson (1962). The same investigators assign the weak absorption band near 2100 A in carboxylic acids as n + A * ; it is uncertain whether an n + A* transition is an unresolved component of the long-wave carboxylate band. The absorptivity of the a-ammonium group is quite small in this range, perhaps of the order of magnitude of the difference between lysine and leucine as seen in Fig. 2. The higher absorptivities reported by Ley and Arends (1932) for the hydrochlorides of various amines were probably due to uncompensated chloride ion. The spectra of arginine in Fig. 2 were obtained in two ways: in the presence of the transparent perchlorate counterion, and with absorbing chloride counterion present, subtracting the absorbance contribution of the chloride. If we accept that the small difference in the two results is beyond experimental error, it may be the result of quantitatively different counterion perturbations of the ground level energy of either the absorbing cation (arginine) or the absorbing anion (chloride). The spectra of the isoelectric amino acids can be represented in a good approximation by the sum of the absorptivities of the carboxylate group and the side-chain. For a better approximation, one might attempt to account for the mutual perturbations arising from the a-ammonium, the a-carboxylate, and the side-chain group, as discussed in Section VI.
E . The Peptide Bond 1 . Model Compound Studies Due to developments in the past decade, it is now possible to discuss the spectroscopy of the peptide bond. This discussion can reasonably begin with the papers of Saidel and Goldfarb, who reported extensive investigations on the spectra of most of the common amino acids (Saidel et al., 1952) and a systematic sampling of simple peptides (Goldfarb et al., 1951; Goldfarb, 1953; Saidel, 1955 a,b,c; and Saidel and Lieberman, 1958). These studies were supplemented at an early stage by Ham and Platt’s (1952) results with a vacuum spectrograph on aqueous solutions and on solid films of a few amino acids, peptides, and bovine serum albumin. Ham and Platt showed that the peptide bond exhibits an absorption peak at -1850 A. This conclusion was further secured by the vapor-phase spectra and tentative transition assignments of simple amides by Hunt and Simpson (1953). Preiss and Setlow (1956) also studied the absorption of solid films of amino acids and peptides in the range 1450-2400 A. These studies (a) verified and extended the observations of Ley and
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
327
Arends (1932) on the spectra of the common amino acids; ( b ) showed that proteins have a high-intensity absorption band in the neighborhood of 1900 A; (c) showed a similar band in simple peptides, with absorptivity (on a weight concentration basis) increasing with increasing chain length in oligopeptides; and (d) showed the existence of a similar spectral band for N-acetyl or N-glycyl derivatives of almost all of the common amino acids. The 1900 A band is not seeii with the aliphatic amino acids. These facts taken together constitute strong evidence for associating the 1900 A band with the peptide bond in proteins, and further show that, in a first approximation, peptide bonds behave as isolated chromophores. These conclusions are somewhat obscured by the wealth of detail (specific Nsubstitution effects, etc.) to be found in these papers. It should be noted that the bulk of the data published by Goldfarb and Saidel was obtained “the hard way,” on a Beckman DU spectrophotometer in the wavelength range 2000 to 2400 A, making corrections (Saidel et al., 1951) for stray light where necessary. We defer discussion of certain details of these papers to a later part of this section. 2. Amide Transition Assignments
Next we shall consider the paper of Peterson and Simpson (1957) on the absorption spectra of myristamide and N ,N’-diacetylhexamethylenediamine in the crystalline state. The importance of this work for protein chemistry is that it provides definite electronic transition assignments for the major absorption bands of simple amides, and, with high probability, for peptide bonds as well. If the crystal structure of a substance is known and if similar groups have similar orientations, measurements of absorption of polarized radiation by the appropriately oriented crystal can be used to identify the transition moment accompanying the absorption. These conditions are satisfied for crystalline myristamide except that the crystal structure does not allow a unique solution for the direction of polarization of the strong absorption band a t 1850 A; i.e., two possible orientations are obtained. Similar dichroism measurements on crystals of N ,N’-diacetylhexamethylenediamine did permit an unambiguous choice of orientation. The choice was in gratifying agreement with theoretical results obtained by a molecular-orbital treatment of the r-electrons of an amide bond. Since a detailed discussion of the studies of Peterson and Simpson (1957) is beyond the scope of this review, we will simply present the polarization spectra (Fig. 5 ) and the transition assignments (Fig. 6). Figure 5 shows the polarized spectra of myristamide, with the subscripts indicating orientation of the a or b crystal axis with the polarization direction of the polarizing prism. I n Fig. 6, N --f V1 corresponds to the strong-
328
D. B. WETLAUFER
est absorption band (peak about 1880 A) which includes components associated with electronic coupling found in the crystalline but not in the vapor phase (formamide). N -+ Tiz is assigned to the region below 1600 A (estimated 1520 A). RS is the first Rydberg transition ( N -+ R), assigned to the 1660 A absorption. Finally, the weak absorption around 2200 A is given the assignment n -+ ?r*.
3. Conformation-Dependenceof Peptide Absorption Since the spectrum of the amide groups shows electronic resonance coupling in the crystal, one could guess that the presence of orderly arrays
*
0-\ #
P I
Y
I
rn
I
2.0
I I
\
\
\ \ \
\
W
cl
u
1.0
U
P
PI 0 0
1600
1700
1800
4 1900
2000
2100
2200
2300
W A V E L E N G T H I N A FIG.5 . Polarized spectra of myristamide. Broken lines denote uncertainty, The curves above 2100 A me magnified tenfold. (Peterson and Simpson, 1957.)
of amide groups in proteins might also be reflected in the spectrum. Moffitt (1956a), on the basis of an exciton model, actually predicted absorptionband splitting for helical polypeptides and proteins before Peterson and Simpson’s (1957) experimental demonstration of band splitting in myristamide. In 1959 Imahori arid Tanaka showed that, for poly-L-glutamic acid, the peptide spectrum is indeed conformation-dependent. Now, Goldfarb (1951) had shown that the absorbance of bovine serum albumin solutions a t 2050 A increases after short heating at 100°C, and suggested that absorptivity at this wavelength “. . . can be correlated with the peptide structure of proteins.” This absorbance increase was complicated by kinetic effects (scattering?) and appeared to be of dubious generality, since only one protein was studied. Imahori and Tanaka’s choice of
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
329
polyglutamic acid provided a more convincing demonstration of a conformation-dependent amide spectrum, since the helix-coil transition in this material had already been well established (Doty et al., 1957). Polyglutamic acid is well suited in another respect, in that its change in sidechain absorptivity due to the ionization of the carboxyl group between pH 4 and pH 7.3 (in which range the helix-coil transition is essentially complete) can be corrected for with some assurance. A conformationdependent spectrum has been demonstrated for polylysine and polyglutamic acid by Tinoco et al. (1961). Further confirmation has come from Rosenheck and Doty (1961), who studied several native and denatured
t
-4*3m - 2.7
- 10.9 - 12.5
3s
2pY (OXYGEN)
n o nt
FIG.6. Orbital energy diagram for myristamide: numerical scale, electron volts; intensities are depicted by varying widths of the arrows representing the various transitions. (Peterson and Simpson, 1957.)
proteins in addition to the aforementioned polyamino acids. Figure 7 (Rosenheck and Doty) shows the essential features of conformation-dependence of poly-L-lysine spectra. Note first of all the strongest absorption band, ( N V,), a t 1900 A. The most obvious feature of the results summarized in this figure is the decrease in absorptivity shown by the a-helical conformation, compared either with the random or the @-conformation. Further, the spectrum of the a-helical conformation shows evidence of band splitting: note the appearance of a new band at -2050 A, not evidenced in the other two conformations studied. This band splitting, which Imahori and Tanaka failed to observe, is of special interest because it was predicted by Moffitt (1956 a,b) in his exciton treatments of the anomalous rotatory dispersion of a-helices. Not only did he predict ---f
330
D. B. WETLAUFER
splitting of the N 3 V1 band, but also that the two band components should show different polarizations with respect to the helix axis.
4. Dichroimn of Helical Polypeptides The matter has been carried further in the studies of Gratzer et al. (1961) with measurements of the absorption of polarized radiation by oriented films of a-helical polypeptides. Their polarization spectra of poly-L-alanine, and of polyr-methyl-L-glutamate (Fig. 8) clearly show the predicted opposite polarization for the two peptide absorption bands of the a-helix. Taking these band positions as 1910 and 2060 A, we obtain a band separation of 3800 cm-I. [The value of 2700 cm-l in the paper of Gratzer et al. 8000 7
\ 't' ,Random Coil
O 180
L 190
L
-
200
250
Xmu FIG.7. Ultraviolet absorption spectra of poly-L-lysine hydrochloride in aqueous solution: random coil, pH 6.0, 25°C; helix, pH 10.8, 25°C; 8-form, pH 10.8, 52°C. (Rosenheck and Doty, 1961.)
(1961) is a misprint.] This is probably something of an underestimate of the true band separation, however, since bands overlapping to the extent seen in Fig. 8 will cause the observed peak separation to be less than the true separation. Nonetheless, the experimental results can be considered to be in good agreement with the predicted value of Moffitt (2800 cm-l) as well as the recent figure of 2590 cm-' resulting from the pointmonopole calculations of Tinoco et al. (1961). Figure 8 also shows dichroism in the weak band in the 2200-2300 A range, which may correspond to the weak amide absorption assigned as an n -+ a* transition by Hunt and Simpson (1953). This may quite possibly be the transition involved in an apparent Cotton effect seen in the optical rotatory dispersion of a-helical structures (Simmons et at., 1961). If this is true, and it appears possible, it raises a serious question about the
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
33 1
-
adequacy of Mofitt’s assumption (Moffitt, 1856 a,b) that the rotatory V1 and dispersion of a helical polypeptides derives solely from the N N 3 Vo electronic transitions. Until measurements of optical rotation, or perhaps better, circular dichroism, can be extended well into the N 3 V1 transition region, this will likely remain a matter for conjecture. 5. Estimation of Peptide-Chain Conformation
Rosenheck and Doty (1961) have employed protein absorptivity values = 1900 A) to estimate the in the strong peptide absorption band (A,
X(rnpL)
FIG.8. Polarization spectra of oriented poly-y-methyl-L-glutamate film. The baseline due to prism and quartz support versus gauze has been subtracted. (Gratzer et al., 1961.)
a-helical content of proteins. Absorptivity is corrected for estimated side-chain contributions (which of course requires knowledge of the amino acid composition of the protein), and per cent helicity (helical content) assigned on the basis of a linear interpolation between a residue extinction coefficient for a “random-coil” conformation and the coefficient for the a-helical conformation. Their results for several proteins are shown in Table I, where comparison is also made with the estimates of helical content from optical rotatory dispersion studies (Yang and Doty, 1957). There is seen to be good to fair agreement between the estimates by the two methods. Rosenheck and Doty note that their estimates contain two nonexperimental uncertainties, the first arising from the assumption
332
D. B. WETLAUFER
12,000
1
I
I
I
I
1
1
1
1
I
I
-
-
-
UJ
- +40 - +20
a
- -200
- -40 I I
2100
2300
I
2500
I
I
2700
I
1
2900
I
I
3100
t
3300
FIG.9. Spectra of tyrosine in various states of ionization. Above, spectra of isoelectric tyrosine (pH 6), and the divalent anion (pH 13) which is dominated by the phenolate ion. Center, the spectral change from pH 6 to pH 13 represented as a difference spectrum. Bottom, for comparison with center, the difference spectrum generated by measuring tyrosine a t p H 6 versus tyrosine a t p H 0.5. Note the disparity of ordinate scales for the three levels of the figure. The scale for the bottom figure above 2430 A is on the right. (S. Malik, 1961.)
333
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
that amino acids are adequate models for determining side-chain absorptivities, and the second from the fact that they have not attempted to account for conformations other than “random” and a-helical. It appears quite certain that the absorptivity of the N + V1 band of proteins does indeed reflect the main-chain conformation. It remains to be ascertained to what extent useful correlations can be obtained. It was necessary to assume the absence of environmental influence on side-chain absorptivity, although this was recognized by Rosenheck and Doty (see also Section VI) to be not strictly true. This important work represents an attempt to set up a new empirically based scale for estimating helical content in proteins. At its present preliminary stage of development the method certainly has some serious TABLEI Comparison of Estimated Helical Contents of Several Proteins by Ultraviolet Absorption and Rotatory Dispersion Methods0 Ultraviolet Protein
Paramyosin Myoglobin Insulin Ribonuclease Ribonuclease, oxidized &Lactoglobulin, pH 6.4 @-Lactoglobulin,pH 8.7 a
mp
197 mp
205 mp
Average
105 99 96 58 18 33 23
93 67 44 35 18 25 14
102 81 57 27 19 31 12
100 82 66 40 18 30 16
190
Rotatory dispersion 100 74 >51 > 17 14 11 12
Rosenheck and Doty (1961).
ambiguities. But so, for that matter, do the other, independent, methods that are assumed to reflect protein conformation in solution: kinetics of deuterium-hydrogen exchange, optical rotation and rotatory dispersion, and infrared spectra of characteristic amide bands. One interesting aspect of this helix-based hypochromicity is that equimolar amounts of right- and left-handed helices will mutually cancel in optical rotatory effect, but that this will not be the case for absorptivity. Indeed, Rosenheck and Doty deduce the existence of appreciable helical content in poly-m-glutamic acid at pH 3.0 from the decrease in absorptivity (corrected for side chains) vis-d-vis a pH 8 solution (random conformation). The question of whether the amide-associated weak transition around 2200-2300 A can be correlated with conformation is considered in Section VI,H.
334 6.
D. B. WETLAUFER
Basicity of the Amade Bond
The absorpt,ivity of simple amides and peptides, in the range 20002100 A, decreases in strongly acidic solutions, according to Goldfarb et al. (1955, 1958). These investigators have interpreted this spectral change as reflecting protonation of peptide bonds. Ionization constants calculated from spectral measurements were in good to fair agreement with independently determined values. Values of pK’ for the first protonation ranged from -3 to -4.5 for simple peptides and proteins. This is a surprisingly large difference from values around -0.5 for N-methylacetamide and other model amides. Another puzzling result of these studies is that serum albumin and ovalbumin showed absorptivity increases, in contrast to the decreases observed with peptides; gelatin, on the other hand, showed no significant absorptivity change as a function of acid concentration. Moreover, the spectral change with protein solutions was shown to be reversible. No adequate interpretation of these observations is apparent.
V. SPECTROPHOTOMETRIC TITRATIONS A . General In Section V wc will discuss acid-base equilibria. I t is, of course, quit,o possible and often very useful to study other reactions of proteins by spectrophotometric titration methods. The reaction of thiol groups with heavy metals and the binding of a coenzyme or an inhibitor by an enzyme represent familiar examples. However, it will be most convenient to limit our present discussion to hydrogen ion equilibria. When the acidic and basic forms of a substance differ in absorptivity, the variation of absorptivity with acidity will be a kind of titration curve. A familiar case is the change of color of phenolphthalein as a function of pH. When applied to the titration of ionizing groups of proteins, this is called a spectrophotometric titration. Ideally, the procedure may permit selective study of the protonation of a particular kind of basic group in a mixture of bases. Practically, spectrophotometric titrations have usually focused on tyrosyl groups, although studies involving sulfhydryl and carboxyl titrations have been possible in a few favorable cases. Tyrosyl groups have been so extensively studied because they present an easily isolated spectral band in their 2950 A phenolate peak (see Fig. 9). The ionizations of sulfhydryl, carboxyl, and amino groups are also accompanied by detectable absorptivity changes, but these occur in spectral regions which, in most proteins, are occupied by a multitude of strong bands. This has made exploitation of these latter groups much less attractive than for tyrosyl residues. In the following we will discuss spectrophotometric titrations of protein
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
335
model systems, and then summarize spectrophotometric H+-titrations of proteins .
B. Protein Models 1. Tyrosyl Groups
A thorough study has been made by Martin et at. (1958) of the ionization of tyrosine, coupling electrometric and spectrophotometric titration studies. Evaluation of all twelve “microscopic ionization constants” was achieved from studies on tyrosine, tyrosine ethyl ester, O-methyltyrosine, and O-methyltyrosine ethyl ester. The microscopic ionic species and their interrelationships are indicated schematically in Fig. 10. In spectrophotometric titrations, it is customary to determine the extent of ionization from a relationship of the form a = (e - eO)/(el - E,,) where t is the molar absorptivity a t some fractional degree of ionization, a ; €0 is the absorptivity when a = 0, and tl is the absorptivity when a = 1. (We imply here that e is measured a t a constant wavelength, and that Beer’s law holds.) Then, if the equilibrium involves only two microscopic species, as in the case of phenol (phenol and phenolate ion), one can calculate the apparent ionization constant, pK’ as follows: pK’ = pH - log [a/(l - a)]and the value found for pK’ is invariant with a,within experimental error. With the amino acid tyrosine, this is not so; the value of pK’ (calculated from the photometrically measured phenolic ionization) rises slowly, but definitely, as a increases. When we consider the fact that deprotonation of the a-ammonium group of tyrosine also occurs in the same p H region as the phenolic ionization, we now recognize an equilibrium involving H+ and the four microscopic species: ( - O + ) , (- - +), (-00), and (- - O ) , using the nomenclature employed in Fig. 10. I n the second and fourth of these the phenolic hydroxyl is ionized; in the first and third it is not. These species are seen (in the upper right half of Fig. 10) to be related to one another by the microscopic equilibrium constants, k I 2 , k 1 3 , k 1 2 3 , k I a 2 , which are in turn related to the over-all dissociation constants Kz and K3, as follows : K2K3
=
K2
klZkl23
=
hz
=
k13k132
+ kia
1/K3 = l/kiz3 -t1 / h 3 z For this situation, defining aOH as the fraction of all the tyrosine molecules in which the phenolic group has ionized, we have
336
D. B. WETLAUFER
Defining MoE
=
(Hf)aoE/(l - aOH)it follows that
where pMoH is seen to be an apparent pK for the phenolic ionization, variable with aOH . A plot of pMoH versus aOHgives a smooth curve connecting the two limit values of pMOH]ooH+~ = phz = 9.63 and P M O H I ~ O= ~-+I phi32 = 10.04.
i'
i'
OH NH3 COOH
'
(OO+)
OH: 2 NH3' 3 COOH :1
k3\
5 1-,-q
j23i
COOH
COOH
FIG.10. The eight microscopic forms of tyrosine and tjhe twelve ionization constants which interrelate them. Parentheses enclose symbols indicating the charge on each of the ionizing groups, in the sequence: carboxyl, phenolic hydroxyl, amino. (Martin el al., 1958; redrawn with permission.)
Methods for evaluating the microconstants are discussed in the paper by Martin el al. (1958), and by Edsall et at. (1958). Martin et al. (1958) found pklz = 9.63 f 0.02 (phenolic ionization constant for tyrosine carrying ammonium and carboxylate ions) and plC132 = 10.04 & 0.03 (phenolic ionization constant for tyrosine carrying uncharged amino group and carboxylate ion), a t 2,5"C and ionic strength 0.16. It must be noted that for estimating aOH the authors found it necessary to assume the equality of E(- -+) and E(- -0) . As they point out, there is no direct evidence on this question, but in 0-methyltyrosine, for example, the absorptivity a t the maximum changes only slightly with ionization of the a-amino group; the assumption is therefore probably good, if not perfect. The value of E(--o) can be determined from absorbance measure-
ULTRAVIOLET SPECTRA O F PROTEINS AND AMINO ACIDS
337
ments at pH > 12.5, where tyrosine is almost wholly in the form of the divalent anion. But there appears to be no simple way to evaluate c(- - +) , since at pH values between 12.5 and 7.5, a tyrosine solution contains a mixture of at least three microscopic species, none in negligible quantity. A similar problem arises in the spectrophotometric titration of cysteine (Section V,B,2). The study by Martin et al. is of interest not only for the rationalization of the electrometric and spectrophotometric measurements in terms of the microconstants, but also because the spectrophotometric titration of tyrosine relates so closely to similar studies in proteins. In particular, the multiple H+-equilibria of tyrosine result from the close juxtaposition of amino and phenolic groups in the same molecule; under these circumstances the ionizations are mutually interacting. We suggest that some of the anomalies seen in tyrosyl ionization in proteins may arise in a similar fashion, but in terms of magnitude, this mechanism clearly cannot account for such anomalous tyrosyl groups as those seen in ribonuclease or ovalbumin. Katchalski and Sela (1953) studied the spectra and the spectrophotometric titration of polytyrosine (number average D.P. = 30). The behavior of these polymers was consistent with that of tyrosyl residues in a polyelectrolyte molecule. In a later paper, the same authors (Sela and Katchalski, 1956) reported spectrophotometric titrations of copolymeric polyamino acids containing tyrosyl and acidic, neutral, or basic residues in an attempt to demonstrate the often-hypothesized tyrosyl-carboxylate bond. The copolymers studied and their residue ratios were L-Tyr, L-ASP (1:1, 1:3, 1:9), L-Tyr, L-LYS(1:3, 1:9), and L-Tyr, DL-Ala (1:9). The results showed that tyrosyl groups were freely and reversibly ionizable in all the polymers, and that the shift of apparent pK for tyrosyl groups was up in the aspartic acid copolymers and down in the lysine copolymers, a result consistent with the simplest electrostatic considerations. No evidence was found for tyrosyl-carboxylate bonds. One may infer either that tyrosyl-carboxylate bonds do not exist in these model compounds, or that these experiments were inadequate to demonstrate them. The question of putative tyrosyl-carboxylate bonds is further discussed in Sections VI,c, and V1,D. Katchalski and Sela also studied (1953) poly-3,5-diiodotyrosine, finding here an inexplicably high intrinsic pK of 7.7 (after applying a reasonable electrostatic correction). An apparently adequate model compound, 3,5-diiodotyrosine, has a phenolic pK’ = 6.4 (Crammer and Neuberger, 1943; Gemmill, 1955). 2. Cysteinyl Groups and Thiols
Dissolved oxygen rapidly oxidizes simple thiols, through a mercaptide ion intermediate, to disulfides, in the presence of traces of certain metallic
338
D. B. WETLAUFER
ions. Because of this difficulty it was not until the work of Benesch and Benesch (1955) that reliable spectra of the mercaptide form of cysteine were reported. These are shown in Fig. 11, for varying degrees of thiol ionization. The progressive long-wave shift of the absorption peak with increasing pH is a secondary effect, due to the overlapping ionizations of the thiol and amino groups. By measuring absorptivity as a function of pH, these authors were able to supplement conventional titration data in such a way as to obtain four “microconstants” for relating the equilibria between hydrogen ion and the four main ionic species existing in cysteine solutions between p H 7 and pH 12. These species can be represented as
&;:I
0.8’ 0.6
0.D.
p H 12.0 p H 11.04
0.2 0.1
p H 9.85 p H 9.03
-
220
PH
230
240
as1
250
X(mp) FIG.11. Absorption spectra of cysteine; concentration of cysteine, 1.70 X 10-4 M (Benesch and Benesch, 1955.)
HSCysNHt , -SCysNHZ , HSCysNHz, and -SCysNH2. Despite the reasonably good self consistency of the results, two points in this work may be questioned. First, uncertainty was introduced by not operating a t constant ionic strength, and second, the somewhat uncertain assumption was made that the molar absorbance is the same for -SCysNHt asfor -SCysNH2. The first question has been answered by Gorin and Clary (1960), who find that the ratio of -SCysNHZ to HSCysNH2 does not change with ionic strength, and approximately confirm the Beneschs’ results on the absolute value of this ratio. The second criticism is more difficult to evaluate in terms of probable error. The studies with tyrosine and O-methyl tyrosine (Sections IV,A,2 and V1,C) show a small but real effect of the ammonium ionization on the phenolic chromophore, both in peak height and
ULTRAVIOLET SPECTRA O F PROTEINS AND AMINO ACIDS
339
peak position, but there is no necessary reason for expecting a close parallel between the n -+u* transition of cysteine and the T -+ ?r* transition of tyrosine. As support for the validity of the assumption of an equal absorptivity for -SCysNHZ and for -SCysNH; , Benesch and Benesch note that the absorptivity for cysteamine (pK’ = 8.35 for the --SH proton) is essentially unchanged a t its peak from pH 10 to pH 12 (pK’ = 10.7 for the -NH$ proton), although the peak position does shift from 2320 to 2360 A. We must tentatively accept this working assumption, which is supported by the consistency between the spectrophotometric and electrometric titration results. DeDeken and co-workers (1956) have reported spectrophotometric studies of the ionization of cysteine and related thiols, but have not accounted for multiple ionic species. In addition to using spectral measurements for determining ionization microconstants, Benesch and Benesch (1955) also determined the heat of ionization of the sulfhydryl group of thioglycolic acid. The result from their absorbance measurements, 6.5 kcal/mole, was in good agreement with their value of 7.0 kcal obtained from electrometric measurement of apH/dT for a thioglycolic acid/thioglycolate buffer. It may seem odd that the technique of spectrophotometric titration, so extensively applied to the tyrosyl residues of proteins, has not been used in the study of protein sulfhydryl groups. The only study of this sort known to the author is the determination of the ionization constant of -SH groups of thiolated gelatin (introduced by reaction with N-acetylhomocysteine thiolactone). By measuring the change in absorptivity as a function of pH at 2380 A, Benesch and Benesch (1958) found the apparent acidity of the thiol groups of the modified gelatin (pK’ = 9.8) to be nearly the same as that of an appropriate model compound, N-acetylhomocysteine (pK’ = 10.0). The very low content of aromatic amino acids in gelatin provides a favorable case for studying sulfhydryl ionization in a protein. The usual extent of interference by aromatic groups is suggested by a consideration of their absorptivities. The increment in molar absorptivity on thiol ionization at 2380A is about 4.5 X lo3. For tryptophan, at the same wavelength, E M 2.5 X lo3, and is relatively constant over the pH range of interest. (It must be noted, however, that since de/dX is large for tryptophan at 2380, E will be particularly sensitive to perturbations, as discussed in Section V1,B.) In contrast, the presence of tyrosine in a protein poses the problem of a large variable absorptivity, varying from -0.6 X loa a t pH 7 to -10 X lo3at pH 12. Thus, there is a large and variable absorptivity of tyrosine and tryptophan in the 2400 A region which is otherwise suitable for measuring thiol ionization. For these reasons, the prospects for spectrophotometric titrations of protein sulfhydryl groups seem generally poor.
340
D. B. WETLAUFER
3. Carboxyl and Amino Groups
Although carboxyl and amino groups both undergo absorptivity changes on ionization, their absorption bands are so weak (Ley and Arends, 1932) in the usual practical wavelength range (>ZOO0 A) as to offer little possibility for spectrophotometric titrations directly. Such titrations can sometimes be carried out if the ionizing group is situated close to a longer wavelength chromophore. It may then be possible to visualize the titration of an amino or carboxyl group by its perturbing influence on a more easily accessible chromophore. Saidel (1955a) demonstrated both the carboxyl and ammonium ionizations of glycylglycine in the pH-dependence of the peptide absorptivity a t 2050 A. The possibility of a spectrophotometric titration of the amino and carboxyl groups of 0-methyltyrosine by a perturbation method (using difference spectral measurements at 2840 A) was briefly indicated by Wetlaufer et al. (1958). For tryptophan, Hermans el al. (1960) not only reported the spectrophotometric titration of the amino and carboxyl groups, but also determined the heat of ionization of these two groups from the temperature-dependence of their apparent pK’s. The spectrophotometric titration of poly-L-glutamic acid was first done by Imahori and Tanaka (1959). The primary interest in this titration is the conformation-dependence (Section IV,E,3) of the peptide-bond absorptivity. Since measurements were made well below 2000 A, the spectral change due to carboxyl ionization was only one-quarter of the total change. The titration curve obtained by Imahori and Tanaka is biphasic in shape, unlike simple titration curves, but this is perhaps not unexpected with a conformational transition accompanying the titration. A complete rationalization of the titration of polyglutamic acid has not yet been achieved, although good progress has been made with the electrometric studies of Wada (1960).
C. Proteins 1. General Anomalous tyrosyl ionization in proteins was first demonstrated in the pioneering study of Crammer and Neuberger (1943), who showed by spectrophotometric titration that most of the tyrosyl groups of ovalbumin did not ionize a t all below p H 12, and that their eventual ionization at higher alkalinity apparently occurred only with concomitant denaturation of the protein. These results were in contrast with those obtained for insulin, where the phenolic pK’ appeared high, compared with that of tyrosine, but tyrosyl ionization was reversible and no protein denaturation occurred. Following the study of Crammer and Neuberger, numerous investigations on protein tyrosyls by the spectrophotometric titration technique have been published. These are summarized in Table 11. It
TABLE I1 Tabular Rum.mary of Spectrophotometric Titrations Substance
Finding
Bovine serum albumir
( a ) Most Tyr’s ionize a t high pH with irreversible denaturation ( b ) Tyr’s titrate reversibly Tyr pK high but ionization rc veraible 3 of 6 T y r ionize only at high pH and with denaturation Tyr pK high, ionization reversibl
T, ysozyme
Tyr PIC high, titration curve flat
Ribonuclease
Confirm and extend findings o Shugar (1952) pK’s for part of Tyr’s high; also titrations of insoluble films Substantial absorptivity changes a t 2050 A follow N-terminal and C-terminal ionizations Carboxyl pK’s determined from difference spectrum asf(pH) Determined “microconstants” for the multipIe -SH dissociation pathways; AH for -SH ionization Assignment of pK‘ values for amide protonation in strong HzS04 2 of 4 Tyr’s ionize only a t high p H and irreversibly Extent of T y r ionization a t pH li increases after urea denaturation Tyr pK high, but titration reversible; expanded protein conformation indicated a t pH > 10 No difference in the T y r ionizatioi pattern
( a ) Ovalbumin ( b ) Insulin Lysozyme R i bonuclease
Fibrinogen, silk fibroin, and r-globuli Glycylglycine and acetylglycine Tyrosine, AcTyrOEt, and BzArgOEt Cysteine and related thiols Proteins, model amides, and peptides Chymotrypsinogen Fibrinogen, trypsin, 7-globulin, and bovine fraction I11 8-Lactoglobulin Ribonuclease compared with guanidinated RNase Ribonuclease Ribonuclease
Thiolated gelatin Chymotrypsin and chymotrypsinogon Poly-L-glutamic acid
All 6 Tyr’s Litrate as one class in 8 M urea All Tyr’s titrate as one class in glycol; 3 anomalous Tyr’s regenerated on glycol removal Thiol pK apparently normal
Confirm and extend Wilcox (1957)
Reference Crammer and berger (1943)
Fromageot and Schnek (1950) Shugar (1952) Tanford and Roberts (1952) Tanford and Wagner (1954) Tanford et al. (1955) Schauenstein (1955) Saidel (1955a)
Schwert and Takenaka (1955) Benesch and Benesch (1955) Goldfarb et al. (1955, 1958) Wilcox (1957) Ungar et al. (1957)
Tanford and Swanson (1957) Klee and Richards (1957) Blumenfeld and Levy (1958) Sage and Singer (1958)
Benesch and Benesch (1958) Chervenka (1959)
Peptide absorption spectrum fron Imahori 1900-2400 A is conformation-de(1959) pendent 341
Neu-
and Tanaka
TABLE 11-Continued ~~~
~
~~
Substance
Finding
-
Itiboliuclease, oval - Performic acid oxidation "normalizes" all Tyr's; heat-denabumin, p-lactoglobturation normalizes only a part ulin, serum albumin Pepsin-inactivated RNase has 1 Ribonuclease anomalous Tyr; Oxidized RNasi has none anomalous Myosin and mero- Some Tyr's in myosin, and most in light meromyosin titrate onl! myosins a t high pH, irreversibly Ribonuclease in urea- All Tyr's titrate as one group; electrostatic factor high guanidinium chloride solution Three classes of tyrosyl ionizaTrypsinogen tion, one class irreversible Lysozyme, insulin, At high pH, irreversibly titrating Tyr's found in all three proteins and catalase Conalbumin with and 11 of 18 Tyr's titrate normally; rewithout Fe"' mainder a t high pH, irreversibly; with Fe"', 5 of 18 Tyr's titrate normally Analysk of low-temperature, low Ribonuclease pH transconformations from Tyr A-spectra Three classes of Tyr ionization; Papain titration irreversihle above pH
Reference Tranicr
arid
Shugsr
(1959)
Bigelow (1960) Stracher (1960) Cha and Scheraga (1960) Smillie
and
Kay
(1961) Inada (1961)
Wishnia el al. (1961)
Hermans and Scheraga (1961a, b) Glazer
and
Smith
(1961h)
12
3 of 4 Tyr's ionize a t pH <12.5 Edelhoch (1961) but not all reversibly Irreversibly acid-de- 2 of 3 abnormal Tyr's are nor- Bigelow (1961) natured ribonucleast malized Poly-L-lysine and Peptide absorption peak split for Tinoco el al. (1961) poly-L-glutamic acic a-helical form; sharp transition a t pH 9 for poly-Lys (random S helical) Chymotrypsin and Tyr's are the same for both forms Havsteen and Hess DIP-chymotrypsin of enzyme; electrostatic factor (1962) high in denaturing solvent in 0.14 M KCI and guanidine hydrochloride-urea solven Angiotensin and pep- Used phenolate band a t 2450 A for Paiva and Yaiva (1962) increased sensitivity; Tyr's nortide analogs mal in all compounds tested Myoglobins and hem0 Three classes of Tyr groups in M t Hermans (1962) globins two classes in Hb; correlation with X-ray structural informa tion Ribonuclease in ethyl Extension of previous (1958) work Sage and Singer (1962) ene glycol
Thyroglobulin
342
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
343
appears that few proteins are completely “normal” with respect to tyrosyl ionization. No single method for protein study gives much information by itself and spectrophotometric titrations also have been best exploited in conjunction with auxiliary studies. For example, Tanford has routinely coupled his studies with results obtained from electrometric H+-titration studies, to show with certainty that only part of the anomalous phenolic titration of proteins can be explained by a general electrostatic argument. The review by Tanford in this volume discusses these problems in detail. I n studies on proteins, it has been customary to treat electrostatic effects in terms of a model that assumes a uniform distribution of the net charge over the surface of a simple solid, such as a sphere. The ionization of tyrosyl groups in proteins is often so anomalous, (in comparison with model compounds) as to be obviously unaccountable solely in terms of a diffusedcharge electrostatic model. Certain models involving discrete charge distribution could probably account for some anomalous tyrosyl ionizations, but unless the three-dimensional structure of a protein is known, such treatments would necessarily rest on ad hoc assumptions about the locations of tyrosyl and other ionizing groups. Alternative schemes for rationalizing such anomalies (hydrogen bonding, location of ionizing groups in a nonsolvated part of a protein, etc.) are discussed in Section VI,C, and by Tanford in this volume. 2. Particular Examples
A most interesting case of anomalous tyrosyl groups is seen in conalbumin and iron-conalbumin. Conalbumin binds two molecules of ferric ion with the simultaneous displacement of six H+ from the protein. The pHdependence of this reaction provided the basis for the suggestion that three tyrosyl phenolate groups are involved in the binding of each ferric ion (Warner and Weber, 1953). Spectrophotometric titrations by Wishnia et al. (1961) showed six more tyrosyl groups to be normally titratable in conalbumin than in iron-conalbumin, thus confirming the suggested association between iron and ionized phenolic groups of the protein. Both conalbumin and iron-conalbumin were shown, by spectrophotometric titration, to have seven additional tyrosyl groups titratable only a t abnormally high pH and with concomitant structural changes in the protein. Iron-conalbumin, not unexpectedly, proved to be more resistant to structural changes a t high pH than the metal-free protein. I n summary, both conalbumin and iron-conalbumin showed seven ‘Linaccessible’ltyrosyl groups, conalhumin showed eleven normally reactive tyrosyl groups, and iron-conalhumin showed five normal tyrosyl groups. Of particular interest is the attempt by Hermans (1962) to correlate
344
D. B. WETLAUFER
H+-reactivity of heme protein tyrosyl groups with three-dimensional structural features obtained by X-ray diffraction methods (Kendrew et ul., 1961; Perutz et al., 1960). Hermans carried out spectrophotometric titrations on sperm whale myoglobin a t 2450 A (cf. Fig. 9) to eliminate interference from the heme absorption which overlaps the 2950 A phenolate band. The titrations were carried out by the difference-spectral technique. Hermans found that, of the three tyrosyl groups of myoglobin, one titrated normally (pK’ = 10.3), another titrated with a rather high pK’ of 11.6, and the third showed no ionization a t p H 12.8, the most alkaline solution examined. Hermans made similar studies on human and horse hemoglobin, and also on the corresponding heme-free globins. The globins show a normal reactivity in all twelve tyrosyl groups (pK’ = 10.5). I n contrast, eight of the tyrosyl groups of the hemoglobins titrate with a pK’ of 10.6, while the remaining four groups ionize with timedependence between p H 11.4 and 12.3. Hermans’ neglect of the absorptivity increase a t 2450 A (see Fig. 11) due to the ionization of the sulfhydryl groups of globin and hemoglobin fortunately does not alter this interpretation. Since the structures of myoglobin and the hemoglobin quartermolecule are very similar a t low resolution, Hermans has suggested that the same structural feature is responsible for the strongly anomalous tyrosyl groups in both proteins, and has postulated that this is the tyrosyl group which, by X-ray evidence, appears to be H-bonded to a peptide carbonyl oxygen. It will be of great interest to see what insights can be developed from the study of the properties of groups in known protein structures. 3. Technical Notes
The first experimental criteria to be met in a spectrophotometric titration are that the absorptivities be time-independent, and reversible. This is so fundamental that it should be obvious, but it still must be stressed. Proteins not infrequently possess marked alkali-sensitivity, as shown by the inactivation of pepsin a t pH 7, the hydrolysis of one particular amide linkage in a-corticotropin at pH 9 (Shepherd et al., 1956), and the irreversible inactivation of bacitracin a t p H > 7 (Weisiger et al., 1955). I n any particular case, temperature control should be considered essential until proven unnecessary. This point is elaborated in Section VI,F. A useful test for spectrophotometric titrations is to compare the apparent tyrosyl ionization from absorptivity versus pH measurements a t several wavelengths. A good illustration is found in Tanford and Wagner’s (1954) study on lysozyme. Their measurements a t 2880,2900, and 2950 A resulted in nonidentical titration curves. From these results, they concluded that ‘‘. . the observed changes in light absorption are not a true
.
ULTRAVIOLET SPECTRA O F PROTEINS AND AMINO ACIDS
345
representation of the ionization of the phenolic groups.” The studies of Donovan et al. (1961) on lysozyme offer a reasonable explanation for Tanford and Wagner’s observations. Donovan et d.(1961) showcd that there is a variable absorptivity contribution in this wavelength region due to perturbation of the tryptophyl groups, and that more nearly idcntical titration curves are obtained after accounting for these perturbations. AH+, in which both A and AH+ For a reaction of the type, A H+ absorb radiation, there will generally be some wavelength a t which = eAH+ . This wavelength and the corresponding absorptivity are commonly said to define an isosbestic point; that is, a common point of intersection of all absorption spectra composed of mixtures of A and AH+, in all practical ratios. Other components in the system may absorb a t this wavelength and not interfere with the existence of the isosbestie point. The existence of an isosbestic point in protein tyrosyl spectra is occasionally used (see, for example Shugar, 1952) as a test of the validity of the assumption that two and only two absorbing components are changing in a spectrophotometric titration. It is a less direct test of this question than comparison of the apparent degree of ionization a t different wavelengths, as done by Tanford and Wagner (1954), but, if a recording spectrophotometer is accessible, demonstration of an isosbestic point can be a simple and useful test. The ionization of tyrosyl groups can, of course, be measured a t wavelengths other than the customary 2950 A peak. Absorption bands from conjugated groups may overlap this region, as with the heme proteins. The most obvious spectral region (see Fig. 9) is in the 2450 A band, where the absorptivity change accompanying tyrosyl ionization is five times as great as a t 2950 A. The greater sensitivity can in itself provide a reason for preferring this spectral region-only one-fifth as much material is required for absorbance measurements a t 2450 A as a t 2950 A. Careful attention should be given to possible interferences from sulfhydryl ionization and from tryptophyl perturbations (Section V,B,2). I n connection with spectrophotometric titration of tyrosyl groups, the author recommends a more general use of denaturing solvents to estimate the maximal tyrosyl ionization. The purpose is, of course, to normalize all ionizing groups by disrupting the protein structure. This approach has been used by Blumenfeld and Levy (1958), Cha and Scheraga (1960), and by Inada (1961). In general, aqueous guanidinium chloride would appear to be a better choice than aqueous urea, since it is a more effective denaturing agent. (Anson, 1941, describes the purification of guanidinium salts.) I n certain cases, as in the incisive study of ribonuclease in ethylene glycol by Sage and Singer (1958, 1962), other solvents may be preferable. When ribonuclease is titrated with base in aqueous solution, three of its six tyrosyl groups titrate normally, but the other three ionize a t high p H
+
346
D. B. WETLAUFER
and only with concomitant irreversible denaturation of the protein (Shugar, 1952; Tanford et al., 1955). Sage and Singer showed that all six tyrosyl groups of ribonuclease ionize reversibly in glycol solution, and that catalytically active enzyme could be obtained by dilution of the reversibly titrated glycol solution with water. This finding supports the conclusion that there is no obligatory connection in ribonuclease between tyrosyl ionization and irreversible base denaturation. The use of buffer solutions is usually advisable for accurate control of the pH of solutions used in spectrophotometric titrations, since the solutions will usually be so dilute (because of the relatively high protein or peptide absorptivity) as to offer little self-buffering. The usual buffers for the pH range from 9 to 13, i.e., borate, glycinate, phosphate, lysine, a-aminocaproic acid, etc., are generally transparent through the 2950 A phenolate band. Buffer systems composed of piperidine (pK, ‘v 11) and its hydrochloride have been used to avoid the use of multivalent anions in the spectrophotometric titration of ribonuclease (Klee and Richards, 1957), but no other advantage is apparent for this system. Another caution against light-scattering contributions to apparent absorbance may be in order. One can easily test for the presence of scattering effectswith absorbance measurements at wavelengths >3300 A.
VI. DIFFERENCE SPECTRA AND CHROMOPHORE ENVIRONMENT^ A . Introductory The aromatic residues in proteins absorb ultraviolet radiation in approximately, but not exactly, the same way as the free amino acids (Sten strom and Reinhardt, 1925; Beaven and Holiday, 1952). The study of their spectral perturbations has undergone almost explosive growth in the past decade. One reason must certainly be the increasing evidence from titration studies that most proteins contain some “abnormal” tyrosyl groups. Another stems from the introduction of a new mode of representing these small spectral changes: the difference spectrum. Several factors are capable of inducing small spectral perturbations in proteins. I n a general sense these all amount to changes in the environment of protein chromophores, where ‘Lenvironment”includes all near atoms, irrespective of whether they are constituents of the solvent, or part of the molecule to which the chromophore is attached. We will not attempt to credit the originators of the study of difference spectra, but will simply suggest that the work of Bastian (1949), Hiskey (1949), and Chance (1952) has played a major role in the popularization of the technique among chemists and biochemists. It was first applied to a
* Discussions of this subject from somewhat different points of view have recently appeared by Scheraga (1961), and by Harrap et al. (1961).
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
347
study of peptides by Schwert and Takenaka (1955), and to proteins by Laskowski et al. (1956). A large number of studies have since appeared, both on proteins and on simpler, model compounds. These are summarized in Table 111.
B. Difference Spectral Technique 1. Experimental
Before discussing mechanisms responsible for difference spectra we will describe briefly the technique used for determining difference spectra. In the original papers of Hiskey (1949) and Bastian (1949), the point was simply to increase analytical precision by measuring absorbance photometrically versus a reference solution of moderately high absorbance (say A = 1.5) whose concentration is very precisely known, rather than versus the solvent. This is a valid gambit, and achieves the desired increase in analytical precision. It can be appreciated, however, that for determining the concentration of an absorbing species in aqueous solution by difference spectrophotometry, no distortion of the spectrum is allowable, since distortion would be falsely interpreted as a concentration change. NOW,instead of using differential photometry at a fixed wavelength to determine the concentration of an absorbing species, we may compare two solutions of identical concentration whose spectra differ slightly (because of a difference in the solvent, for example). Now, using one (either one, arbitrarily) of these solutions for the reference path and measuring absorbance as a function of wavelength through an absorption band, we obtain a difference spectrum. An example is shown in Fig. 12. Figure 12a (upper) shows acid difference spectra of the glycyl peptides of phenylalanine, tyrosine, and tryptophan. Another example is shown in Fig. 12b, which presents three protein difference spectra generated by comparing neutral protein solutions with acidified solutions. The peaks in the protein difference spectra are seen to be qualitatively accountable in terms of contributions from peptide difference spectra. It may clarify matters to point out that difference spectra can also be obtained from very precise conventional measurements of two nonidentical spectra at separate times, by simply subtracting absorbance values of one from absorbance values of the other, wavelength by w a v e r e m . In this fashion one obtains the same result, but with a (necessarily) much larger experimental error (Hiskey, 1949; Bastian et al., 1950) than by measuring the difference spectrum directly (sample in the reference beam). In general, if stray light and fluorescence errors can be excluded, direct measurement of a difference spectrum is highly preferable. The technique of difference spectrophotometry as usually practiced in protein work, consists of photometrically measuring the absorbance of a
TABLE I11 Difference Spectral Studies Related to Proteins Materials studied
Findings
Tyrosine, AcTyrOEt, Ester hydrolysis -+ A-spectrum. and BzArgOEt Rate of AA made the basis for proteolytic enzyme assay Tyrosyl A-spectrum results from Insulin acid titration or tryptic digestion Aqueous phenol and Tyrosyl A-spectra shown in model sodium acetate system, interaction energy estimated Ribonuclease Acid versus neutral --* A-spectrum AE versus pH abnormally steep Ribonuclease A-Spectral and optical rotation changes are qualitatively parallel in several environments Serum albumin and A-Spectra with urea, acid;A-specovalbumin tral rate studies with urea denaturation TMV protein A-Spectrum results from pH 7 versus p H 2 solutions Ribonuclease Correlation of A-spectra with enzyme activity; specific anion effects shown Pepsin, and Tyr mode Alkaline denaturation of pepsin studies .--t A-spectrum; dioxane and HOAc perturb Tyr spectrum compared with HzO T y r and 0-MeTyr Ionization, non H-bonding memodel studies dium effects -+ A-spectra Chymotrypsinogen Activation of zymogen + Tyr Aand chymotrypsin spectrum; autolysis or urea treatment of enzyme + Try A spectrum A-Spectra involving both Tyr and Pepsin T r y from acid or autolysis versus pH 4.6 Trypsin and trypsino- Autolysis, urea denaturation -+ Tyr and Try A-spectra; pregen liminary kinetic studies Ri bonuclease Heat inactivation of RNase shifts acid-induced A-spectrum to higher pH Aromatic hydrocar- Model studies: spectra of deterbons gent-solubilized aromatic hydrocarbons Bovine plasma albu- Correlation of Tyr A-spectra with min various H+-linked parameters Amino acids and ri- Model studies focusing on the role bonuclease of refractive index of solvents (sucrose, LiX, urea) 348
References Schwert and Takenaka (1955) Laskowski et al. (1956) Wetlaufer (1956) Scheraga (1957) SeIa and Anfinsen (1957) Glazer et al. (1957) Fraenkel-Conrat (1957) Sela et al. (1957) Edelhoch (1958)
Wetlaufer et at. (1958) Chervenka (1959)
Blumenfeld and Perlmann (1959) Smillie (1959) Tramer and (1959)
Shugar
Riegelman et al. (1958) Williams and Foster (1959) Bigelow and Geschwind (1960)
TABLE111-Continued Materials studied
-
-
Insulin
Ribonuclease and modificationis Bovine plasima albumin Chymotrypsiaiogen Myosin and mlbromyosins Model studievll with amino acid derivatives and several proteins RNase, S-S cleaved RNase, and bovine serum albun in Several proteins and amino acida
P
Lysosyme, ribonuclease, c h y m t r y p sin, and aromatic amino acids Chymotrypsinogen Lysozyme and amino acids Ribonuclease
Bovine serum albumin Chymotrypsin Several proteins
Chymotrypsin Serum albumin Bovine serum albumin and ribonuclease
Findings
References
A-Spectrum of T y r and Phe shows complex pH-dependence; trypsin digestion -+ A-spectrum Discrimination between individual abnormal Tyr’a Perturbation of spectrum of protein-coupled anthracene by acic titration Kinetics of urea denaturation Urea denaturation, trypsin digestion of myosin + Tyr Aspectrum Micelle, variable solvent studies, acid-generated A-spectra of proteins, analysis of mechanisms Solvent-exposed groups estimate( by A-spectral response t o cosolutesof higher refractive index AA maximum near 2350A on change of chromophore environ ment “Temperature-difference” spect r a with proteins are similar t o solvent difference spectra with amino acids Further urea-denaturation studies Acid A-spectrum of lysozyme is sensitive t o ionic strength, does not involve transconformation Intensive studies on thermal- and acid-generated transconformations Solvent-perturbation study of detergent binding AA-peak a t 2450 A is not imidazole-based Connection of peptide-chain conformation with AA peak near 2300 A T r y AA-peak a t 2900 A is connected with catalytic site Classification of tyrosyls by solvent perturbation method h-spectra show different modes of detergent-protein interaction a1 low and high detergent concentrations
Laskowski et al. (1960b)
~
349
Bigelow (1960) Williams (1960)
and
Foster
Chervenka (1960) Stracher (1960)
Yanari (1960)
and
Bovey
Herskovits and kowski (1960)
Las-
Glazer and Smith (1960)
Foss (1961)
Chervenka (1961) Donovan et al. (1961)
Hermans and Scheraga (1961a, b) Leonard and Foster (1961) Wootton and Hess (1961) Glazer and Smith (1961a) Wootton and Hess (1962) Herskovits and Laskowski (1962) Bigelow and Sonenberg (1962)
D. B. WETLAUFER
350
TABLE 111-Continued Finding
Material studied Ribonuclease Ovalbumin and bovine serum albumin Conalbumin
1
Reference
Kinetics of unfolding in urea, and of its reversal, followed by Aspectra Kinetic and equilibrium studies of the acid and urea denaturations were followed by A A ~ o by U , by 7 , and by s I n acid and urea denaturation, UV A-spectral changes are more rapid than those in a,7 , and the visible spectrum I
I
I
Nelson and (1962)
Hummel
Glazer and McKenzie (1962a) ,
Glazer and McKenzie (1962b)
I
I
I
0.2 0.1
0 O.?
0.2 0.1
n
o
0
0.1
a
0.1
0 0.1
0 1
I
I
I
I
I
250
260
210
280
290
300
v
FIG.12. Difference spectra of the individual aromatic amino acid residues (Fig. 12a) and of three proteins (Fig. 12b) ;solution of pH 6 to 7 in sample beam and solution of pH 1 to 1.5 in reference beam. Curve 1 : lo-* M glycyl-IJ-phenylalanine; curve d: 2.5 X 10-a M glycyl-L-tyrosine; andcurve 3: 5 X 10-4 M glycyl-L-tryptophan. (Yanari and Bovey, 1960.)
protein solution in a test solvent (variable pH, salt, denaturing agent, temperature, etc.) against (in the reference beam) precisely the same concentration of protein in the reference solvent. Since difference spectrophotometry increases precision, it will be appreciated that more than customary care is demanded in the volumetric manipulations to arrange
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
351
identical sample concentrations in the sample and reference cell. Any difference between the absorbance of the reference and test solvents requires a correction. To obtain a solvent-corrected absorbance difference in a single measurement, two cells can he arranged in series in both the reference and the sample beams (Remily and Wolfe, 1960; Laskowski et al., 196Ob). In the sample beam, the first cell contains absorber in the test solvent and the second cell contains the reference solvent. I n the reference beam, the first cell contains the test solvent and the second cell contains the absorber in the reference solvent. The experimental arrangements for obtaining corrected difference spectra directly are much simplified by use of the tandem two-compartment cells devised by Herskovits and Laskowski (1962). Since in difference spectrophotometry one usually works with solutions of high absorbance, stray-light errors are more probable. Setting the monochromator wavelength scale must be a precision operation when dA/dX is large, and these are the very regions of an absorption band which give difference spectral maxima. The magnitude of AA will also be somewhat dependent on the effective band width (determined by the monochromator slit width and resolving power), as shown by Hiskey and Young (1951); therefore, use of a constant slit width is advisable in a given set of measurements. 2. Representation of Results
In the actual plot of a difference spectrum one has two choices as to which solution will be called the reference solution. The two curves generated by these two choices will obviously be mirror images of each other. There is no succinct generalization as to which solution should represent the reference solution, and the choice is a matter of convenience. To reduce confusion, it is important to keep an orientation toward the conventional absorption spectrum-this can be done simply by speaking of a short-wave or a long-wave spectral shift (equivalently: blue-shift or red-shift). 3. Generation of Diference Spectra; Differential Spectra
If, as a first approximation, the spectral perturbation only shifts the absorption curve slightly along the wavelength scale without change in size or form, the differences between the two curves will be greatest at wavelengths of where the slope dA/dX is the greatest. A brief inspection of Figs. 13 and 14 shows examples of such cases. I n particular, the absorption spectra of 0-methyltyrosine (Fig. 14) show steep slopes near 2770 and 2840 A. These are, indeed, the locations of the maxima in the difference curve in Fig. 14. To summarize, a characteristic difference spectrum is really charac-
D. B. WETLAUFER
352
I I
0
D 0,
0.
Q
AD
-Q
-0.
-0. I
220
1
1
240
1
I
260
I
I
280
-
I 3 300
X (mp) FIG.13. Difference spectra of tryptophan a t 25°C. The solutions are 1.0 X M , the ionic strength, 0.15. The spectrum of tryptophan a t pH 7 is shown i n the upper part of the figure, on the same wavelength scale, and also a t a concentration of 1.0 X 10-8 M , for comparison. (Donovan et al., 1961.)
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
353
teristic of the parent spectrum, so long as the spectral change has predominantly the quality of a simple wavelength shift. Under these conditions the relationship of difference to parent spectrum is approximately that of a first derivative. True derivative or differential spectra are ob-
-200
'
: 2800
L
I
I
2700
I
I
1
I
2800
W A V E L E N G T H
L-50
2600
I N
A
FIG.14. Spectra and difference spectra of O-methyltyrosine at various p H values. The absorption spectra are the family of four curves. At pH 1.08,0 ; a t p H 5.7,A ; a t p H 3.6, 0 ; a t pH 11.5, 0. Difference spectra are A , p H 5.7 versus p H 1.08; 0 , p H 11.5 versus pH 1.08. Left, ordinate for absorption spectra; right, ordinate for difference spectra. (Redrawn, with permission, from Wetlaufer et al., 1958.)
tained by rather different experimental techniques (Section 111, F ) ; they have apparently not been applied in studies of proteins. Further discussion of difference spectra arising solely from wavelength shift may be found in the papers of Chervenka (1959), Donovan et al. (1961), and Kagarise and Whetzel (1961). The condition of simple wavelength shift often appears approximately
D. B. WETLAUFER
354
to obtain in the difference spectra of proteins and amino acids, but numerous exceptions have appeared, which may represent band intensification, fine-structure changes, band broadening, or larger changes in the form of the absorption band, or combinations of these effects. Because there is no simple test to prove that a difference spectrum originates solely from wavelength displacement, we see no advantage in further elaborating the first-derivative argument, which has this assumption as its basis. Correlation is often good enough between AA and slope to predict that maxima in difference spectra will fall near wavelengths of greatest slope, but quantitative correlation is uncertain. Yanari and Bovey (1960) have submitted the criticism of difference spectra as being . . . not a reliable measure of solvent-induced spectral shift because of the varying contributions of spectral broadening and over-all increase in absorption in different solvents.” This is true when substantial changes in form of the absorption envelope are involved, as was the case in some of Bovey and Yanari’s model compound studies. However, such is not generally the case in protein studies. In studying protein spectral perturbations one can almost always determine the shift of an absorption band-edge by difference spectral measurements more precisely than one can locate an absorption maximum. We can agree with Yanari and Bovey’s criticism only as qualified here. ((
C. Interpretation of Spectral Perturbations Perturbations in the spectra of proteins are of interest only if some useful answers can be found to the question, “What do they mean?” As we shall show shortly, useful answers do exist, although no unique answer is available. In the renaissance of interest in spectral perturbations in proteins the first publication was that of Laskowski et al. (1956). In their studies on insulin spectra, these authors revived the suggestion of Crammer and Neuberger (1943) that the tyrosyl groups of the protein are involved as hydrogen-bond donors with some basic protein groups, perhaps carboxylate. The pH-dependence of the insulin difference spectrum suggested a pK of 3 for the group(s) involved in the spectral transition. As Laskowski et al. pointed out, this lower-than-normal carboxyl pK is consistent with an H-bonded carboxylate group. At about the same time, Wetlaufer (1956) showed that the spectrum of phenol in strong urea or sodium acetate solutions is perturbed in a fashion similar to that of the tyrosyl groups in insulin. While this could have been taken as evidence of phenol-carboxylate hydrogen bonding, Wetlaufer pointed out that it was impossible to exclude a nonspecific medium effect as the cause of the spectra1perturbation.
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
355
Further model studies on tyrosyl derivatives (Wetlaufcr et al. , 1958) showed two modes of perturbing tyrosyl spectra, in addition to hydrogen bonding. One derives from the finding that O-methyltyrosine, (whose spectrum is almost identical with that of tyrosine) showed a very similar difference spectrum to that of tyrosine when strong urea or sodium acetate solutions of the amino acids were compared with isoelectric aqueous solutions. Since the hydrogen of O-methyltyrosine is replaced by a methyl group, it is clear that it cannot participate in a hydrogen-bonding mechanism as a donor. This does not, of course, prove that tyrosyl groups do not sometimes serve as H-bond donors, but it does show that H-bonding is not a unique way of generating tyrosyl difference spectra. In the same study, Wetlaufer et al. (1958) pointed out explicitly what was already implicit in the paper of Fromageot and Schnek (1950), and the review of Beaven and Holiday (1952): that ionization of the a-carboxyl or a-amino group of an aromatic amino acid perturbs its spectrum. That is to say, a difference spectrum results from comparing tyrosine at pH 1 with tyrosine a t pH 7 (pK for the a-carboxyl = 2.3). With tyrosine itself, any small spectral effect of ionization of the amino group is overwhelmed by the phenolic ionization. With O-methyltyrosine (Fig. 14), however, there is no ionizable phenolic hydrogen, and the perturbing effect of the amino ionization is clearly seen. Comparing the ionization-induced difference spectra of glycyl-o-methyltyrosine and O-methyltyrosine, Wetlaufer et al. (1958) showed that while the effect of the carboxyl ionization is the same in the two compounds, the effect of the amino ionization is markedly different. In O-methyltyrosine there is a substantial spectral perturbation due to the amino ionization, but there is almost no spectral perturbation when the free ammonium group of glycyl-0-methyltyrosine is neutralized. This difference appears to mean that the perturbing effect of a nearby ionic group falls off very rapidly with increasing distance. This conclusion is supported by the finding of Donovan et al. (1961) of a tenfold reduction of the difference spectral peak due to amino ionization in glycyltryptophan, compared to tryptophan itself. Wetlaufer et al. (1958) remark that it is not clear how much of the ionogenic perturbation is transmitted through the solvent and how much through the amino acid or protein framework. They suggest three mechanisms in which an ionic group might contribute to a protein difference spectrum: (a) simple H+ titration of an ionizing group located close to a chromophoric side chain; (b) changing the location of an ionic group in the near neighborhood of a chromophoric side chain; (c) generating (as by peptide-bond hydrolysis) a new ionizing group near a protein chromophore. (Pepsin, by its preference for hydrolyzing peptide bonds adjacent to aromatic residues, should be especially effective by route c.)
356
D. B. WETLAUFER
A different approach to the basis of difference spectra in proteins came from the studies of Williams and Foster (1959) on bovine plasma albumin. In a thorough and careful study of the perturbation of the tyrosyl spectrum throughout the neutral and acid region, these workers related the magnitude of the spectral difference to the protein titration curve, to the calculated molecular radius of the acid-expanding protein, and to net charge on the protein (accounting for ion binding). They concluded that: . . . general structural alterations [of the protein] are in all probability responsible for the low-pH change in tyrosyl absorption and that carboxylate anions are not necessarily the acceptor groups.’’ While the evidence did not permit a clear choice between the alternative mechanisms already mentioned, Williams and Foster showed that still another mechanism is consistent with their data. In summary, their argument concerns the change in energy between the ground- and electronically-excited states of a chromophore. As Williams and Foster have presented it, it involves both the polarity and the polarizability of the environment of the chromophore. [Several different expressions have been given for the polarizability, a. One of these is a = T N ) (M/p) (n2- l)/(nz a), where N is Avogadro’s number, A4 the molecular weight of the solvent, p the density of the solvent, and n its refractive index at a particular wavelength.] In their brief analysis, both effects would operate to produce a short-wave shift of tyrosyl spectra on protein denaturation, which is qualitatively what is observed. Williams and Foster (1960) further pursued their suggestion that nonspecific environmental effects can account for spectral shifts in proteins, with a study of the perturbation of the spectrum of mercurated anthracene coupled to the sulfhydryl group of bovine plasma albumin (BPA). It appeared that anthracene was an ideal indicator group, being nonpolar and having absorption bands well out of the wavelength region of protein absorption, which makes it convenient for study. Summarizing the results, the authors concluded that “The nature of the effect of pH on the differential [sic] spectrum [of BPA-coupled anthracene] is such as to suggest that the spectral perturbation is a manifestation of nonspecific interaction of the anthracene with its protein environment, the character of the environment being dependent on the molecular configuration of the protein at a given ~€1.” The authors suggested further that similar effects are involved in tyrosyl difference spectra in proteins. Since anthracene has no ionic groups or hetero atoms, it appeared to Williams and Foster that it would not participate in H-bond formation with the protein. Anthracene clearly cannot be an H-bond donor, but it should be noted that it is a strong electron donor (much stronger than benzene), and quite conceivably could act as an H-bond acceptor. With this as a real possibility, the support for their conclusion is less compelling than it seemed at first (Williams, 1962).
(x
+
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
357
The acid transformation of serum albumin has been studied further with the solvent-perturbation technique (Section V1,G) by Leonard and Foster (1961), and by Herskovits and Laskowski (1962). The results of these studies support the idea that the tyrosyl groups of serum albumin do become progressively more exposed to solvent, both in the N-F transformation and in the molecular expansion at slightly lower pH. However, it is not yet possible to make unique assignments for the causes of the spectral perturbations observed by Williams and Foster (1959). Thus far, difference spectra in proteins had been mainly concerned with tyrosyl residues, probably because the first proteins to be studied had been low or lacking in tryptophan. Tryptophyl difference spectra in proteins did not wait long to make their appearance, and when they did appear, the hydrogen-bonding mechanism for difference spectra received serious re-examination. This was, of course, because the indole nitrogen is so weakly polar that nobody would seriously suggest its participation in hydrogen bonding. (On more sober reflection, however, there appears to be no formal reason that indole could not be an H-bond acceptor.) The developments in interpretation of protein difference spectra followed along the course indicated by the work of Williams and Foster (1959, 1960). Bigelow and Geschwind (1960) published extensive model studies on the aromatic amino acids and related compounds, wherein they point out a generally good qualitative correlation between the spectral perturbation and the refractive index of the aqueous solutions. Long-wave spectral shifts resulted when the refractive index was increased by additions of urea, sucrose, sodium acetate, or inorganic salts. An exception, apparently a specific interaction, was found in the short-wave shift produced with tyrosyl groups and lithium halides. Sodium chloride and bromide produced distorted difference spectra with tyrosine: peaks of about equal height appear a t 2730 and 2800 A; this is in contrast with the usual pattcrn of peaks at 2780 and 2850 with a peak ratio of about N (see Pig. 12h and Fig. 14). The critical paper of Yanari and Bovey (1960) greatly helped to bring the matter of protein difference spectra into reasonable perspective. As these authors point out, solvents affect absorption bands by altering the energy of electronic transitions. This means that the solvent-solute interactions in the ground state, and in the excited state, will all contribute to the effect of the solvent. The nature and extent of the interactions will depend on the kind of electronic transition (n + T * , T 3 T * , etc.), the polarity of the solvent, the dipole moments of the ground state and the excited state, and the polarizability of the solvent. Yanari and Bovey report spectral studies on benzene, phenol, and indole in solvents ranging in polarity and polarizability from perfluoropentane (CSF12, nz5 = 1.239) = 1.486). through water (n:5 = 1.333) to petrolatum
358
D. B. WETLAUFER
Their results can be summarized by saying that the spectra of indole and phenol behave much the same as that of benzene (a general long-wave displacement, red-shift, is observed with increasing solvent refractive index), supporting their conclusion that the spectral shifts of all three chromophores are induced by a similar mechanism. They consider the most likely mechanism to be the interaction of solvent with a dipole associated with the actual electron-excitation event, called the transition dipole. This follows since benzene has no permanent dipole moment either in its ground state or in its first excited state (corresponding to the absorption band at 2500 A). Spectral-shift studies with fluorobenzene and benzene trifluoride, which have permanent dipoles but cannot hydrogen-bond, showed similar behavior to indole, phenol, and benzene. These results indicate that hydrogen bonding is relatively unimportant in determining the location of the spectral peaks of the aromatic amino acids in water. Yanari and Bovey point out further that the interpretation of Wetlaufer et al. (1958) of the basis of sodium acetate- and urea-induced difference spectra in tyrosyl model compounds was inaccurate : these perturbations are due to the polarisability of the solvent, and not to ion-dipole or dipoledipole interactions with the ground state. In another phase of their study, Yanari and Bovey (1960) demonstrated that difference spectra similar in many respects to those observed in protein denaturation can be generated by comparing protein chromophores in aqueous solution with the same concentration of chromophore in a micellar detergent solution. The interior of the detergent micelles, which will contain a large part of the chromophores of its solution, has a higher polarizability than the aqueous solvent. In this respect the chromophore-containing micelle is a good spectral model for the chromophore-containingprotein interior. An auxiliary point was raised by Yanari and Bovey (1960). It is that charge effects of the sort observed in the ionization of the aromatic amino acids cannot account for the magnitude of the difference spectra observed in proteins. This contrasts with the estimate of Wetlaufer et al. (1958), who found, in the protein data then available, shifts per tyrosyl group of the same order of magnitude (Am70 = 60 to 180) as those accompanying the carboxyl and amino ionizations of tyrosine or O-methyltyrosine. On the basis of more recent studies, (Bigelow, 1960, 1961) it now appears that the magnitude of the extinction coefficient change at the corresponding 2870 A difference peak in proteins can be as large as 800 to 1000 per tyrosyl group. This validates the specific criticism of Yanari and Bovey. But such high A6 values in proteins do not exclude all mechanisms involving interactions of charged groups with chromophores, only those arising from charged groups on the a-carboxyl or a-amino groups of residues bearing
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
359
the perturbed aromatic side chains. The suggestion of Wetlaufer et al., (1958) that charged side chains from other amino acids in a protein may perturb aromatic chromophores still appears t,o he a real possibi1it)y. Indeed, a recent, study of the t,ryptophyl difference spectra resulting from the acid titration of lysozyme (Donovan et al., 1961) appears to present a case in point. In this elegant study, these authors not only showed that mechanisms other than ionic perturbation are improbable, but that the large spectral perturbation of tryptophyl groups was ionic-strength dependent. This seems to imply that the perturbed chromophores and the perturbing ionic groups are at a protein-solvent interface, a situation which permits the solvent to transmit at least part of the perturbation. Leach and Scheraga (1960a) have presented a summary which appears to formally cover most, if not all, of the types of interactions in which protein chromophores may be involved.
D. Phenolic Hydrogen-Bonding Model Studies Interpretation of tyrosyl spectral perturbations has broadened from an early conception of a hydrogen-bonding mechanism, to the current view, which must accommodate ionic perturbations and changes in polarizability of the environment of tyrosyl groups as additional possible mechanisms. Since the hydrogen-bonding hypothesis still represents a possible mechanism, model studies specifically related to phenolic hydrogen bonding merit some discussion. We will briefly report here, both nonspectral studies on the strength of phenolic H-bonding and other interactions, and a few leading references on spectral studies connected with phenolic hydrogen bonding. Of the first sort, we may begin by mentioning that the distribution coefficient of phenol between benzene and water is 2/1 in favor of the organic phase. This provides a crude basis for estimating the free energy connected with transporting an un-ionized phenolic group from water into some nonpolar environment (for example, a cluster of phenylalanine residues). At room temperature, this free energy amounts to about -0.5 kcal/mole. This can be taken as a very crude model for a “hydrophobic bond” (Tanford et al., 1955; Kauzmann, 1954, 1959) stabilizing one phenolic residue someplace in the interior of a protein molecule. Another crude model, this time for estimating the strength of tyrosylcarboxylate interactions, was proposed by Wetlaufer (1956), who showed (1) a qualitatively weak interaction in the miscibility of phenol and aqueous sodium acetate solution, but (2) on the basis of the comparison of the solubility of tyrosine in sodium chloride and sodium acetate solutions, a maximum association constant no greater than 0.10 (assumed binary association) between tyrosine and acetate. By a comparison of salt effects
3GO
D. B. WETLAUFER
on the solubilities of the N-acetyl ethyl esters of tyrosine and O-methyltyrosine, Laskowski and Hrones (1961) have arrived a t a more satisfactory estimate of the strength of a tyrosyl-acetate association constant, which is coincident with Wetlaufer’s estimated maximum value of 0.10. This leads to a standard (1 M standard states) free energy of formation of such a bond of +1.4 kcallmole. If we choose the effective concentration of carboxylate ion to he 20 M (a generous estimate) in a small volume element containing a protein-water interface, the resulting free energy of formation of a tyrosyl-carboxylate bond will be -0.4 kcal/mole. A reasonable conclusion from these studies is that tyrosyl-carboxylate H-bonds are very weak in aqueous media, and that such interactions on the surface of a protein can contribute very little to the stabilization energy of a protein conformation. There is a strong likelihood that such an interaction would be strengthened in a medium less polar than water, such as we might expect to find in the interior of a protein. Indeed, most (proteinprotein) donor-acceptor interactions would be thus strengthened. However, it is misleading to say that a stronger tyrosyl-carboxylate bond is formed in the interior of a protein than on its surface. To clarify this, we must remember that free energy values are assigned not to bonds themselves, but to the processes of making or breaking them. When the donor and acceptor are on the surface it may be quite possible for the bond to form and break without changing the remainder of the protein structure. In contrast, when the donor and acceptor groups are in the interior of the protein, it seems that such a bond could break only with substantial alteration of the three-dimensional protein structure, i.e., as part of a concerted reaction. The free energy of formation of this cooperative interaction can be analyzed into three parts, consisting of (a) the formation of a nonpolar region in the protein molecule from a more random assembly of nonpolar side chains in contact with solvent, (b) the transport of polar groups into this nonpolar region from a condition of free contact with solvent, and (c) the subsequent interaction of the polar groups in the nonpolar region. I n general, it is easier to measure the free energy change for a complete concerted process than for one of its component processes. However, in addition to this practical consideration, it is conceptually misleading to speak of the strength of donor-acceptor interactions being increased in nonpolar regions of proteins, since this focuses attention only on (c) as an active process, and neglects the fact that processes (a) and (b) are equally active . Returning now to matters that are more properly within the spectral scope, we refer to the studies on phenolic H-bonding of Tsubomura (1952), who reported that with phenol and a variety of H-bond acceptors (in dilute hydrocarbon solution), there was no direct relation between the wavelength
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
361
shift and hydrogen-bond strength. More recently, Baba and Suzuki (1961) have studied effects of H-bonding on both the N 4 VI and N 4 V Z transitions of phenols and naphthols. Their results show the existence of systematic relationships between H-bond strength and the electronic spectra in the series of compounds studied. In a novel approach to distinguishing between hydrogen bonding and less specific solvent interactions with phenols, Ito (1960a) has devised criteria based on the temperaturedependence of phenolic spectra over the temperature range -70” to 60°C. He studied the temperature-induced spectral changes of the lowestenergy transitions of phenol, aniline, and diphenylamine, as examples of typical A -+ A* transitions; acetone, pyrazine, and crotonaldehyde spectra were studied as representative n -+T * transitions. All T -+ T* transition spectra show a large long-wave shift and an increase in the total absorption intensity when the temperature is lowered in the presence of H-bonding solvent. In the absence of H-bonding solvent, the only appreciable spectral change seen was a sharpening of the fine structure of the absorption envelope. The n 4 A* transition band of acetone showed a decrease in oscillator strength (peak area) at reduced temperature, and a short-wave shift when the temperature was reduced in the presence of an H-bond donor (ethanol). These examples indicate the simplicity and the power of criteria based on the temperature-dependence of the spectra of simple systems. Ito (1961b) has also used this approach in a spectral study on the selfassociation of phenol and the three cresols. When spectra of the side chains of tyrosine, tryptophan, and phenylalanine are observed in solvents increasingly less polar than water, more and more of the fine structure is resolved. Although there are strong reasons for believing that the aromatic chromophores are often in the nonpolar interior of proteins, one never sees an increased resolution of fine structure in protein spectra. The actual experimental findings are therefore a bit surprising. We tentatively suggest that perhaps the chromophores in nonpolar regions of a protein are not well insulated from the spectral effects of the solvent, nor from the multipolar character of the protein itself. Finally, we must mention the interesting report of Badger and Greenough (1961). They find that the self-association of phenol in CCL solution is facilitated by small amounts of water, and present several kinds of evidence that the predominant associated species is a “hemihydrate dimer.” By infrared spectroscopy it appears that the water hydrogens are not involved in H-bonding, and the authors suggest that the dimer is stabilized by “bonds formed through the agency of the hydrogen atoms of the phenol hydroxyls.” Of course, there is absolutely no evidence for anything of this sort in a protein. But, since components for a similar system clearly
362
D. B. WETLAUFER
do exist in proteins, the writer submits this interesting structure as a bizarre and improbable, but not quite impossible, item for conjecture. It appears from these model studies on spectra of phenols that we cannot, by spectral means alone, discriminate between a hydrogen bonding, a hydrophobic bonding, or an electrostatic mechanism for perturbations of protein tyrosyl groups.
E. Analysis of Di$erence Spectra in Quantitative Terms 1. Analysis of Multiple Factors in Absorbance Changes
It was recognized early in the development of difference spectral studies on proteins that more than one factor may be operating to influence a given AE measurement. This can be illustrated with a plot of absorbance versus urea concentration, from Bigelow and Geschwind (1960). Figure 15 shows that up to about 4 M urea, the increase in absorptivity is linear with urea concentration. This can be understood as resulting from the interaction of the three “normal” tyrosyl groups of ribonuclease with solvent of increasing refractive index. Linear extrapolation of this segment of the experimental curve to 8 M urea concentration gives f400, the absorptivity increase expected for three tyrosyl groups at this solvent composition. Above 4 M urea, the experimental curve changes slope and rapidly drops to a minimum around 8 M urea. We can understand this segment of the curve as a reflection of the transfer of the three anomalous tyrosyl groups of ribonuclease from regions (containing charged groups, hydrogen-bond acceptors, or simply regions of higher refractive index) not previously accessible to solvent, to free contact with the solvent. (For 8 M urea, 260 nz760 = 1.452, compared with n% = 1.366 for water.) The observed absorptivity change with 8 M urea is, of course, -1750, but this number is composed of three parts: (1) the absorptivity increase (+400) of the three normal tyrosyl groups going from water to the higher refractive index of 8 M urea, (2) the absorptivity decrease of t,he three anomalous tyrosyl groups on normalization and exposure to the aqueous medium, and (3) the absorptivity increase (another +400) resulting from the exposure of these normalized tyrosyl groups to 8 M urea, rather than to the reference solvent, water. To estimate that part of the absorptivity change due purely to the transfer of the three anomalous tyrosyl groups in the native protein to free contact with water, one finds the difference algebraically between the corrected final state in 8 M urea (-1750 -400) and the hypothetical initial state also in 8 M urea (+400) , which turns out to be -2550. The hypothetical absorptivity increment curve for a protein with six accessible tyrosyl groups, sketched in Fig. 15, is actually derived from measurements on performic acid-oxidized ribonuclease, where there
ULTRAVIOLET SPECTRA O F PROTEINS AND AMINO ACIDS
363
is no evidence of inaccessibility of m y of the tyrosyl groups. Oxidized ribonuclease in 8 M urea gives A6 = +880, in satisfactory agreement with the less precise value just employed (2 X 400 = 800).
M -
U R E A
FIG.15. The effect of urea concentration on the molar absorbance of ribonuclease a t 2870 A. Reference solution, ribonuclease with no urea. Solid line, experimental; lower dashed line, linear extrapolation of experimental curve; upper dashed line, experimental curve for oxidized ribonuclease. (Redrawn, with permission, from Rigelow and Geschwind, 1960.)
These points arc illustrated by the elegant studies of Nelson and Hummel (1962) on the kinetics of denaturation of ribonuclease by urea. As is shown in Fig. 16, the difference spectrum generated with ribonuclease using 7 M urea versus aqueous reference solvent is markedly time-dependent. Difference spectra taken shortly after mixing protein and urea solvent reflect the refractive index red-shift of urea on the tyrosyl and phenylalmyl groups of the protein in its native conformation. As the urea denaturation
364
D. B. WETLAUFER
proceeds, the difference spectra contain increasing contributions from the denaturation blue-shift. After denaturation is essentially complete (2450 sec) , the difference spectrum reflects complete exposure of the phenylalanyl and tyrosyl groups to 7 M urea; the deep trough a t 2870 A corresponds to the large negative (equilibrium) value of At a t 7 M urea seen in Fig. 15. Nelson and Hummel also demonstrated parallel changes in optical rotation with time and showed that the kinetics of denaturation as determined by either measurement, could be described by a first-order rate coefficient. rconning poriod seconds
40
20
0
Wovelength in m p
FIG.10. The change in the difference spectrum of 1.8 X 10-4 M ribonuclease with time in 7 M urea, 0.04 ionic strength imidazole, pH 7.0 a t 23'-24"C. Each spectrum was scariried starting a t the tinie shown a t 300 m p and was completed a t 250 mp 50 sec later. Each division on the vertical axis represents 0.1 absorbancy unit. Values above each baseline represent increased absorbnncy of ribonuclease in urea over t h a t in its absence. (Nelson and Hummel, 1962.)
They have studied the variation of this coefficient with urea concentration, temperature, and pH, and have also studied the ultraviolet absorption and kinetics of the reversal of denaturation which occurs when a solution of ribonuclease in concentrated urea is diluted with several volumes of aqueous buffer. Similar kinetic studies on other proteins should afford data of great value. 2. Discrimination between Particular Chromophores in a Protein
A further extension of the utility of difference spectra has been proposed by Bigelow (1960, 1961) in his attempts to relate difference spectral changes
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
365
to particular tyrosyl residues in ribonuclease. Table IV summarizes the quantitative spectral-shift information for ribonuclease. It should be understood that the identifications of the residues A, B, and C, are derived from the magnitudes of AezB7,,for particular normalizing operations. HerTABLEIV Denaturation Blue-Shifts Observed f o r Denatured Forms and Derivatives of Ribonuclease Compared t o Native Ribonuclease at 26"C, Neutral p H Q Ribonuclease Denatured forms
Treatment Alkali 8 M urea 5 M LiBr 10 M LiBr Low pH, temp. <17°C Low or neutral pH, temp. >43"C
Derivatives
I'crformic acid Complete pepsin digestion Partial pepsin digestion Production of RNase-S-Prot
d'3870
Referenceb
-2660' Shugar (1952) ; Tanford et al. (1955); Bigelow (1961) -26Wd Sela and Anfinsen (1957) - 1700d Bigelow and Geschwind (1960); Harrington and Schellman (1956) -27Wd Bigelow (1960) -6Wd Hermans and Scheraga (1961a) (1961); FOBS - 1700 Bigelow (1960b); Hermans and Scheraga (1961%) -250W Harrington and Schellman (1956) - 2500 Sela and Anfinsen (1957) - 1700
Sela and Anfinsen (1957)
?
From Bigelow (1961).
* References are t o original observers of
the spectral shift under t h e given conditions. c Value corrected for light scattering. Values corrected for effect of medium on normal tyrosyl residues. Calculated value. (Bigelow, 1960.) F.M. Richards, private communication.
mans and Scheraga (1961a,b), have also made comprehensive studies of the tyrosyl groups of ribonuclease. These results seem to offer hope of correlating the anomalous tyrosyl residues of ribonuclease with some kind of information about their environment. It is therefore important to examine the assumptions employed in these studies. The first is that the tyrosyl residues of ribonuclease exist in discrete environments, insofar as their spectra reflect their environments. Put differently, we assume that the small spectral changes occur in discrete
366
D. B. WETLAUFER
steps, and not in a continuous gradation of a large number of closely related states. When the perturbation was carried out with different solvents and on different ribonuclease derivatives, Bigelow found reproducible values for the h a 7 0 (in the range 700-1000) assigned t o each anomalous tyrosyl group. This is reassuring, if not compelling, evidence supporting the validity of Bigelow’s procedure. The point would appear to require testing with each protein examined. Perfect additivity cannot be expected when one is dealing with measurements not made a t strictly constant wavelength and constant effective band width; indeed these two factors present considerable barriers to quantitative interlaboratory comparisons. Aside from experimental considerations, however, we must question the assumption of the additivity of the difference spectra and their corrections. Here we are somewhat reassured by the consistency of the results for ribonuclease, but this cannot serve as a general guarantee. A situation in which nonadditivity of effects will obtain can be imagined as follows: suppose that by heat or strong acid treatment, an irreversible change of conformation occurs. On restoring the protein to the reference solvent, some previously anomalous chromophoric groups are expected to bo “normalized.” But suppose the protein aggregates, as denatured proteins often do. Corrections for scattered light can be applied by conventional means (Section II1,C) but if there should be a change in the environment of chromophoric residues in the protein as an additional consequence of the aggregation, this would also contribute to an observed difference spectrum, and by an uncertain amount. Before closing our discussion for this section, we should mention the tabulation by Donovan et al. (1961) of quantitative ionogenic difference spectral data on tryptophan, phenylalanine, histidine, 0-methyltyrosine, and some of their simple derivatives. Donovan et al. (1961) note that the relative difference spectral effects of a-amino and a-carboxyl ionizations are nearly the same in tryptophan, histidinc, 0-methyltyrosine, and phenylalanine. This, along with the fact that these ionogenic difference spectra are essentially independent of ionic strength, supports the idea that the spectral effect of the ionization in amino acids is transmitted largely through the molecule (including “bound” solvent, as qualified by Donovan et aE.), and not through the medium. On the other hand, in a t least one protein, lysozyme, an ionogenic spectral perturbation appears to be substantially transmitted through the solvent (Section V1,C). It would be premature t o attempt to generalize on this point.
F. Thermal Effects Increasing the temperature of water decreases its refractive index slightly (n2Do”= 1.333; n D1000 = 1.318)) and its dieiectric constant substantially
ULTBAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
367
(D20‘ = 80; D1O0‘= 48). In light of what has already been said about the mechanism of environmental effects on spectra (Section VI,C), we are not surprised to find that increasing the temperature of an aqueous solution of an aromatic amino acid perturbs its spectrum (Foss, 1961). As a first approximation, four effects will contribute to an apparent spectral change with temperature: (1)change of the intrinsic solvent effects, (2) change of solute concentration due to thermal expansion of the solvent, (3) change in the real optical path length due to thermal expansion of the cell, and (4) change of the degree of ionization of certain groups. Effect (3) will probably be negligible over usual temperature ranges, but effects (l), (2), and (4) can be appreciable. All four effects vary slowly with temperature, and their sum will likewise vary slowly. Again, this perturbation is most conveniently followed by the difference spectral method. If we were to plot absorbance versus temperature for tyrosine or tryptophan, we should obtain a slowly changing function. On the contrary, when we repeat this operation on a protein, we observe (Fig. 17) a rapid change, corresponding to a conformation change in the protein throughout this temperature range. That the region of rapid spectral change does indeed result from a change in three-dimensional protein structure is supported by parallel measurements of optical rotation as a function of temperature for lysozyme (Foss, 1960a), and for ribonuclease (Hermans and Scheraga, 1961a; Foss and Schellman, 1959). Indeed, Hermans and Scheraga showed, by normalizing ordinates to “per cent transformed” that plots of optical rotation and A€ versus temperature were superimposable at several pH values. Interestingly enough, Foss (1961) found one case in which an absorbance change is not paralleled by a change in optical rotation. This occurs with lysozyme at pH 2.2. The spectrally seen transitions occur at about 55’ and 80°C while the polarimetrically seen transition occurs only at 80°C (Fig. 18). This suggests that there are indeed two separable thermal transitions in lysozyme, the first involving little more than perturbations of the tryptophyl chromophores, perhaps with only minor changes of conformation, and the second involving major structural features of the protein. Such an interpretation is strengthened by the evidence of Donovan et al. (1961), and of Herskovits and Laskowski (1960b), that most of the tryptophyl residues are at or near the surface of the protein. Quite minor changes in the three-dimensional structure of lysozyme could be enough to give rise to the spectral perturbation if the perturbed groups are already at the surface of the molecule. Thus far, we have only discussed difference spectra generated between sample and reference solutions maintained at different temperatures. Additional useful observations have been made on heat-denatured proteins
P m
0
10
20
30 40 50 T E M P E R A T U R E
60
70
80
(OC)
FIG.17. Optical densities of solutions at 1.90 mgiml, ionic strength 0.16, as a function of temperature, when compared with a reference solution of zcro optical density at, pH 6.83 and low teniperature (<43’C). The pH’s of Lhe solutions are indicated. (Hemans and Scheraga, 19Bla.)
369
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
by Tramer and Shugar (1959) who found, for example, that a plot of Atza,o versus p H had a midpoint at p H 2.6 for heat-denatured ribonuclease, com-
940 0
-80"0
4
3
-
E
u)
lz
E .960
(u
0,
c 0
h c ..->
-700 -
(u
c 0
0
Q
0
c
4-
2 -0
0
n L
.980 2
.-
n
0 c
a. -60.
a
-
I .Ooo
-50°-
I
I
40
I
I
60
I
I
1
80
Temperature OC
FIG.18. Temperature-dependence of absorbance and optical rotation of lysoeyme, pH 2.2,O.lO M KCl (after Fosa, 1961; redrawn by Foss, 1962).
pared with a value of p H 1.8 for the unheated protein. A heat-denatured biopolymer may not, however, be in an easily reproducible state. This follows from the early studies on reversible and irreversible heat-denaturation of trypsin (Kunitz and Northrop, 1934) as well as from the more recent
370
D. B. WETLAUFER
demonstration that kinetic factors influence the course of reversal of thermally denatured nucleic acids (Doty et al., 1960). Bigelow (1961) has studied spectral changes of heat-denatured ribonuclease, measuring heated solutions before they have cooled sufficiently to show spectral reversions, but such a technique may be applicable only in special cases. Continuous temperature control of the sample in the spectrophotometer appears to be a more generally useful way to study thermally induced spectral changes. A final point may be made about thermal conformation changes: they may occur even at room temperature. We may profitably recall the viewpoint of Levy and Benaglia (1950) that there is a continuity (and in some cases identity) in acid- and heat-denaturations. A good recent illustration of this is seen in the studies of Hermans and Scheraga (1961a) on ribonuclease (Fig. 17). Plotting absorbance change versus temperature, they obtained a family of S-shaped curves corresponding to different acidities of the protein solution. As the pH is raised, in steps, from 0.9 to 6.8, the temperature for half-conversion increases from 25" to 65°C. Another example of a low-temperature thermal transition is provided by Smillie and Kay (1961) who show, by spectrophotometric titration, that the abnormal tyrosyl groups of trypsinogen are distinctly more abnormal a t 10" than at 25°C. The anomalous tyrosyl residues are titratable reversibly, but not instantaneously, at 1O"C, while at 25" and 37°C their titration is neither reversible nor instantaneous. This seems to be the first instance recorded in which an anomalous time-dependent ionization of tyrosyl groups has proven to be reversible. It is also possible that a temperature factor may be involved in the apparent discrepancies between the findings of Inada (1961), working in a laboratory at 16"-17"C and those of two occidental groups (Tanford et aZ., 1955; Donovan et al., 1961) working in laboratories some 10°C warmer. Inada found a distinctly anomalous tyrosyl group in lysozyme, while the latter two groups found mild anomalies, at most. It must be recognized that temperature can be a significant experimental variable in protein studies, even in the neighborhood of room temperature. G . The Solvent-Perturbation Technique
As Fig. 15 suggests, the difference spectrum of solvent-accessiblechromophores in a protein is approximately a linear function of the refractive index of the solvent, up to the concentration where a change of conformation occurs. If the refractive index is increased by adding an inert substance such as sucrose, instead of using urea, no conformation change occurs in the protein, and the magnitude of the spectral change can be used to measure the number of solvent-exposed groups (or the average extent of exposure). This is the basis of the solvent-perturbation tech-
371
ULTRAVIOLET SPECTRA O F PROTEINS AND AMINO ACIDS
nique introduced by Herskovits and Laskowski (1960a, 1962). Results of their solvent-perturbation studies on ribonuclease and reductively cleaved ribonuclease are shown in Fig. 19. Here the cleaved material shows no pH-dependence of its difference spectrum generated with solvent containing 20 % sucrose (or 20 % ethylene glycol) versus simple aqueous ribonuclease solution at pH 6. Native ribonuclease, on the contrary, shows the same
T '
O5I-.
2.2 -
rn --+I
'*-
RC RNarr
0
0
240
320
260
A in my
14-
Lo
30
50
7.0
I
9.0
PH
FIG.19. Difference in absorbance a t 2860 to 2880 A between solutions of ribonuclease in water and in 20% polyhydroxy solvents relative to the difference for native
a,
, W, 20 yosucrose; 0 , 0,2070ethylene glycol; 20 7' ribonuclease at p H 5 t o 6. glycerol. Insert, difference spectra of native RNase (pH 5.5) (--); RNase denatured in 8 M urea (. . . . ) ; and reduced, carboxymethylated ribonuclease (R.C. ItNase) (- - -). Twenty per cent sucrose versus water, r/2 = 0.25, c = 3 X lo-' M , 25°C. (Herskovits and Laskowski, 1960.) kind of pH-dependent change of conformation by this criterion as is seen in a simple plot of Aezs,O (reference: € 1 7 0 at pH 6) versus pH. Another important feature of Fig. 19 is that it shows a twofold increase in the solvent-accessibility of the tyrosyl groups of oxidized ribonuclease (and acid-denatured) compared to the native protein. This result fits nicely with the findings of Shugar (1952) and of Tanford et al. @955), who showed by spectrophotometric titration that only three out of a total of six tyrosyl groups of ribonuclease can be titrated reversibly. It appears
372
D. B. WETLAUFER
probable that the same three tyrosyl groups are relatively unreactive both to hydroxyl ion and to spectrally perturbing solvents; if this is so, we can physically locate the abnormally titrating tyrosyl groups inside the molecule, rather than on its surface. With ribonuclease, the reduced refractive index perturbation seems to be the same for sucrose, glycol, and glycerol. (Note that the ordinate for Fig. 19 is a reduced absorbance difference, i.e., the ratio of Ae, to Ae at pH 6. This is a simple way of comparing different polyhydroxylic solutes whose refractive increments are not identical.) With spectrophotometric (low pH) titration of tyrosyl groups in serum albumin, however (Herskovits and Laskowski, 1962), there is a difference in the number of tyrosyl groups perturbed by a small molecule (ethylene glycol) and a large molecule (sucrose). The smaller perturbant affects a larger number of tyrosyl groups than does sucrose, leading to the suggestion that the difference has a steric basis, i.e., that some tyrosyl groups in serum albumin are located in situations analogous to crevices, which can exclude a large perturbant (sucrose) but not a small perturbant (glycol). In another aspect of their solvent-perturbation study of serum albumin, Herskovits and Laskowski (1962) studied the well-known acid conformation change of serum albumin. A sharp transition was observed on lowering the pH from 4.2 to 3.8, apparently resulting in exposure of an additional 20 % of the tyrosyl groups to the sucrose perturbant. At low ionic strengths, decreasing the pH below 3.5 results in progressive exposure of additional tyrosyl groups, approaching the same perturbant-accessibility as is seen in 8 M urea. Returning to the factor of perturbant size, it is notable that the acid transition (at ionic strength 0.25) is observed only with large perturbants: sucrose, glycerol, and polyethylene glycol. With the smaller perturbants, ethylene glycol and dimethyl sulfoxide, the tyrosyl groups are perturbed to the same extent throughout the whole range of pH from 8 to 2. There is no hint of this transition in the acid region with thc smaller perturbants. Herskovits and Laskowski (1962) have critically discussed the necessary assumptions of the solvent perturbation technique, and the experimental limitations imposed thereby. In summary, these are (a) a quantitative measure of the chromophore-perturbing ability of each perturbant must be in hand. This requires specific determinations with adequate model compounds. (b) The perturbant must not change the protein conformation, i.e., it must be inert in all respects except in perturbing “exposed” chromophores. It would clearly be of no value if it changed the degree of chromophore exposure. (c) It is an oversimplification to class protein chromophores as either “completely exposed” or “completely inaccessible.” Gradations betwcen these extremes certainly do exist.
ULTRAVIOLET SPECTRA OF PROTEINS A N D AMINO ACIDS
373
Although this spectral approach has had but limited application thus far, it appears to have great promise for exploring the topology of proteins.
H . Difference Spectra in the 2200-2.400 A Range In a preliminary communication, Glazer and Smith (1960) pointed out that difference spectra resulting from denaturation of several proteins show a much more prominent peak in thc neighborhood of 2300 A than in the 2800-2900A region. They attribute this 2300A peak to conformation-dependence of the peptide-bond absorptivity. Comparing the magnitudes of the changes in proteins induced by acid denaturation, with those accompanying ionization of the aromatic amino acids, they conclude that the effects in proteins are too large to result solely from the aromatic residues. In this connection, we must remark that the magnitude of the long-wave difference spectrum is about 10 times as great (per residue) in a protein conformation change as for the carboxyl ionization in an aromatic amino acid (see Section V1,E). Glazer and Smith also measured the absorptivity difference resulting from the pH-dependent helix-coil transition of poly-L-glutamic acid, and found a difference peak at 2250 A with AE = 85 per glutamic acid residue. Of the various arguments and evidence presented by Glazer and Smith, the following offer the strongest support, in the writer’s opinion, for the possibility of absorptivity contributions in the 2300 A region from peptide bonds. (a) Alkaline-denatured pepsin (at pH 7.3) shows perturbation of tyrosyl and tryptophyl residues at 2850 and 2920 A respectively, but no difference peak between 2300 and 2700 A. The authors have not shown this difference spectrum below 2300 A, which is puzzling, in view of the significance they attach to the polyglutamic acid difference peak at 2250 A. (b) In the guanidinium chloride denaturation of ovalbumin, the spectral changes at 2325 A continue to a small extent after the absorbance at 2870 A and at 2925 A has ceased to change, suggesting that independent processes are represented by changes in the two spectral regions. (c) The conformation-dependent absorptivity differences first observed by Imahori and Tanaka (1959) with poly-L-glutamic acid in the 2150-2350 A range probably do originate largely in the pcptide bond. There is some uncertainty about choice of an adequate model compound to provide correction for carboxyl ionization, but a similar absorptivity difference between helical and random conformations of poly-L-lysine (Rosenheck and Doty, 1961) strengthens the conclusion. Glazer and Smith (1960, 1961a) point out that there is no correlation between the content of tyrosyl and tryptophyl groups of the several proteins they studied, and the magnitude of the 2300-2350 A denaturation
374
D. B. WETLAUFER
difference spectral peak. They submit this as evidence against a large contribution by these chromophores to this spectral effect. Since there is also no correlation hetween the aromatic amino acid content, of proteins and the magnitude of their 2700-3000 A difference s p e c h , which unquestionably arise from tyrosyl and tryptophyl groups this argument must be rejected. Some aromatic side chains behave normally and some do not. Moreover, the fraction of those “normalized” in a structural transition depends, even for a single protein, on the particular change in the medium that brings about the transition. Under these circumstances, one could hardly expect a general correlation between total amino acid composition, and the magnitude of difference spectra arising from conformational transitions. In spite of our criticism of some of Glazer and Smith’s supporting arguments, we can agree with the following summarizing statements from their (1961a) paper: “Finally, it should be emphazised that we do not feel that the difference peak in the region of 230 mp produced by the denaturation of proteins can be attributed to a single phenomenon. There are, undoubtedly, contributions produced by changes in carboxyl group ionization and in aromatic absorption; however, some of the changes appear to represent contributions from alterations in protein conformation other than those mentioned. Clearly, further study on model compounds is necessary before the structural features giving rise to the difference peak in the 230 mp region can be unequivocally identified.” Before concluding this discussion, it is of interest to note the work of ~ ~ Aezs70for human serum albumin, Eisenberg (1961) who compared A E - B and subjected to a variety of denaturing conditions. In summary, Eisenberg found a complete parallel between the magnitude of the long-wave and short-wave difference spectral peaks as a function of pH between 1 and 6. It should be remarked that the long-wave absorption spectrum of human serum albumin is dominated by tyrosyl groups (eighteen per molecule as compared with only one tryptophyl group). An examination of the spectra (Fig. 1) of tryptophan and tyrosine shows that &/dX is large for both of these amino acids near 2300 A; on this basis alone we might expect strong contributions to a difference spectrum by perturbations of these chromophores. Absorption changes in this spectral region due to the ionization of sulfhydryl and tyrosyl groups have been discussed in Section V,B;see also Figs. 9 and 11. Whether an adequate sorting out of the multiple effects contributing to AE in the 2300 A region in proteins is possible, remains to be seen.
I . Diflerence Spectra Summary The aromatic amino acids in proteins absorb radiation at slightly longer wavelengths and often with a slightly higher peak height than do the free
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
375
amino acids. This effect can be accounted for in several ways which appear compatible with our knowledge of protein structure. The most general explanation is that, for those chromophores in the interior of the protein molecule, the higher refractive index of the surroundings is responsible. Hydrogen bonding is another possible mechanism for perturbing the aromatic chromophores, although this suggestion is usually limited to tyrosyl residues. Interaction with a nearby group carrying a discrete charge is another possible mechanism for a spectral perturbation. The peptide bond may change its absorptivity concomitant with a conformation change in the 2300 A region, in addition to the well-documented changes in the 1900 A band. A few criteria exist for discriminating between possible sources of spectral perturbations, but they are usually insufficient to identify a definite source of perturbation. Generation of a difference spectrum on comparing a protein in two solvents does not prove the existence of a change of conformation. Supporting evidence is necessary, as matters now stand. Nonetheless, the use of difference spectra in the near ultraviolet retains considerable utility as an indicator of changes in protein conformation, even though we often may not know precisely how the indicator works. The difference spectral method is applicable to the study of proteins in a wide variety of environments, permitting variation of acidity, temperature, presence of denaturing solvents, or presence of cosolutes which perturb only the spectra of solvent-accessible chromophores without altering the three-dimensional structure of the protein. VII. ANALYTICAL APPLICATIONS
A. Accounting for Protein Absorptivity as the Sum of Its Parts Suppose we try to account for the absorption spectrum of a protein on the basis of its absorbing amino acids, as was the purpose of the study of Fromageot and Schnek (1950) on lysozyme. Let us proceed to make such a calculation, and then critically examine the assumptions. We shall begin with bovine pancreatic ribonuclease, because it is one of the most thoroughly characterized proteins; it has the additional advantage of containing no tryptophyl residues, which will simplify the calculations. We further simplify the problem by attempting to compute absorptivity at only one wavelength, the absorptivity maximum around 2800 A in proteins containing tyrosine and/or tryptophan. We take for molar absorptivities the values selected by Beaven and Holiday (1952): tyrosine (at 2745A max., e = 1340 at pH I ) , tryptophan (at 2780 A max., e = 5550), cystine (at 2800 A, e = 150). Ribonuclease is known by amino acid analysis to have a molecular weight of 13,680 and to contain six tyrosyl and four
376
D. B. WETLAUFER
cystinyl residues. From absorbance measurements and concentration determinations, we also know that EX,,,^^ = €2776 = 9800 (a value obtained in several laboratories). This observed molar absorptivity of 9800 can be compared with that calculated from the components: 6 Tyr, 6 X 1340 = 8040, plus 4 Cys, 4 X 150 = 600, gives a total of 8640 = t e a l o . The ratio of the observed to the calculated absorptivity is 9800/8640 = 1.13. How are we to understand this discrepancy? Is it solely because some of the chromophores are in a more polarizable environment in the protein, as discussed in Section VI? This circumstance does contribute an absorptivity increase but it is not the only reason for protein absorptivities being generally higher than calculated. It is probable, although adequate supporting data are not at hand, that simple peptide linkage of an aromatic amino acid increases its peak height by a small fraction, in addition to a definite red-shift in the peak position (Beaven and Holiday, 1952). It seems likely that the acid forms of the amino acids give a low estimate of the absorptivity of the corresponding residue in a protein. This is strongly suggested by Fig. 14, which shows that, of the three main ionic species of 0-methyltyrosine, the species predominating at pH 1 has the lowest abby some 6 % than that of the strongest absorbing sorptivit,y; lower at A,, species. Which of the ionic species is the best model for an endotyrosyl peptide? Probably none is as satisfactory as acetyltyrosine ethyl ester or amide but regrettably, we have precise spectra for neither of the latter compounds. Re-examination of Fig. 3 shows that we are in no better situation for really adequate cystinyl absorptivities. Further uncertainty in the choice of a suitable cystinyl model derives from Fromageot and Schnek’s (1950) finding that the absorptivity of cystine at 2750 A is about twice as large in base as in acid. Although tryptophyl residues do not concern us in ribonuclease, the same lack of a suitable model compound exists for protein tryptophyl groups. We may reassure ourselves that we are justified in neglecting phenylalanine contributions by noting in Fig. 1 that, the molar absorptivity of this amino acid is only 10 at 2710 A; even if all phenylalanine residues underwent a 40 A long-wave shift in a protein (probably an overestimate), this would contribute a negligible absorptivity at 2750 A. An additional factor to be considered in computing a calculated absorptivity is that A,, varies from one protein to another, and we do not have complete tables of residue absorptivities at frequent wavelength intervals. Therefore we approximate the residue absorptivity at the wavelength of the protein peak. A common approximation, as in the ribonuclease example above, is simply to use the value of t a t A,, for the model compound and hope that it will not be too poor an approximation. The question of analytical accuracy must, of course, be considered in an
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDS
377
accounting of the sort that concerns us here. We mentioned that the average value of e~~~~ = 9.8 X lo3for ribonuclease. For an appreciation of the variability encountered in practice, we now present the individual values. For ribonuclease, €2776 has been variously found to be 10.4 X lo3 (Shugar, 1952; recalculated for M = 13,680), 9.9 X lo3 (Tanford et al., 1955), 10.6 X lo3 (Harrington and Schellman, 1956), 9.8 X lo3 (Sela et ul., 1957), 9.0 X lo3 (Foss, 1960b)’ 9.8 X lo3 (Bigelow, 1961). We must acknowledge that in several instances we have computed these values from graphical representations of spectra in the quoted references; this may sometimes amount to quoting out of context. In any event, reproducibility appears to be better with a stable compound like tyrosine: €2746 = 1340 a t pH 1 (Beaven and Holiday, 1952), 1360 a t pH 1 and 1380 a t pH 7.2 (Shugar, 1952), 1380 a t pH 3.5 (Wetlaufer, 1956), 1400 a t pH 6 and 1380 at pH 1 (Wetlaufer et al., 1958). Admittedly the data quoted may not be statistically representative, but they do show much larger variation for ribonuclease (-15 %) than for tyrosine (-3 %). This suggests that some particular problems are associated with absorptivity determinations for proteins. We may consider, in suggested order of general importance, the following errors. (1) Inaccurate protein concentration determinations. In the author’s opinion, this is one of the most general errors in protein studies. Concentration can rest reliably on dry weight determinations (dry in vucuo a t 105°C to constant weight, and correct for ash) or on well-controlled nitrogen determinations. ( 2 ) Variability in protein preparations. It is clearly more difficult to obtain reproducible preparations of proteins than of amino acids. (3) Failure to test and correct for light scattering, as outlined in Section II1,B. What, then, is the outlook for the general accounting procedure suggested in the beginning of this section? While we are prevented by several small uncertainties from obtaining an exact accounting, there is one application in which we can a t present usefully apply the approach outlined. This is the case where an amino acid analysis of a protein is available. Here it is possible to make a rough check for consistency by comparing the observed absorptivity with that calculated from the composition. It should be clear that a molecular weight determination is not necessary, since both measured absorptivity and measured composition are primarily expressed on a weight basis. Using the absorptivities of Beaven and Holiday (1952) for cystine, tyrosine, and tryptophan, mentioned earlier in this section, we have calculated for several proteins the ratio e o ~ s . / ~ c s l c . The results are shown in Table V, which is not meant to be exhaustive, but rather a somewhat random sample of data in the literature. Chemical analysis for tryptophan is still less certain than for most of the other amino acids, and this must contribute some variability to a compilation of this sort. Also
378
D. B. WETLAUFER
in a few cases, tryptophan analysis was done photometrically, which produces a circular argument. Despite all our criticisms, direct and oblique, we maintain that calculations of the sort assembled in Table V have a real value, which is to point out inconsistencies between compositional analyses ~ ~ . , is lower than and intact-protein absorptivities. A ratio, E ~ I , ~ . / G ~which 1.00 means that something must be wrong either with the determination of absorptivity or with the values for tyrosine and/or tryptophan content. We cannot as easily say how high such a ratio must be before it again becomes suspect. We can be quite sure, because of several independent TABLE V Comparison of Observed and Calculated Absorptivities for Several Proteins Protein
_______~
__---
Carbonic anhydrase Carboxypeptidase A Chymotrypsinogen a-Lactalbumin @-Lactoglobulin Lysozyme Papain Ribonuclease Bovine serum mercaptalbumin Human serum mercaptalbumin A6-3-Ketosteroid isomerase
2800 2780 2820 2800 2800 2810 2780 2775 2800 2790 2770
49,600 66,700 50,200 32,500 36,600 14,700 51,800 9,800 43,600 35,000 18,300
28,000 34,400 25,100 15,550 37,700 38,900 20,700 13,680 66,000 66,000 40,800
6 6 7 5 5 6 5 0 2 1 0
8 20 4 5 8 3 17 6 20 18 10
0 2 5 4 4 5 0 4 18 18 0
44,000 60,100 44,800 35,000 38,100 37,700 50,000 8,640 40,600 32,300 13,400
1.13 1.10 1.12 0.93 0.96 1.06 1.04 1.13 1.07 1.08 1.37
a b,c d e,f g,h i,j
k 1 m m
n
References. a . Rickli and Edsall (1962). b. Neurath (1960). c . Smith and Stockell (1954). d. Chervenka (1959). e. Wetlaufer (1961). f. Gordon and Ziegler (1955). g. Gordon et al. (1961). h . Piez et al. (1961). i . Fromageot and Schnek (1950). ’ Wetlaufer and Stahmann (1958). k. Glazer and Smith (1961b). 1 . See text. m . kdsall and Spahr (1962). n . Kawahara et al. (1962).
determinations, that the ratio of 1.13 is nearly correct for ribonuclease. Remembering that half of the tyrosyl groups of ribonuclease are in contact with the solvent (see Table 111),we can expect that this value could certainly be higher if all the tyrosyl groups happened to be in the interior of the molecule. At this stage of consideration, we suggest that a comparison of a test ratio with those shown in Table V will indicate whether the test value is “in range.” For example, the ratio value 1.37 for the ketosteroid isomerase appears to be so high as to be suspect.
B . Tyrosine and Tryptophan Analysis Use of quantitative spectral measurements to analyze the composition of proteins for their ultraviolet-absorbing components was thoroughly reviewed by Beaven and Holiday (1952). Analysis has been limited to the
ULTRAVIOLET SPECTRA OF PROTEINS AND AMINO ACIDB
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tyrosine and tryptophan content. Holiday’s (1936) procedure and Goodwin and Morton’s (1946) modification employ measurements of absorptivity at two wavelengths in alkaline solvent, and solution of two simultaneous equations expressing the Beer-Lambert law, to obtain molar concentrations of tyrosine and tryptophan. Bencze and Schmid (1957) suggest an alternative procedure for determining the content of these two amino acids from (a) the slope of a line connecting absorption-curve maxima at about 2820 and 2920 A; and (b) the value of the maximum absorptivity in the same wavelength range. The correlation between these data and the content of the two amino acid residues in a protein is based on calibration measurements with tyrosine and tryptophan. The method of Bencze and Schmid has been less extensively tested (Burgi and Schmid, 1961; Glazer and Smith, 1961b), than that of Holiday, but good agreement with chemical analyses has been achieved in a number of cases. Accepting that the methods are of real utility as they stand, the author submits the following suggestions for their improvement. The first suggestion pertains more to the intrinsic nature of proteins: the pure amino acids per se are not the best models for the amino acid residues in proteins. This conclusion is inescapable from a consideration of Section VI. It seems that the N-acetylamino acid amides (or N-methylamides) should provide better models for estimating residue absorbance than the free amino acids themselves. The use of alkaline solvent for these analyses has two functions: first, it permits greater resolution of the tyrosyl and tryptophyl absorptions ; and secondly, it presumably “normalizes” both kinds of residues by bringing them into intimate contact with solvent. The assumption of normalization is generally valid, but occasionally one encounters phenolic groups in a protein which ionize slowly, even a t pH 13. For example, the recent study of Glazer and Smith (1961b) on tyrosyl ionization in papain suggests that some tyrosyl groups in this protein ionize slowly in 0.1 N NaOH, the solvent employed in both the analytical procedures described above. Glazer and Smith found that the apparent Tyr/Try ratio increased substantially with time in this solvent. Indeed, time-dependent ionization of tyrosyl groups was studied and discussed by Beaven and Holiday (1952). It would be convenient to achieve immediate normalization of protein chromophores. Removal of time-dependence should also increase the reliability of such analyses. As our second specific proposal for improvement of these analyses, we suggest routine use of a solvent composed of 0.1 N NaOH and 8 M urea to obtain a more complete and uniform normalization of tyrosyl and tryptophyl residues. Such a high urea concentration is also likely to suppress the turbidity that frequently develops in alkalidenatured protein solutions. Analysis of spectral mixtures of more than two absorbers in a protein
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has not generally been attempted, but may be feasible by a matrix analysis method outlined by Ainsworth (1961).
C. Determination of Protein Concentration by Peptide-Bond Absorptivity Coldfarb et al. (1951) found that in several proteins the molar absorptivity per peptide bond at 2050 A falls in the range 2600 to 3100, and that this contribution represents about 70 % of the total absorptivity. Following this lead, several suggestions have appeared for determining protein concentration from absorbance measurements in this spectral region : Waddell (1956), Bendixen (1957), Tombs et al. (1959), and Murphy and Kies (1960). Such procedures appear to have two advantages over the frequently employed measurement of absorbance at 2800 A: a considerably greater sensitivity, and a smaller variation in absorptivity from protein to protein. Note, however, that the peptide absorptivity is conformationdependent (Rosenheck and Doty, 1961): t Z 0 6 0 = 3200 (random conformation); t 2 0 6 0 = 2000 (a-helical). The absorption of biological materials in general has not been sufficiently investigated to permit any certain statement about the probability of interference by nonprotein absorbers in this region. As with all relatively untried methods, frequent monitoring by independent means appears advisable. We may also mention that peptide-bond absorption at somewhat longer wavelengths has been employed by Mitz and Schleuter (1957), and by Schmitt and Seibert (1961) to estimate the rates of digestion by peptidases acting on simple peptides. Schwert and Takenaka (1955) describe difference-spectral methods for assaying chymotrypsin and trypsin with acetyl-Ltyrosine ethyl ester and benzoyl-L-arginine ethyl ester as the respective substrates.
VIII. MISCELLANEOUS PROBLEMS A . Spectral Transformations in Renin The peculiar spectral effects observed by Haas et al. (1953) with renin, remain unexplained. These investigators describe three forms of this protein, each form differing radically in its ultraviolet spectrum. Spontaneous transformations of one form to another were observed-for example, the a-form changes to the y-form simply on standing in the cold at pH 4. This transformation involves about a sevenfold increase in absorptivity at 2750 A. This absorptivity change is reversible following salt precipitation of the protein, whereas the spontaneous inactivation of the enzyme is not reversible. It appears that aggregation and light scattering could be involved in some of these changes, but this can hardly explain all the observations. The work of Haas et al., has been criticized
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by Passananti (1959), but since the latter’s preparations were of much lower specific activity, his criticism can be given little weight. The behavior of renin remains an intriguing puzzle.
B. Efects of Metal Ions on the Ultraviolet Spectra
Proteins This is an area which has received almost no systematic study, in spite of occasional reports indicating that metals can have substantial effects. We may mention the general spectral intensification shown when conalbumin binds two moles of Fe”’ (Warner and Weber, 1953)’ which may be explained in part by the broad-band ultraviolet absorptivity of ferric ion with various ligands (Buck et al., 1954). We have already noted (Fig. 4) the substantial spectral effects of cadmium and zinc binding by thionein (Kagi and Vallee, 1961). The strong absorptivity of the cupric complexes of amino acids around 2300 A (Spies, 1952) is also pertinent here. of
C. Spectral Intensi$cation at High pH Spectrophotometric titration of protein tyrosyl groups often gives an isosbestic point (for example, see Shugar, 1952) around 2780 A, through which curves from pH 8 to pH 12 all pass. At somewhat higher pH (>13), protein absorption spectra sometimes show a general intensification, which is clearly indicated by the increase in 2780 A absorptivity to some value above the isosbestic point. Observations of this sort have been published for insulin (Crammer and Neuberger, 1943), for ribonuclease (Shugar, 1952), and for serum albumin (Tanford and Roberts, 1952). It is not clear whether such changes are generally reversible on lowering pH. Tanford and Roberts (1952) observed a time-dependent absorbance increase with serum albumin, which they suggested might be attributed either to slow release of “bound” tyrosyl groups, or an aggregation process. The departure from an isosbestic point argues against the former explanation, which was also rejected by Tanford and Roberts, but for a different reason. They found that the rates of increase of apparent absorbance, A’ showed the following order for the indicated wavelengths: (dA’/dt)2660 > (dA’/dt)2780 > (dA’/dt)ze60. As Tanford and Roberts point out, this ranking is clearly inconsistent with a slow ionization of tyrosyl groups. They rejected aggregation as an acceptable interpretation because the change of apparent absorptivity with wavelength in the 2650-3000 A region was greater than predicted by the Rayleigh relationship [turbidity = lc(l/A4)]. This latter is a less compelling argument, and the author suggests that an independent estimate of turbidity (Section II1,C) would be useful in such a case. In addition to the above mechanisms, Beaven and Holiday (1952) suggested that alkaline cleavage of -S-Sbonds and various oxidative
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processes may contribute additional absorptivity. Also, Fromageot and Schnek (1950) imply that irreversible spectral changes occur with lysoayme a t pH 13 when air is not excluded. The particular suggestion that baseaccelerated air oxidation of phenolic groups in proteins yields quinones, was offered by Zein-Eldin and May (1958) to explain similar spectral changes in solutions of tyrosine and proteins which result from autoclaving. However, no adequate test of either mechanism (disulfide cleavage or phenolate oxidation) appears to have been made.
IX. SUMMARY The simplest way of accounting for the absorption spectrum of a protein is as the sum of the spectra of its components. This gives results which are often good approximations to the observed protein spectrum. The assumption of additivity is basic for useful analytical applications of spectral measurements. One example is the estimation of the concentration of tyrosyl and tryptophyl residues in a protein from determinations of absorbance and protein concentration, employing tryptophyl and tyrosyl residue extinction coefficients. Another such example is the determination of protein concentration from a measurement of the absorbance in the peptide absorption band, employing a mean peptide extinction coefficient. In another perspective, the failure of perfect additivity of the component absorptivities affords the possibility of obtaining structural information about proteins. The absorptivity of the peptide bond can change by as much as a factor of two with a change in the conformation of a peptide chain. Since the absorptivity of aromatic side chains is much less sensitive to environmental change, correspondingly more sensitive techniques are required to measure the small changes which do occur. The nature and limitations of the structural information resulting from both peptide-bond and side-chain absorption are discussed. Study of the ionization of protein groups by measuring the associated spectral changes is commonly called a spectrophotometric titration. Measurements of the (usually) smaller spectral perturbations induced by change of solvent are usually carried out by the difference spectral technique. We have examined both of these methods of study from an interpretative as well as an experimental point of view. Absorption spectra can occasionally be a useful tool in the identification of unusual structural features in proteins and polypeptides. Several such applications are discussed. While studies on the ultraviolet spectra of proteins can be pursued at a rather sophisticated level, a large amount of basic work remains to be done. For much of this work, the technical limitations have been overcome. Adequate instrumentation is now commercially available, and many
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specialized techniques have been developed to cope with particular problems. k. Much has already becn accomplished in the study of t,he ultraviolet spectra of proteins and related substances. It is equally clear that much obviously useful work remains to be done. Otherwise dissimilar aspects of protein chemistry are often brought together in spectral studies. Many opportunities remain for imaginative use of this meeting ground.
ACKNOWLEDGMENTS The author is indebted to Drs. C. C. Bigelow, J. T. Edsall, J. Foss, W. B. Gratser, G. Hess, M. Laskowski, Jr., H. A. Scheraga, and W. T. Simpson for access to manuscripts before publication, and for transmittal of unpublished results. He also gratefully acknowledges useful discussions with Drs. S . Basu and M. Kasha. This review was prepared during the author’s tenure of United States Public Health Service Senior Fellowship (SF-505). Figures 5,6,11,13, and 17 are reproduced with permission from the Journal of the American Chemical Society.
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Author Index Numbers in parentheses are reference numbers and are included to assist in locating the reference where the authors' names are not mentioned in the text. Numbers in italics refer to the page on which the reference is listed.
A Abraham, E. P., 324, 387 Abul-Haj, S. K., 231, 301 Adair, G. S . , 233, 246, 256, 257, 262, 264, 267, 280, 286, 287, 288, 301 Adelman, M. B., 35, 67 Ahrendt, M. E., 309, 387 Ainsworth, S., 380, 383 Akesson, A., 137, 138, 164 Album, H. E., 281, 282, 899 Alex, M., 245, 248, 258, 263, 271, 273, 289, 300 Alexander, A. E., 256, 899 Allawala, N. A., 348, 388 Allen, D. W., 140, 166 Alway, C. D., 313, 387 Ambrose, E. J., 47, 48, 66 Ames, B. N., 221, 224 Anderer, F. A., 170, 190, 206, 215, 226 Anderson, J. W., 321. 386 Ando, T., 170, 806 Anfinsen, C. B., 50,51,66,67,172,206,212, 824, 348, 365, 377, 388 Annau, E., 383 Ansevin, A. T., 60, 67 Anson, M. L., 383 Aoki, K., 161 Arends, B., 326, 327, 340, 387 Arens, J. F., 7 Armstrong, S. H., Jr., 98, 130, 164 Asadoorian, A., 35, 37, 66 Aschheim, E., 341, 389 Ashworth, J. N., 64, 66 Astbury, W. T., 237, 249, 250, 253, 297, 308
Audrieth, L. F., 4, 7, 66 Augenstine, L. G., 305, 383 Austin, J. H., 81, 139, 161 Ayer, J. P., 259,276, 897
Ayers, J. J., 119, 163 Asegami, M., 170, 206
B Baba, H., 361, 383 Badger, R. M., 361, 383 Bagdy, D., 283, 308 Baglioni, C., 193, 206 Baha, G. F., 231, 237, 298 Bailey, A. J., 249, 898 Balo, J., 238, 249, 277, 279, 283, 898 Balls, A. K., 62, 66, 67 Bamford, C. H., 17,26,27,31,35,48,86 Banga, I., 238,249,267,277,279,282,283, 898 Bardawil, W. A., 278, 279, 301 Barnafi, L., 170, 806 Barnes, E. E., 326, 383 Barnes, R. B., 306, 386 Barnett, L., 204, 806 Barrow, G. M., 8, 66 Bartsch, R. G., 170, 184, 194, 206 Basch, J. J., 145, 162, 378, 386 Basset, E., 215, 826 Bastian, R., 346, 347, 383 Bates, R. G., 24, 66, 82, 161 Baumann, P., 252, 298 Bayaer, H., 309, 388 Bear, R. S., 237, 898 Beaver, G. H., 33, 66, 304, 305, 308, 311, 312, 314, 346, 355, 375, 376, 377, 378, 379, 381, 383 Bechet, J., 339, 384 Beckman, A. O., 308, 386 Bedell, H. M., 306, 386 Behrens, 0. K., 170, 806 Beiser, S. M., 215, 286 Bell, P. H., 170, 806 Bell, R. H., 306, 344, 386, 388
391
392
AUTHOR INDEX
Bello, J., 64,66 Benaglia, A. E., 370, 387 Bencxe, W. L., 379, 383 Bendall, J. R., 249, 262, 298 Bendixen, G., 380, 38s Benesch, R., 338, 339, 341, 383 Benesch, R. E., 338, 339, 341, 383 Bennich, H., 216, 218, 226 Benson, A. A., 309, 388 Berger, A., 39,68, 74, 16s Bergmann, M., 187, 206 Bernal, J. I)., 59, 66 Berns, D. S., 14, 66 Rertelsen, S., 268, 270, 298 Bertolin, A., 275, 298 Bettelheim, F. R., 315, S83 Beychok, S., 92, 93, 140, 161, 164 Bielschowasky, M., 229, 298 Bier, M., 51, 64, 68, 161, 162 Bigelow, C. C., 156,161,315,317,318,342, 348, 349, 357, 358, 362, 363, 364, 365, 370, 377, 383 Bill, A., 212, 226 Birkner, M. L., 278, 302 Bjork, W., 221, 226 Black, E. S., 78, 129, 130, 164 Blombiick, B., 170, 206 Bloom, B., 309, 587 Bloom, W., 235, 301 Bloot, E. R., 3, 26, 28, 29, 31, 35, 37, 66, 68, 329, 330, 384,389 Blumenfeld, 0. O., 155, 156,161, 341, 345, 348, 383 Bodansky, A., 124, 163 Boeri, E., 138, 161 Bonjour, G., 257, 298 Borsy, J., 282, 299 Bovey, F. A , , 34, 68, 314, 315, 317, 318, 349, 350, 353, 357, 358, 383, S90 Bowes, J. H., 248, 263, 298 Bradbury, J. H., 10, 66 Branson, H. R., 26, 67 Braude, E. A , , 384 Braunitzer, C., 170, 180, 193, 194, 606 Bray, R. W., 274, 302 Brenner, S., 204, 206 Bresler, S. E., 42, 66 Breslow, E., 81, 150, 151, 161 Brice, B. A., 306, 386 Brimley, R. C., 293, 301 Brink, N. G., 277, 278, 281, 300
Brinton, C. C., Jr., 60, 67 Brode, W. R., 306, 386 Broekhuysen, J., 339, 384 Bromer, W. W., 170, 206 Bronx, D., 231, 232, 249, 251, 299 Brown, G. L., 311, 383 Brown, H., 170, 206 Brown, I., 20 Brunauer, S., 255, $98 Bruning, W., 47, 66 Buc, G. L., 306, 386 Buck, R. P., 313, 381, 384 Buckley, C. E., 111, 42, 47, 63, 68 Buddecke, E., 268, 298 hettner-Janusch, V., 170, 206 Hugarseky, S., 151, 161 Bunbury, H. M., 7 Bunville, L. G., 94, 96, 103, 130, 144, 145, 163, 164 Burgi, W., 379, 384 Burton, D., 248, 298 Butts, H. A., 260, 8@9 Buzzell, J. G., 108, 164 C
Cacciola, A. R., 344, 388 Calvin, M., 309, 324, 384, 388 Cammarata, P. S., 278, 238 Campbell, D. H., 62, 67 Canada, A. H., 306, 386 Cannan, R. K., 144, 152, 153, 161 Cannon, C. G., 17, 66 Carlstrom, D., 246, 298 Carr, C. W., 98, 146, 161 Carton, R. W., 232, 298 Cartwright, T. E., 60, 67 Cassidy, H. G., 12, 66 Cecil, R., 324, 384 Cejka, J., 90, 142, 164 Cemhrano, J., 244, 298 Cesari, M., 26, 30, 67 Cha, C. Y., 33, 66, 155, 161, 342, 345, 384 Chance, B., 346, 38.4 Charlwood, 1'. A., 95, 162 Chervenka, C. H., 315,318, 341, 348,349, 353, 378, S8.4 Child, R. G., 344, 588 Chung, D., 170, 206 Churchill, J. R., 306, 386 Citi, S., 242, SO2 Clark, R. E., 74, 81, 163
393
AUTHOR INDEX
Clary, C. W., 338, 386 Clegg, J. B., 172, 173, 174, 198, 201, 207 Coates, J. H., 54, 65 Coffin, R. L., 318, 390 Cohen, B. B., 278, 279, 283, 284, 300, 301 Cohen, C., 26, 28, 29, 30, 31, 66, 68, 330, 389 Cohen, E., 132, 166 Cohen, L., 315, 318, 388 Cohn, E. J., 10, 12, 22, 35, 64, 66 Cole, R. D., 136, 169, 170, 206 Cole, R. H., Y' Coleman, J. E., 89, 130, 162 Coleman, J. S., 98, 157, 164 Colvin, J. R . , 206 Condliffe, P., 223, 226 Cook, W. H., 206 Cordon, T. C., 282, 898 Corey, E. J., 315, 318, 384 Corey, R. B., 26, 6Y Cornick, J., 170, 193, 206 Coryell, C. D., 81, 139, 162 Courts, A., 249, 256, 298 Cowling, E., 221, 225 Cox, R. C., 298 Craig, C. P., 280, 281, 284, 300 Craig, L. C., 59,66,211,218,225,226, 324, 344, 389 Cramer, R. H., 38'7 Crammer, J. L., 32, 66, 80, 152, 162, 337, 340, 341, 354, 381, 384 Crestfield, A. M., 224, 226 Crick, F. H., 249, 301 Crick, F. H. C., 204, 206 Croft, H. J., 224, 226 Cullis, A. F., 344, 388 Cunningham, L. W., 323, 384 Curme, G. O., 20, 46, 66 Curtain, C. C., 212, 226 Caerkawski, J. W., 279, 280, 289, 296, 299
D Dainauskas, J., 232, 298 Daisley, K. W., 214, 226 Danckwerts, P. V., 136, 162 Dandliker, W. B., 316, 317, 390 Danehy, J. P., 9, 68 Danielli, J. F., 58, 66 Dannenberg, H., 304, 384 Dannhauser, W., 7 Davie, E. W., 145, 163, 378, 388
Davies, D . S., 170, 206 Davies, M. C., 344, 388 Davies, S. B., 170, 206 Davis, H. F., 231, 233, 246, 248, 256, 257, 262, 264, 267, 271, 280, 286, 287, 288, 290, 292, 295, 296, 301 Dawson, L. R., Y De, P. K., 31, 41, 42, 47, 54, 63, 68 Debye, P., 305, 384 DeDeken, R. H., 339,384 De La Haba, G., 278, 298 Dempsey, E. W., 230, 255, 258, 271, 273, 289, 298, 299, 300 de Robertis, E., 238, 299 Desnuelle, P., 257, 298 Deuel, H., 209, 226 Devenyi, T., 271, 299 Dexter, R. N., 74, 81, 163 Dickerson, R. E., 23, 26, 33, 35, 6Y, 123, 149, 163, 170, 193, 206, 344, 386 Dintais, H., 78, 162 Dixon, G. H., 325, 384 Dixon, J., 136, 163 Dixon, J. S., 170, 206 Doljanski, L., 247, 298 Dolnick, E. H., 230, SO1 Donohue, J., 26, 30, 66 Donovan, J., 384 Donovan, J. W., 82,120,147,148,149,162, 317, 318, 325, 340, 345, 349, 352, 353, 355, 359, 366, 367, 370, 386 Doty, P., 5, 10, 26, 28, 29, 30, 31, 36, 37, 40, 45, 51, 54, 63, 66,68, 304,313, 317, 329, 330, 331, 333, 370, 373, 380, 384, 385, 388, 390 Douglas, T. B., 7 Downie, A. R., 37, 66 Doyle, P., 283, 302 Drabkin, D. L., 81, 139, 161 Duff, G. L., 246,298 Duke, J. A., 161, 162 Dumi Tru, E. T., 253, 301 Dus, K., 170, 184, 194, 206 Dvonch, W., 281, 282, 299
E Eaker, D., 156, 162, 220, 225 Eastoe, J. E., 229, 248, 299, 302 Eberg, R. V., 275, SO1
394
AUTHOR INDEX
Edelhock, H., 154,160,162,315,342,348, Fisher, E. R., 277, 299 Fitton-Jackson, S.,247, 299 384 Editorial, 306, 984 Fitts, D.D., 26, 67 Edmundson, A. B., 150,162,170, 193,806 FitzPatrick, M.,J., 248,261, 299 Edsall, J. T.,10, 12, 22, 34, 66, 68, 70, Fjellstrom, K.E., 216, 226 72, 120, 130, 162, 163, 314, 315, 324, Flodin, P.,210, 211, 212, 213, 215, 222, 335, 336, 340, 348, 353, 355, 358, 359, 226 Flory, P. J., 253, 299, 301 377, 378 384, 387, 388, 390 Edwards, B. G., 318, 319, 384 Fock, W., 20 Edwards, J. O., 305, 384 Folk, J. E., 145, 163, 378, 388 Efrom, M.,193,206 Folsch, G., 74, 162 Ehrenberg, A., 137, 138, 151, 161, 163, FOBS, J. G., 51, 63,66, 315, 318,349, 365, 367, 369, 377, 384 164 Ehrlich, G., 81,162 Foster, J. F., 108, 159,161, 162, 166,348, Eigner, E. A., 170,206 349, 356, 357, 387, 390 Eigner, J., 370,984 Fothergill, J. E., 63, 68 Eirich, F., 10,66 Fraenkel-Conrat, H.,3, 64,66, 134, 263, Eisenberg, D.S., 374, 384 170, 203, 204,SOY, 322,348,386 Elliott, A,, 17, 26, 27, 31, 35, 37, 47, 48, Franchi, C. M., 238, 899 66,66 Frankel, S.,279, 301 Elliott, D. F., 206 Franks, H.S., 19,66 Elliott, R. G., 248, 898 Franzen, J. S., 16, 17,67 Elmqvist, A., 215, 226 Fredericq, E., 60,66, 95, 142, 143, 268 Elsden, D.F., 262, 265, 266, 899 Freed, S.,64,66, 312,386 Emmett, P.H., 255, 298 French, C.S . , 312, 386 Engfeldt, B.,246, 898 Fridovich, I., 308, 386 Engle, R.L., Jr., 230, 299 Frieden, C.,59, 66 English, J. T., 344, 388 Frohwirt, N.,137, 163 Engstrijm, A.,246, 898 Fromageot, C.,341, 355, 375, 376, 378, 382, 386 Ens, A,, 95, 168 Epstein, J., 109, 142, 143, 164 Fruton, J. S., 278, 898 Epatein, W.V., 216, 226 FUOSS, R. M., 7, 14, 66,66,67 Estabrook, R.W., 312, 384, 387 Fiizi, M., 282, 299 Evans, D. F., 307, 384 G Evans, M.W . , 19,66 Evans, R.J., 260,699 Gamow, G., 201, 806 Everett, A. L.,282,298 Garrod, M.,249, 253, 255, 259,300 Evers, E.C.,2, 10,66 Garry, B. J., 221, 224 Geffner, J., 306, 386 F Gehrring-Muller, R.,170, 193, 194, 806 Farkas, W., 309, 386 Geiduscheck, E. P., 9, 25, 53, 54, 55, 66, Fasman, G.,37, 66 68, 304, 384 Fawcett, J. S., 383 Geko, S., 271,899 Ferrari, C . , 277, 802 Gelotte, B.,212, 215, 223, 226 Ferri, E., 253,301 Gemmill, C.L., 315,337, 386 Field, E. O., 59, 66 George, P.,113, 120, 139, 151, 162 Fiess, H.A., 324,386 Gerald, P. S., 193,306 Findlay, V. H., 250, 299 Gergely, J., 271, 299 Finn, B.M., 344,988 German, B.,139, 168 Fischer, E., 806
395
AUTHOR INDEX
Geschwald, I. I., 136, 163 Geschwind, I. I., 170, 206, 206, 315, 317, 318, 348, 357, 362, 363, 365, 383 Gibian, H., 172, 174, 182, 206 Gibson, K. H., 306, 386 Gierer, A., 204. 206 Giese, A. T., 312, ,986 Gilfillan, R. F., 278, 279, 301 Gilligan, D. R., 258, 273, 300 Gillman, T., 231, 232, 236, 249, 251, 299 Gilmore, J., 306, 386 Ginger, B., 278, 302 Gish, D. T., 170, 207 Gladner, J. A., 145, 163, 378, 388 Glazer, A. N., 153,162,342,348,349,350, 373, 374, 378, 379, 386 Goldblatt, H., 380, 386 Goldfarb, A. R., 308, 322, 325, 326, 327, 328, 334, 341, 380, 386, 588 Goldring, L. S., 308, 386 Good, M., 319, 387 Goodman, M., 37, 66 Goodwin, T. W., 290, 299, 379, 386 Gordon, L., 145, 16.9 Gordon, W., 378, 386 Gore, I., 269, 299 Gorin, G., 338, 386 Gormley, J. F., 7 Gotte, L., 240,241,261,262,265,266,299, 301
Graham, G. N., 283, 299 Granath, K. A., 226 Grandonico, F., 242, 302 Grant, N . H., 261, 277, 279, 299 Grant, R. A,, 62, 66 Gratzer, W. B., 304, 312, 313, 320, 325, 330. 331, ,983. 386 .W.Q Gray, I. 9, 53, 66 Green, D. E., 59, 67 Green, R. W., 255, 299 Greenberg, D. M., 24, 66 Greenough, R. C., 361, 383 Greenstein, J. P., 321, 322, 387, 388 Griffiths, J., 256, 299 Gross, J., 237, 238, 299 Gruen, L., 120, 143, 144,162, 386 Griinig, W., 253, 302 Grunwald, E., 24, 66 Guettel, C. L., 306, ,986 Gundlach, H. G., 33, 66
Curd, F. R. N., 74, 81, 142, 149, 150, 151, 161, 162, 163 Gutbezahl, B., 24, 66 Gutstein, N., 334, 341, 386
H Haas, E., 380, 386 Habeeb, A. F. S. A., 12, 66 Haber, E., 212, 224 Haefele, L. F., 315, 318, 384 Hajek, A., 257, 301 Hale, C. W., 231, 299 Hall, D. A., 228, 230, 244, 245, 248, 249, 250, 256, 263, 267, 279, 280, 282, 289, 296, 298, 299, 302 Halpern, A., 329, 330, 342, 389 Ham, J. S., 326, 386 Hammett, L. P. 5, 66 Hanania, G., 113, 120, 139, 151, 162 Hanby, W. C., 17, 26, 27, 31, 35, 37, 48, 66, 66 Handler, P., 308, 386 Handschuh, D., 215, 226 Hannay, P. W., 250,299 Hanson, L. A., 221, 226 Harber, E., 224 Hare, G. H., 308, 386 Harfeneist, E. J., 59, 66 Harker, D., 35, 67 Harkness, M. L. R., 242,248, 274,299 Harkness, R. D., 242,247,248,274, 299 Harrap, B. S., 304, 386 Harrington, W. F., 38, 39, 66, 68, 152, 162,348,365,377,386,388 Harris, J. I., 136,163, 170, 206,206 Harrison, G. R., 306, 386 Hartley, B. S., 170, 206,249,279,281,302 Hartree, E. F., 309, 312, 386 Hass, G. M., 244,259, 276,297, 299 Hass, M., 232, 298 Hathorn, M., 236, 299 Hauenstein, J. D., 32, 45, 68,92,94,102, 103, 118, 120, 135, 164, 341, 346, 359, 365, 370, 371,377, 389 Haugaard, G., 258, SOB Haupt, G. W., 308, 376 Hausmann, W., 324, 344, 389 Havsteen, B. H., 133, 162, 342, 386 Hawes, R. C., 308, 386 Heidt, L. J., 313, 386
396
AUTHOR INDEX
Heilbron, I., 7 Helfierich, F., 209, 225 Helnikanip, G. K., 55, 06 Heppel, L. A . , 62, 66 Hermans, J., Jr., 81, lP2, 151, 155, 162, 340, 342, 343, 349, 365, 367, 368, 370, 386
Herskovits, T. T., 9 25,47,53,54,55, 66, 349, 351, 357, 367, 371, 372, 386, 387 Hess, G. P., 133, 162, 325, 342, 349, 385, 390 Hilker, I). M., 243, SO0 Hill, R. J., 170, 193, 206, 206, 218, 225 Hill, R. L., 137, 163, 193, 206 Hill, T. L., 107, 153, 162 Hilschmann, N., 170,180,193,194,205 Hilse, K., 170,193,194,205 Hindley, J., 170, 205 Hirose, F. M., 283, 302 Hirs, C. H. W., 150, 162, 170, 193, 205, 206
Hirsch, E., 14, 66 Hirschman, D. J., 134,163 Hiskey, C. F., 308, 346,347, 351, 386 HjertBn, S., 210,211,225 Hobom, G., 170,193,194,205 Hocott, J. B., 275, 301 Hoeve, C. A. J., 253,299 Hoffman, E., 334, 385 Hogness, T. R., 308, 386 Holeysovskf, V., 169, 170, 172, 174, 184, 190, 198, 206, 207 Holiday, E. R., 33, 65, 290, 299, 304, 305, 311, 312, 314, 346, 355, 375, 376, 377, 378, 379, 381, S83, 386 Hollingworth, B. R., 34, 68, 74, 163, 315, 335, 336, 340, 348, 353, 355, 358, 359, 377, 384, 887, 390 Holtzer, A., 47, 65 Holtzer, A. M., 10, 66 Holzwarth, G. M., 313, 330, 331, 386 Horbacaewski, J., 286,299 Horton, E. W., 205 Horvath, M., 279, 298 Howard, K. S., 170, 806, 344, 388 Hospelhorn, V. D., 248, 261, 299 Howes, E. L., 250, 301 Hrenoff, M. K., 348, 388 Hrones, J., 360, 387 Huehns, E. R., 206
Hughes, H. K., 306, 386 Hughes, W. L., ,Jr., 35, 64, 65 Hiihn, K. H., 231, 298 Humason, G. I,., 230, 299 Hunimel, J. I)., 350, 363, 364, 387 Hunt, H. I)., 326, 330, 386 Hunt, J. A , , 193, 206
1 Iniahori, K., 28, 29, 37, 40, 66, 68, 313 328, 340, 341, 373, 386 Inada, Y., 342, 345, 386 Inagami, T., 51, 52, 61, 66 Ingalls, E. N., 139, 140, 165 Ingram, V. M., 175, 193, 204, 205, 296 Ioffe, B. V., 7 Ioffe, K. G., 289, 300 Isemura, T., 389 Ishii, S., 170, 296 Iso, K., 30, 68 Ito, M., 386 Iwai, K., 170, 205
J Jackson, D. S., 248, 300 Jacobs, M. B., 4, 67 Jacobs, S., 248, 300 Jacobsen, C. F., 11, 67, 70, 88, 162, 323, 387
Jakab, L., 271, 299 James, T. W., 26, 67 Jaurequi-Add61, J., 170, 206 Jensen, C. E., 268, 298 Johansson, B. G., 221, 225 Johnson, A. M., 313, S86 Johnson, E. A., 308, 312, 313, 383, 386 Johnson, W. H., 153,162 Johnston, F., 20, 46, 66 Joll&s,J., 170, 206 JollBs, P., 170, 206 Jones, H., 282, 298 Jones, J. T., 170, 193, 206 Jones, R. S., 230, 300 Jordan, D. O., 54, 65 Jurtshuk, P., Jr., 59, 67
K Kagarise, R. E., 353, 386 Kiigi, J. H. R., 321, 322, 381, 386
AUTHOR INDEX
Kahn, J. S., 153, 162 Kalan, E., 145, 162 Kalan, E. B., 378, 386 Kalt, W. B., 308, 327, 388 Kameda, K., 154, 163 Kamen, M. D., 170, 184, 194, 206 Kao, K . T., 243, 244, 300 Kaplan, D., 269, 300 Karkela, A., 291, 300 Karush, F., 110, 162 Kasha, M., 307, 386 Katchalski, E., 10,39,66,68,74,163,337, 386, 387, 388 Katersky, E. M., 258, 273, 350 Katsoyannis, P. G., 74, 163 Kata, J. J., 5, 8, 10, 48, 52, 67 Kauffmann, D. L., 170, 207 Kauzmann, W., 11, 16, 17, 19, 20, 23, 31, 34, 47, 56, 61, 62, 67, 124, 126, 163, 359, 386 Kawahara, F. S., 378, 386 Kawase, O., 240, 300 Kay, C. M., 160, 164, 342, 370, 389 Kay, R. L., 2, 10, 66 Keech, M. K., 248, 298 Keil, B., 169, 170, 172, 174, 180, 184, 185, 187, 190, 194, 196, 198, 206, 207 Keilin, D., 309, 312, 386 Kenchington, A. W., 70, 139, 163 Kendrew, J. C., 23,26,33, 35,67,123,149, 163, 170, 193, 194, 206, 207, 344, 386 Kent, P. W., 229, 302 Kenten, R. H., 248, 263, 298 Kerr, V., 309, 389 Kibrick, A , , 144, 152, 153, 161 Kiddy, C. A., 59, 68 Kies, M. W., 380, 387 Killander, J., 212, 221, 266 Kilpatrick, M., 5, 67 Kimmel, J. R., 137, 163, 170, 206 King, M. V., 35, 67 King, N. R., 232, 235, 301 Kingma, S., 193, 206 Kirkwood, J. G., 26,67,115,121,122,163, 164
Kisliuk, R. I,., 212, 226 Kitsi, R., 170, 206, 206, 280, 301 Klee, W. A., 341, 346, 386 Kleinberg, J., 4, 7, 65 Klemperer, E., 29, 37, 40, 66
397
Klotz, I. M., 16, 17, 67, 119, 163, 324, 386 Knesslouh, V., 169, 172, 174, 184, 198, 207 Knichal, V., 172, 173, 198,207 Knight, C. A., 170, 207 Koch, H. P., 323, 386 Kolpak, H., 236, 300 Kolthoff, I. M., 324, 386 Koltun, W. L., 74, 81, 163 Konigsberg, W., 170, 193, 205, 206, 218, 225 Korngoth, S. E., 321, 386 Kostka, V., 169, 170, 172, 174, 184, 190, 198, 207 Krafka, J., 253, 300 Kreil, G., 193, 206 Krishna-Prasad, Y. S. R., 28, 31, 68 Krompecher, S., 247, 300 Kronick, P. L., 7 Kuby, S. A., 324, 387 Kulonen, E., 291, 300 Kunitz, M., 369, 387 LA Bella, F. S., 271, 287, 290, 291,292,300 Laitinen, E. A., 240, 300 Lamfrom, H., 380, 386 Lamy, F., 277, 280, 281, 284, 300 Landmann, A. A., 170, 207 Lanni, F., 176, 206 Lansing, A. I., 230, 244,245,248,258,263, 271, 273, 277, 289, 298, 299, 300 Lapiccirella, R., 277, 301 Lardy, H. A., 324, 387 Larkey, B. J., 269, 699 Larson, C. E., 24, 66 Laskowski, M., 283, 302 Laskowski, M., Jr., 33, 67, 82, 118, 120, 139, 143, 144, 147, 148, 149, 162, 163, 164, 315, 317, 318, 325, 345, 347, 348, 349, 351, 352, 353, 354, 355, 357, 359, 360, 366, 367, 370, 371, 372, 384, 386, 386, 387
Lathe, G. H., 209, 211, 226 Latimer, P., 309, 387 Lauffer, M. A , , 60, 67 Lavin, G. I., 311, 387 Lawson, W. B., 318, 388 Lea, D. J., 210, 226 Leach, A. A , , 248, 300 Leach, S. J., 309, 349, 351, 359, 387
398
AUTHOR INDEX
Leader, G. B., 7 Lee, T. H., 170, 806 Lengyel, P., 204, 906 Leonard, W. J., Jr., 349, 357, 387 Leone, O., 242, 309 LBonis, J., 70,88, 104, 135, 136, 168, 163, 387 Lerner, A. B., 170, 806 Levedahl, B. H., 26, 67 Levine, s., 10, 67 Levy, M., 155,156,161,341,346,370,383, 387 Lewis, A. L., 230, 300 Lewis, G. P., 806 Lewis, J. C., 134, 163 Lewis, V. J., 277, 278, 281,300 Ley, H., 326, 340, 387 Li, C. H., 104,135,136,163, 170,806,606, 387 Lieberman, H., 326, 388 Liebermann, L., 151, 161 Liesegang, R. E., 300 Light, A., 170, 806 Lighty, P. E., 306, 386 Lillo, M., 244, 898 Lindermeyer, A., 312, 387 LinderstrGm-Lang, K . , 11, 67, 70, 78, 88, 97, 121, 169, 163, 164, 323, 387 Lindner, E. B., 215, 221, 886 Lindqvist, B., 209, 886 Lipp, W., 212, 886 Lisowski, J., 215, 986 Little, K., 898 Lively, E. P., 42, 47, 63, 68 Lloyd, D. J., 249, 253, 255, 259, 300 Loeven, W. A., 267, 277, 280, 282, 500 Logan, M. A., 248, 273, 301 Lohss, F., 271, 301 Loisel, G., 247, 300 Lokgina, L. A., 170, 806 Lonite, R., 221, 696 Loofbourow, J. R., 311, 389 Loomeijek, F. I., 272, 291, 300 Lorinez, A. L., 249, 300 Lorinz, F., 274, 300 Lowry, 0. H., 258, 273, 300 Luborsky, F. E., 5, 67 Lucas, R. V., 230, 898 Luft, J. H., 239, $00 Lund, E,. 78, 164 Lundgren, H. P., 267, 308
Luse, R. A., 305, 387 Lushbaugh, C. C., 230, 889 Luzeati, V., 26, 30, 67
M McCalla, K., 170, 193, 806 McClellan, A. L., 18, 67 MacDonald, C. E., 62, 67 McDonald, D. A., 242, 248, 274, 899 McFadden, M. L., 347, 348,354,387 McGavack, T. H., 243, 244, 300 McGlynn, S. P., 307, 319, 387 McIvor, B. C., 277, 301 McKenzie, H. W., 348,386 Maclaglan, N. F., 380, 389 McLaren, A. D., 305, 387 MacLennan, J. D., 250, 301 McMillan, G. C., 246, 898 McPhee, J. R., 324, 384 Magdoff, B. S., 35, 67 Malcolm, B. R., 37, 66 Malik, S., 310, 332, 887 Mallory, F. B., 230, 300 Mandl, I., 250, 276, 278, 279, 283, 284, 300,301 Marcker, K., 143, 165 Mardones, J., 244, 898 Margolena, L. A., 230, 301 Margoliaah, E., 137, 163,170, 193,806 Markham, R., 62, 66 Marko, A. M., 247, 899 Marmur, J., 55, 67, 370, 384 Marrama, P., 277, 301 Marsden, N., 212, 865 Martin, A. E., 313, 387 Martin, G. R., 74, 133, 166 Martin, R. B., 324, 335, 336, 384, 387 Martin, R. G., 221, 964 Miisiar, P., 169, 172, 174, 184, 198,807 Mason, H. S., 316, 390 Masson, F., 26, 30, 67 Matheka, H. D., 212, 696 Matsen, F. A., 307, 314, 387 Matthaei, J. H., 204, 606 Maximow, A. A., 235, 301 May, B. Z., 382, 390 Mehler, A. H., 308, 309, 387 Meisenhelder, J. H., 344, 388 Mele, A., 334, 341, 386 Melin, M., 64, 66
AUTHOR INDEX
Meloun, B., 169, 170, 172, 174, 184, 190, 198, 207 Mergenhagen, S. E., 133, 163 Metzger, H., 160, 162 Meyer, K., 269, 300 Meyer, K. H., 253, 301 Mihalyi, E., 138, 151, 163, 168, 206 Mikeg, O., 169,170,172,174,184,190,198, 206,207 Mittleman, F. A., 119, 163 Mitz, M., 308, 380, 387 Miyada, D. S., 278, 301 Mizushima, S., 17, 67 Mochel, J. M., 20 Moffitt, W., 26, 31, 67, 328, 329, 331, 387 Moon, H. D., 277, 301 Moore, S., 33, 66, 68, 157, 164, 170, 106, 221, 226, 263, 264, 291, 301 Morhvek, L., 169, 206 Moret, V., 301 Morgan, R. S., 188, 206 Morita, Y., 154, 163 Morris, D. L., 258, SO1 Mortier, A., 339, 384 Morton, R. A., 290,289,379,386 Mosbach, R., 210, 226 Mosovich, E., 326, 380, 386 Moss, J. A., 248, 298 Moyer, A. W., 344, 388 Muir, H. M., 247, 199 Muirhead, H., 344, 388 Mulfond, D. J., 64, 66 Muller, C. J., 193, 206 Muller, K., 212, 226 Mulliken, R. S., 307, 313, 319, 387, 388 Murayama, M., 61, 67, 193, 206 Murphy, J. B., 380, 387 Mutch, M. O., 172, 173, 174,198,201, 207
N Nachmansohn, D., 124, 166 Nagasawa, M., 70, 121, 163 Nag-Chaudhuri, J., 319, 387 Naghski, J., 282, 298 Naughton, M. A., 170, 806,249, 279, 281, 301
Nelson, C. A., 350, 363, 364, 387 Neuberger, A., 32, 66, 80, 152, 162, 242, 247, 2@9,301, 337, 340, 341, 354, 381, 384 Neukom, H., 209,226
399
Neuman, R. E., 248,263,273,301 Neurath, H., 170,207, 325, 378, 384, 387 Newton, G. G. F., 324,387 Nielsen, 8. O., 70, 78, 121, 163 Niemann, C., 187, 206 Nirenberg, M. W., 204,206 Noda, L. H., 324, 387 Nord, F. F., 51, 64, 68, 161,168 North, A. C. T., 344, 388 Northrop, J. H., 311, 369, 387 Nozaki, Y., 94,96,103,130,141, 144,145, 146, 163, 164 Nuenke, B. J., 322,384 0
O'Brien, J. R. P., 59, 66 Ochoa, S., 204, SO6 Olcott, H. S., 3, 64, 66 Olson, E. C., 313, 387 Ona, R., 92, 93, 140, 164 OrechoviB, V. N., 170, 206 Ornes, C. L., 274, 301 Osterberg, R., 74, 162 Osterloh, F., 52, 68 Otey, M. C., 321, 322, 387 Oth, J. F. M., 253, 301 Ott, H., 271, 301 Ottesen, M., 70, 88, 162
P Paiva, A. C. M., 342, 387 Paiva, T. B., 342, 387 PalBus, S., 137, 138, 163 Palilla, F., 347, 383 Palmer, A. H., 144, 152, 153, 161 Palmstierna, H., 221, 226 Parcell, A., 324, 387 Parisoli, V., 277, 301 Partridge, S. M., 231, 232, 235, 246, 248, 249, 256, 257, 262, 264, 265, 266, 267, 271, 277, 278, 280, 286, 287, 288, 290, 292, 293,294,294,295,299,301,108 Passananti, J. T., 381, 387 Patchornik, A., 74, 163,318,387,388 Paul, K. G., 138, 161 Pauling, L., 26, 62, 67,81, 139,162 Pedersen, K., 223, 226 Penn, J., 231, 232, 236, 249, 251, 299 Pepper, K. W., 293, 301 Pepper, W. P., 7 Perlmann, G. E., 348, 383,388
400
AUTHOR INDEX
Remily, C., 351, 388 Repetto, M., 275, 301 Reynolds, W. L., 7 Rhinehart, J. F., 231, 301 Rhodes, D. N., 249, 298 Rhodin, J., 58, 68 Rice, S. A., 54, 68, 70, 121, 263 Rich, A., 201, 205, 249, 301 Richards, F. M., 341, 346, 386 Riekli, E., 378, 388 Riddick, J. A., 4, 7, 68 Riddiford, L. M., 153, 164 Riegelman, S., 348, 388 Rinderknecht, H., 212, 226 Ringertz, N., 246, 298 Robbins, K. C., 261, 277, 279, 299 Robert, L., 279, 283, 302 Roberts, E., 245, 248, 263, 300 Roberts, G. L., 32, 68 Roberts, G. L., Jr., 341, 381, 389 Robertson, T. B., 9, 67 Robinson, G. W., 307, 388 Robinson, R. A., 243, 251, 301 Roddy, W. T., 274, 901 R o d h , L., 212, 225 Rogers, L. B., 313, 381, 384 Rohrbach, R., 253, $03 Roller, D., 306, 386 Romano, D. V., 341, 389 R Romlet, F. C., 247, 298 Rook, A. J., 250, 308 Raacke, I. D., 170, 206 Roos, P., 170, 205 Raacke, I. E., 136, 163 Rosenbaum, E. d., 306, 386 Rabinowitch, E., 309, $87 Rosenberg, A. J., 308, 388 Ramachandran, G. N., 249, 501 Rosenheck, K., 313, 317, 329, 330, 331, Ramachandran, L. K., 316, 319, 388 333, 373, 380, 388 Ramasarma, G. B., 245, 248, 263, 300 Rosenthal, T. B., 245, 248, 258, 263, 271, Randall, J. T., 311, 383 273, 277, 289, 299, 300 Rands, D. G., 32, 45, 68, 92,94, 102, 103, 108, 120, 155, 164, 341, 346, 359, 365, Rossman, M. G., 344, 388 Roox, M., 231, 232, 249, 251, 299 370,371, 389 Rudloff, V., 170, 193, 194, 205 Rasmussen, H., 218, 226 Ruthven, C. R. Y . , 209, 211, 225 Rasper, J., 124, 126, 163 Ryle, A. P., 52,67,170,206 Reardon, G. V., 254, 302 Reddi, K. K., 309, 388 S Redfield, R. R., 172, 205 Reed, R., 248, 249, 250, 267, 282, 289, 298, Sachar, L., 279, 301 Sadek, H., 14, 67 239, 302 Sage, H., 341, 342, 345,346, 388 Rees, E. I)., 3, 8, 9, 48, 51, 52, 60, 62, 67 Sage, H. J., 23, 25, 32, 40, 44, 45, 46, 51, Reinhard, M., 346, 389 60, 67,82,155,164 Reisman, J., 10, 66
Perutz, M. F., 344, 388 Peters, T., Jr., 316, 319, 324, 388 Petersen, R. C., 7 Peterson, I). L., 313,327,328,329,388 Peterson, E. W., 316, 390 Pettersson, G., 221, 225 Phillips, D. C., 23, 26, 33, 35, 67, 123, 149, 163, 344, 386 Phillips, J. P., 308, 388 Phillips, P. H., 274, 308 Philpott, D. E., 259, 286, 297 Pierce, J. A., 275, 301 Pierce, J. V., 218, 285 Piez, K. A., 145, 163, 378, 388 Pimentel, G. C., 18, 67 Platt, J. R., 307, 326, 385, 387 Polson, A , , 211, 285 PontBn, J., 212, 225 Porath, J., 210,211,212,215,216,219,221, 223, 224, 225, 226 Postma, J. C. W., 7 PrBaux, G., 221, 226 Preiss, J. W., 326, 387 Pressman, D., 62, 67 Price, V. E., 387 Proskauer, E. S., 4, 7, 68 Prusfk, Z., 169, 170, $06 Psychoyos, S., 341, 389
AUTHOR INDEX
Saidel, 1,. J., 308, 322, 325, 326, 327, 340, 341, 380, 385, 388 Salak, J., 90, 164 Snlmar, W., 64, 66 Salmore, W., 312, 385 Salvini, L., 242, 302 Samuel, P., 279, 283, 301 Samulsrson, G., 218,226 Sanger, F., 52, 67, 168, 170,205, 206, 249, 279, 280, 281, 301 Ssroff, H. A., 130, 146, 164 Sawada, F., 170, 805 Saxl, H., 231, 248, 298, 301 Sbarra, A. J., 278, 279, 302 Scapinelli, R., 275, 298 Scarselli, V., 280, 301 Scatchard, G., 20,78,98,129,130,157,164 Schally, A., 221, 226 Schauenstein, C., 309, 341, 388 Scheflan, L., 4, 67 Scheinberg, I. H., 98, 130, 164 Schellrnan, J. A., 17, 28, 31, 37, 51, 63, 66,67,68,365,367,377,384,385 Scheraga, H., 33, 65, 67, 81, 82, 118, 120, 139, 143, 144, 147, 148, 149, 153, 155, 161, 162, 163, 164, 304, 309, 317, 318, 325, 340, 342, 345, 347, 348, 349, 351, 352, 353, 354, 355, 359, 365, 366, 367, 368, 370, 384, 385, 386, 387, 388 Schildkraut, C., 370, 384 Schleuter, R. J., 308, 380, 387 Schmid, K., 379, 383, 384 Schmidt, W. R., 137, 165 Schmir, G. L., 315, 318, 388 Schmitt, A., 380, 388 Schmitt, E. E., 37, 66 Schneider, J., 257, 301 Schneider, R., 283, 302 Schnek, G., 341, 355, 375, 376, 378, 382, 386 Schramm, G., 170, 190, 206 Schroeder, W. A., 170, 193, 206 Schuler, J., 267, 298 Schwarte, D., 176, 206 Schwarte, H. C., 193, 206 Schwert, G. W., Jr., 308, 341, 346, 348, 380,385, 388 Scott, D. B., 133, 163 Scott, J. F., 308, 311, 388, 389 Scribner, B. F., 306, 386
401
Sears, P. G., 7 Sehon, A. H., 210, 225 Seibert, G., 380, 388 Sela, M., 39, 51, 67, 68, 74, 163, 337, 348, 365, 377, 386, 387, 388 Serafini-Fracassini, A., 240, 241, 261, 299 Setlow, R., 326, 387 Sevag, M. G., 269, 301 Seeuku, I., 59, 67 Shainoff, J. B., 63, 68 Shakespeare, N . E., 170, 206 Shaw, D. C., 249, 379, 281, 901 Sheldon, H., 243, 251, 301 Shelton, J. B., 170, 193, 206 Shelton, J. R., 170, 193, 206 Shen, A. L., 98, 130, 157, 164 Shepherd, R. G., 170,206,344,388 Shibaeva, S. M., 269, 301 Shibata, K., 309, 388 Shifrin, S., 318, 389 Shimanouchi, T., 17, 67 Shooter, E. M., 206 Shore, V. C., 23, 26, 33, 35, 67, 85, 105, 120, 123, 149, 156, 157, 163, 164, 344, 386 Shore, W. S., 13, 68 Shugar, D., 32, 68,154,164,341,342,345, 346, 348, 365, 369, 371, 377,381, 389 Sicher, N., 279, 301 Sidman, J., 307, 318, 389 Sidwell, A. E., Jr., 308, 386 Simmons, N. S., 31, 68, 330, 389 Simpson, W. T., 313, 326, 327, 328, 329, 330,342,383,386, 388,389 Singer, M., 229, 302 Singer, S. J., 3, 8, 9, 12, 23, 25, 32, 40, 44, 45,46,48, 51,52,53,54, 55,60,62,63, 66, 67, 68, 82, 155, 164, 341, 342, 345, 346, 388 Singhadeja, S., 313,381, 384 Sinsheimer, R. L., 311, 389 Sieer, I. W., 316, 389 Sjoquist, J., 170, 206 Sjostrand, F. S., 58,68 Skrube, H., 212, 226 Slack, H. G. B., 242, 301, 302 Smillie, L. B., 160, 164, 342, 348, 370,389 Smith, A. V., 64, 68 Smith, D. B., 205 Smith, E., 170, 206
402
AUTHOR INDEX
Smith, E. L., 153, 168, 170, 193, 806, 324, Stretton, A. 0. W., 193, 806 342, 349, 373, 374, 378, 379, 386, 380 Strong, L. E., 64,66 Smith, F., 80 Sturtevant, J. M., 51, 52, 54, 61, 66, 68 Smith, L. F.,170,806 Susi, H.,81, f64 Smithies, O.,209, 224, 886 Sussman, R.,320, 326, 880 Snell, N. S., 134, 163 Suzuki, S.,361, 388 Sonenberg, J., 110,16.8 Sutherland, G. B. B. M., 81,168 Sonenberg, M.,349, 383 Swallen, L. C., 9, 68 Splrensen, M.,258, 308 Swanson, S. A., 13, 32, 68, 85, 105, 108, Splrensen, S. P. L., 74, 164 120, 156, 157, 158, 164,341,389 Sorm, F.,169, 170,172,173,174,175, 176, Swenson, R. T., 193,806 180, 182, 184, 186, 187, 188, 190, 192, Synge, R. L. M.,209, 221,886 194,196, 198, 202, 806,807 Szabo, D.,249, 808 Sorokin, V. M.,289,300 Szekely, J., 271, 800 Souter, F.,380, 389 Szent-Gyorgyi, A., 28, 29, 30, 31, 66,68, Spach, G.,26,30, 67 330,380 Spahr, P.,378,384 Szeredy, I., 274, 300 Speyer, J. F.,204,806 Spies, J. R., 381, 389 T Spiro, R.G., 138, 164 Taggart, V. G., 31,41,42,54,68,116,164 Spitnik, P.,10, 66 Takagi, T., 880 Spitzy, H.,212, 286 Takenaka, Y.,341,346,348,380,388 Spurr, 0.K.,263, 301 Talalay, P.,378, 386 Stadtman, E.R.,325, 380 Tan, M.,216,886 Stahmann, M.A., 321, 378, 386,390 Tan, W.,132, 166 Stanley, W.M.,170,807 Tanaka, J., 313, 328, 340, 341, 373, 386 Stark, G. R., 33, 68 Tanenbaum, S. W.,215,886 Staub, A., 170,806 Tanford, C.,12, 13, 31, 32,41,42,44, 45, Steele, 8.D., 306, 886 47,51,54,63,68,70,82,85,92,94,101, Stein, W.A., 170,806 102, 103, 104, 105, 106, 108, 109, 116, Stein, W. H., 33, 66, 68, 157, 16.4, 264, 117, 118, 120, 121, 122, 123, 127, 130, 291, 301 131, 142, 143, 144, 145, 146, 147, 148, Steinberg, D.,168, 169, 172,807 149, 156, 156, 157, 168, 163, 264, 314, Steinberg, I. Z., 39, 68 341, 344, 345, 346, 359, 365, 370, 371, Steinhardt, J., 70, 92, 93, 140, 161,164 377, 381, 389 Stelos, P., 12,16 Tanzer, P., 229, 3OB Stenstrom, W., 346, $89 Tappel, A. L., 278, 301 Stepanov, V., 215, 886 Tattersall, R. N.,249, 250, 308 Stern, P.M.,262, 265, 266,899 Tauber, S.,280,281,283, 300 Stetten, D.,Jr., 309,887 Taylor, H.L., 64, 66 Stickney, M.E., 308, 386 Teale, F. W.J., 315, 317, 318, 389 Stitt, F.,81, 139, 268 Teller, E.,255, 898 Stockell, A., 378, 389 Tews, B.,232, 898 Stolkowski, J., 308,888 Theorell, H., 137, 138, 151, 162, 164 StorgBrds, T., 209, 886 Thomas, E. W. P., 250, 308 Stracher, A., 342, 349, 380 Thomas, J., 249, 277, 278, 292, 294, 296, Stracker, A., 161, 16.4 SO8 Strait, L. A., 348, 388 Strandberg, B. E., 23,26,33, 36,67,123, Thompson, E. 0. P., 280,301,315, 889 Timasheff, S. N.,59,68,81,I64 149,f63, 170, 193,806,344,986 Timmons, M.M.,885 Strehm, J., 28,31,68
AUTHOR INDEX
Tinoco, I., 329, 330, 342, 389 Tiselius, A.,24, 68, 209,226 Tolnay, P.,283,309 TomGek, V., 169, 170,172, 174, 184, 190, 198,206,207 Tombs, M.P.,380, 389 Toops, E.E.,Jr., 4, 7, 68 Toth, M.,282,999 Townend, R.,59, 68 Tramer, Z., 342, 348, 369,389 Treloar, L. R. G., 296, 302 Ts’o, P. 0.P., 55,66,67 Tsuboi, M.,17,67 Tsubomura, H.,307, 313, 360, 389 Tsuda, Y.,17, 67 Tsuqita, A.,170,203, 204, 207 Tunbridge, R.E.,248, 249,250,267,282, 289, 298, 299, 302 Tuppy, H., 137, 138, 163, 193, 206 Turnbull, J. H.,64,66, 312,386
U Uhlig, H., 170, 190,806 Ulfendahl, H.R., 212, 226 Unger, G.,341, 389 Ungnade, H.E.,309, 389 Urquhart, J. M.,324, 386 Urnes, P.J., 26, 28, 68 Usuku, H.,239, 302
V v. Hofsten, B., 224, 226 Val, J., 244, 298 Vallee, B. L.,89, 162, 321,322, 381, 386 van Dam, A. F., 212, 226 van der Scheer, J., 344, 388 VanBEek, J., 169, 170, 172, 174, 184, 190, 198,807 Vaughan, M., 168, 169, 172,207 Vial, J. D., 230, 298 Vincent, J. M.,26, 30, 67 Vinograd, J. R.,64, 66 Viola, S.,242, 302 Virag, S.,271, 299 Vodmazka, Z.,90, 142, 164 von Hippel, P.H., 38, 39, 66 Vratsanos, S. M.,51, 64,68
W Wada, A., 329, 340, 384, 389 Waddell, W. J., 380,389
403
Wagner, M. L., 32,68, 104, 105, 147,148, 149, 164, 341, 344, 345,389 Wake, R. G., 348,586 Waldman, S.,322, 325, 326, 388 Walford, R. L.,283,302 WallBn, P., 170,206 Walsh, A. D., 319, 389 Walsh, K.A., 170,207 Wang, C.-C., 387 Wang, H.,278, 302 Wang, S-F.,378, 386 Ward, A. G.,139, 163 Ward, W. H.,267, 309 Warga, M.,306, 386 Warner, R., 342, 343, 390 Warner, R.C., 115,120,133,134,166,343, 381,389 Watson, H. C . , 23,26,33,35,67, 123,149, 165, 170, 193, 194,906, 207, 344, 386 Watts-Tobin, R. J., 204, 906 Weare, J. H., 36, 66 Weber, E.,170, 190, 906 Weber, H., 124,166 Weber, I., 115,120,133,134,166,305,315, 317, 318, 342, 343, 389, 390 Weber, R. E., 42, 44,45, 51, 68 Weberling, R.,347,383 Webster, M.E.,218,226 Weigert, C.,229, 302 Weir, C.E., 278, 809 Weisiger, J. R., 324, 344, 389 Weiss, J., 230,302 Weiss, R. H., 7 Weissberger, A.,4, 7, 68 Weissmann, G., 246, 50.2 Weissmann, S.,246, SO2 Weitnauer, H., 253, 302 West, W., 308, 389, 390 Wetlaufer, D.B., 31, 34, 68, 74, 80, 81, 163, 166, 315, 318, 330, 335, 336, 340, 348, 353, 354, 355, 358, 359, 377, 378, 387, 389, 390 Whetzel, K. B., 353, 386 White, A.,315, 318, 390 White, W. F., 170,207 Whitfeld, P.R.,62,66 Widom, J. M.,347, 348, 354, 387 Wiederhorn, N.M.,254, 302 Wiener, E.,137, 163 Wilcox, P.E.,86, 118, 131, 132, 143, 162, 166, 215, 226, 341, 390
404
AUTHOR INDEX
Wilhoit, E. D., 7 Will, G., 344, 388 Willeford, B. R., Jr., 324, 386 Williams, A. P., 248, 302 Williams, D. E., 277, 278, 281, 300 Williams, E. J., 348, 349, 356, 357, 390 Williams, J., 172, 173, 174, 198, 201, 207 Wilson, G. D., 274, 302 Wilson, S. D., 170, 206 Windrum, G. M., 229, 3Q2 Windus, W., 282, 298 Winter, K. K., 279, 301 Wishnia, A., 115, 120, 133, 134, 166, 342, 343, 390 With, T., 291, 302 Witkop, B., 315, 316, 318, 319,388, 390 Wittman, G., 212, 226 Wittmann-Liebold, B., 170, 180, 183, 194, 206 Wohlisch, E., 253, 302 Wolfe, R. G., 351, 388 Wolpers, C., 236, 237, 30.2 Wood, G. C., 289, 302 Wood, H. N., 62, 66 Wood, M. J., 248, 298 Wood, S. E., 20 Wootton, J. F., 325, 349, 390 Wu, F. C., 283, 302
Wyman, J., 70, 72, 74, 120, 162, 314, 384 Wyman, J., Jr., 139, 140, f62, 166
Y Yanari, S., 34, 68, 315, 317, 318, 349, 350, 354, 357, 358, ,990 Yanari, S. S., 314, 383 Yang, J. T., 5, 10, 26, 31, 36, 40, 63, 66, 67, 68, 108, 159,166,329,331,384,990 Yasunobu, K. T., 316, 317,380 Y6as, M., 169,172,201,204,206,207 Yokota, H., 239, 309 Young, I. G., 351, 386 Young, J., 170, 207 Youngson, M. A., 221, 226 Youse, E., 309, 389
z Zahn, H., 52, 68 Zaiser, E. M., 70, 92 140, 164 Zein-Eldin, Z. P., 382, 390 Zell, T., 81, 164 Zherebtsov, L. D., 269, 901 Ziegler, J., 378, 386 Zimm, B. H., 30, 68 Zipper, H., 250, 301 Zschiele, F. P., Jr., 308, 386 Zwaan, J., 212, 926
Subject Index A
C
Absorption processes, terms used in, 306307 Acetic acid, properties of, 6 as protein solvent, 8 Acetonitrile, properties of,7 Acetyl imidazole, absorption spectrum of, 325 Alanine , poly-, absorption spectrum of, 330 tetra-, p K of, 73 Amino acids, interchanges in peptide sequences, 176 pK’s of, 73 UV spectra of, 303-390 Amino groups, heat of dissociation of, 120 protein, titration of, 112, 113 Ammonia, properties of, 6 Amphiprotism, 4 Angiotensin, spectrophotometric titration of, 342 Antigen-antibody systems, nonaqueous solvents in study of, 62-63 Arginine, absorption spectrum of, 326 benzoyl-L-, ethyl ester, solution behavior of, 61 pK of, 73 Arteries, calcification of, 245-247 elastic tissue of, 233-236 elastin content of, related t o age, 244 Asparagine, absorption spectrum of, 325
Carbonic anhydrase, absorptivity of, 378 Carboxy groups, heat of dissociation of, 120 protein, titration of, 112, 115 Carboxypeptidase, absorptivity of, 370 titrat,ion curve of, 89 or-Casein, gel filtration of peptides of, 218, 220 Cellulase, gel filtration of, 221-223 2-Chloroethane, helical contents of proteins in, 30 polypeptide behavior in, 37, 38 properties of, 6, 10 protein behavior in, 3, 40, 41, 44 Chromatium, heme protein structure studies on, 184-185, 194, 198 Chymotrypsin, absorption spectra of, 325 difference spectra of, 348, 349 spectrophotometric titration of, 341, 342 titration of, 133 Chymotrypsinogen, absorption spectrum of, 341, 378 difference spectra of, 348 phenolic groups of, 85, 86, 90, 110-111 pK’s of groups in, 112, 113, 118 spectrophotometric titration of, 342 structure of, 170, 173 titration of, 93, 95, 131-133 volume change in, 126 Chpeine Z, structure of, 170 Collagen, amino acids of, 248 compared t o elastin, 228, 247-251 denaturation changes in, 249 fibrils, titration of, 133 Collagenase, action of, 250 Conalbumin, difference spectra of, 350
B Benzene, unitary free energy change of, 20 N-Bromosuccinimide, use i n protein studies, 318-319
405
400
SUBJECT INDEX
pK’s of groups in, 112, 113 N, N-Dimethylformamide, spectrophotometric titration of, 342, properties of, 7 343 as protein solvent, 3, 41, 43 titration of, 95, 133-135 Dimethylsulfoxide, Connective tissues, components of, 228as nucleic acid solvent, 55 229 properties of, 7 a-Corticotropin, Dioxane, pK’s of groups in, 112, 113 properties of, 7, 18 structure studies of, 170, 190 as protein solvent, 42, 56, 62-63 peptide sequences in, 191 titration of, 95, 102, 104, 105,106, 135E 137 “Elacin,” 250 Cotton effect, 31 El ast ases , m-Cresol, properties of, 6 action of, 280-284 Cysteic acid, absorption spectrum of, assay of, 279-280 325 biological inhibitors of, 283 Cysteine, effect on collagen, 249 absorption spectra of, 319-321 effect on elastic fibers, 238 oxidized, absorption spectra of, 321effect on epidermal cells, 282 323 occurrence of, 276-280 pK of, 73 properties of, 280-284 spectrophotometric titration of, 337specificity of, 283-284 339, 391 Elastic cartilage, 236 Cystine, Elastic tissue, absorption spectra of, 319-321 age changes in, 242, 243, 251, 275 oxidized, absorption spectra of, 321of arteries and veins, 233-236 323 degenerated, 231-232 Cytochrome c, electron microscopy of, 237-242 structure studies on, 170, 178, 193 structure of, 232-236 compared to other heme proteins, submicroscopic structure of, 236-242 195 Elastin, 227-302 of peptides, 186 a- and &proteins of, 286-290 titration of, 137-138 amino acids of, 248, 265 D age variation of, 245 in arteries and veins, 233 Denaturation, see also Proteins association with lipids and lipoprodefinition of, 86 Deoxyribonucleic acid, teins, 270-272 association with mucopolysaccharides, solubility of, 9 solution behavior of, 53-55 267-270 chemical properties, 228 Deuterium exchange, in protein study, chemical structure of, 284-297 34-35 chromophoric nucleus of, 294-296 Dextrans, cross-linked, as molecular comparison with collagen, 228,247-251 sieves, 209-226 determination in tissues, 272-276 N , N ’-Diacetylhexamethylenediamine, from different tissues, 262-267 absorption spectrum of, 327 elasticity of, 251-254 Dichloroacetic acid, properties of, 6 “enzyme,” 261 fluorescence of, 290-292 as protein solvent, 5, 41 histochemistry of, 229-232 N,N-Dimethylacetamide, properties of, hydration of, 254-256 7
407
SUBJECT INDEX
hydrolysis of, relation t o properties of, 261 isolation of, 257-276 acid and base extraction of, 258-260 neutral extraction of, 257-258 metabolic turnover of, 242-243 microscopy of, 229-232 occurrence of, 228-229 peptides from hydrolysis of, 292-297 physical properties of, 251-256 properties, relation t o isolation procedure, 260-262 solubility of, 254-256, 286-290 UV absorption of, 290-297 “Elastoblast,” 247 Elastolytic enzymes, 276-284 Electrometric titration, of proteins, 7678 Enzymes, behavior of, in nonaqueous solvents, 61-62 Ethanol, properties of, 6 as protein solvent, 41 Ethylene glycol, properties of, 6 as protein solvent, 44-47, 56 Ethylenediamine, properties of, 6 as protein solvent, 8 Exciton theory, 307
F Ferriheme water group, heat of dissociation of, 120 protein, pK’s of, 114 Fetuin, titration of, 138 Fibrin, peptide sequences in, 188 solution behavior of, 4 titration of, 139 Fibrinogen, spectrophotometric titration of, 341, 342 titration of, 138-139 Fibrinopeptides, structure of, 170 Fibroin, solution behavior of, 40, 48 spectrophotometric titration of, 341 Formaldehyde, use in proteins studies, 319
Formamide, as nucleic acid solvent, 55 properties of, 7 as protein solvent, 3, 47, 56 Formic acid, properties of, 6 as protein solvent, 13 G
Gel filtration, 224 in mixed solvents, 221 of protein, 216-218 Gelatin, liquefying reagents for, 256 thiolated, -SH group study on, 339 spectrophotometric titration of, 341 titration of, 139 Gliadin, solubility of, 9 Globin H , helical content of, 30 Globulin, Y-1
solution behavior of, 48, 52 spectrophotometric titration of, 341 gel filtration of, 221-222 Glucagon, structure of, 170 Glutamate, poly-y-benzyl-L-, solution properties of, 35-36 Glutamic acid, p K of, 73 POlY-, absorption spectrum of, 328-329,340 spectrophotometric titration of, 341 Glutamic dehydrogenase, quaternary structure in solution, 59 Glycerol, absorption spectrum of, 325 Guanidyl group, protein, titration of, 113
H Helixes, in proteins in solution, 30, 35, 40, 43 Heme peptide, structure of, 170 Hemoglobin(s), absorption spectra of, 312 ferri-, isoionic point of, 130 titration of, 92, 93, 139 fetal, 193 in molecular sieve studies on, 213 pK’s of groups in, 112,113
408
SUBJECT INDEX
quaternary structure in, 59 sickle cell, 61 spectrophotometric titration of, 314, 342 structure studies of, 170, 173, 175, 181182, 184-185 comparison t o myoglobin, 194, 197 pathological substitutions and, 193, 203 of peptide, 181-182,186,192-193,199, 200, 201, 203 titration of, 95, 139-142 volume change in, 126 Henry’s law, 19-20 Histidine, absorption spectra of, 325 poly-, p K of, 73 Hist one, gel filtration of, 224 helical content of, 30 Hydrazine, properties of, 6 as protein solvent, 8, 13 Hydrofluoric acid, properties of, 6 as protein solvent, 5, 8 Hydrogen bonding, in protein solution, 16-18 Hydrogen ion curves of proteins, 69-165 p - Hydroxybutyric apodehydrogenase, extraction of, 59 Hydroxylapatite, in atherosclerotic plaques, 246-247 Hydroxyproline, absorption spectrum of, 328 Hypertensin, structure studies on, 190
I Imidazole group, heat of dissociation of, 120 protein, titration of, 112, 114-115 Infrared absorption, in protein titration, 81 Insulin, aggregation of, 109 difference spectra of, 348, 349 helical content of, 30, 333 pK’s of groups in, 112, 113 quaternary structure in, 59 solubility of, 9
solution behavior of, 47, 60 spectrophotometric titration of, 341 , 342, 381 structure of, 170, 180, 181, 183 titration of, 95, 142-144 zinc binding in, 90 Isoelectric solution, definition of, 78 Isoionic point, of proteins, 128-130 Isoionic solutions, definition of, 78 Isoleucine, absorption spectrum of, 325
K Keratin, swelling of, 256 Ketosteroid isomerase, absorptivity of, 378
L a-Lactalbumin, absorptivity of, 378 &Lactoglobulin, absorptivity of, 378 helical content of, 333 isoionic point of, 130 pK’s of groups in, 112, 113, 11&117 quaternary structure in, 59 solution behavior of, 4142, 47, 48 spectrophotometric titration of, 342 titration of, 77, 79, 87, 90, 94, 95, 96, 102, 103, 144-147 tyrosines in, 32 Ligamentum nuchae, elastin from, 228, 229 fiber structure of, 232-233 Light scattering, in protein systems, 309 Linderstrpim-Lang equation, 95-99, 117118 deviations from, 106-111, 121 Locust bean gum, cross-Iinked, as moiecular sieve, 211 Lyophobic interactions, in protein solutions, 18-22 Lysine, poly-, absorption spectrum of, 329, 330 spectrophotometric titration of, 341 Lysozyme, absorptivity of, 378 difference spectra of, 349, 359, 367, 375-376 electrostatic interaction in, 110 helical content of, 30
409
SUBJECT INDEX
isoionic point of, 79 pK’s of groups in, 112, 113, 117, 118 spectrophotometric titration of, 341, 342, 343 structure of, 170 titration of, 90, 95, 104, 105, 147-149 volume change in, 124, 126 tyrosine in, 32
M Magnetic susceptibility, use in protein titration, 81 Melanophore-stimulating hormones, gel filtration of, 221 structure of, 170 Methanol, properties of, 6 Methionine, absorption spectra of, 319-321 oxidized, absorption spectra of, 321323 poly-L-, solution behavior, 36-37 Methionine sulfone, absorption spectrum of, 323 N-Methylacetamide, hydrogen-bonded dimers of, 16 properties of, 7 N-Methylpropionamide, as protein solvent, 41 Methylthiazoline, absorption spectra of, 323-324 Molecular sieves, 209-226 Mucopolysaccharides, association with elastin, 267-270 Myoglobin, helical content of, 28, 333 spectrophotometric titration of, 342, 343 structure of, 23, 170, 177, 179, 193 compared t o hemoglobin, 194-197 peptide sequences of, 187 titration of, 81, 95, 112, 113, 123, 149151 Myosin, difference spectra of, 349 para-, 153-154 spectrophotometric titration of, 342 titration of, 151 Myristamide, absorption spectra of, 327328
N Net proton charge, definition of, 78 Nitrobenzene, properties of, 7 p-Nitrophenylacetate, in protein titration, 81 Nucleic acids, in nonaqueous solvents, 53 0
Orcein, as elastin stain, 230-231 Ovalbumin, difference spectra of, 350 helical content of, 30 pK’s of groups in, 112, 113 spectrophotometric titration of, 340, 341 titration of, 95 Oxyhemocyanin, absorption spectra of, 325 Oxytocin, gel filtration of, 221
P Pancreatic enzymes, gel filtration of, 223-224 Papain, absorptivity of, 378 pK’s of groups in, 112, 113 spectrophotometric titration of, 342 structure of, 170 titration of, 153 Paramyosin, helical content of, 333 optical rotatory dispersion of, 29 titration of, 153-154 Pepsin, absorption spectra of, 311 difference spectra of, 348 helical content of, 30 spectral solvent perturbation studies on, 373 titration of, 95, 154 volume change in, 126 Pepsinogen, structure of, 170 Peptide bond, absorption spectroscopy of, 326-334 Peptides, hydrogen bonding in, 26 separation by molecular sieving, 216224 Peroxidase, titration of, 154
410
SUBJECT INDEX
pH-stat, 70, 88 Phenol, properties of, 6 Phenolic groups, heat of dissociation of, 120 protein, titration of, 112, 114, 115 spectrophotometric titration of, 80 Phenylalanine, absorption spectrum of, 310, 314, 317 Phosphatase, gel filtration of, 224 Plasma, gel filtration of, 222 Plasma albumin, difference spectra of, 348, 356 Poly-y-methyl-L-glutamate, absorption spectrum of, 330, 331 Polypeptide, infrared spectra of, 27 in nonaqueous solvents, 36-40 optical rotatory dispersion of, 29 Proline, poly-L-, solution behavior, 3839 1,a-Propanediamine, properties of, 6 L ,2-Propanediol, properties of, 6 1-Propanol, properties of, 6 as protein solvent, 4 %-Propanof,properties of, 6 Propylene carbonate, properties of, 7 Propylenediamine, as protein solvent, 8 Protein(s), aggregation of, 109 binding of ions by, 127 counting of titratable groups in, 82-90 denaturation of, 86, 367-374 affects on titration of, 107-109 desalting, by molecular sieves, 212 determination by peptide-bond absorptivity, 380 homogeneity of, 168-169 hydrogen-bonding studies on, 359-362 hydrogen ion curves of, 09-165 isoionic point of, 128-130 quaternary structure in solutions of, 59-61 lyophobic regions in, 31 in nonaqueous solvents, 1-68 chemical modification of, 64 conformational changes in, 10-59 at low temperatures, 63-64 optical rotatory dispersion of, 28
peptide sequences of, 172173 photo-oxidation of, 90 “salt bridges” in, 118-119 solid-state spectrophotometry of, 313 separation by molecular sieving, 216224 solubility, in nonaqueous solvents, 510 solvents, acid-base properties of, 4-5 classificetion of, 4 requirements of, 2-3 spectral perturbation of, 354-359 spectral solvent-perturbation studies on, 370-373 structure, evolutionary aspects of, 201 mathematical studies on, 198-201 protein synthesis and, 201-205 regularities of, 171-191 theory of, 169-171 titratable groups in, 71-72 titration of, volume changes in, 124126 titration curves of, electrostatic interaction factor in, 101-111 factors affecting, 105-111 intrinsic pK’s and, 112-113 time-dependence of, 90-91 UV spectra of, 303-390 Pseudoxanthoma elasticum, 250 Pyridine, properties of, 6
R Renin, spectral transformations in, 380381 Reticular fibers, microscopy of, 229 Ribonuclease, absorptivity of, 378 gel filtration of, 224 difference spectra studies on, 348, 349, 350, 362 helical content, 30,333 peptides, gel filtration of, 220-221 pK’s of groups in, 112, 113 solution behavior of, 40, 44-46, 51-52, 60
411
SUBJECT INDEX
spectral solvent perturbation studies on, 372, 377 spectrophotometric titrations of, 341, 342, 345, 381 structure studies on, 170, 173, 176, 177 peptide sequences of, 187, 189 titration of, 92,94,95,102,103,154-156 volume changes in, 124, 126 tyrosines in, effect on structure, 32
S Sephadex, structure of, 210 Serum, molecular sieve studies on, 216 proteins, gel filtration of, 221 Serum albumin, absorption spectra studies on, 324-325, 381 difference spectra of, 348, 349, 350, 357 S - (p - Dimethylaminobenzenenazophenylmercuric) derivative of, 119 galactosyl-, purification of, 215 gel filtration of, 223-224 heat of dissociation of, 85 isoionic point of, 130 pK’s of groups in, 112, 113, 117 solubility of, 10 solution behavior of, 48, 52 solvent perturbation studies on, 372 spectrophotometric titration of, 341, 342 titration of, 102, 105, 108-109, 156-160 volume change in, 124, 126 tyrosines in, 32 Serum niercaptalbumin, absorptivity of, 378 Spectrophotometric titration, 334-346 of phenolic groups, 80 Spectrophotometry, absorptivity errors in, 308-309 differential, 312-313, 347-375 fluorescence errors in, 309 instruments for, 307-308 Sulfur dioxide, properties of, 6 as protein solvent, 8, 10
T Tetrabutylammonium picrate, ion-pairing and, 14 Thiaaolines, absorption spectrum of, 323-324 Thioglycolic acid, spectral measurements of, 337 Thiol esters, absorption spectra of, 324 Thiols, spectrophotometric titration of, 337-339 Thionein, absorption spectrum of, 321, 323 Thyroglobulin, spectrophotometric titration of, 342 titration of, 168 Thyrotropin, gel filtration of, 223 Tobacco mosaic virus, difference spectra of, 348 peptide isolation from, 215 structure studies on, 170, 173, 182, 190 peptide sequences of, 192 Trifluoroacetic acid, polypeptide behavior in, 37 properties of, 6 as protein solvent, 5, 8 Tropomyosin, helical content of, 30 optical rotatory dispersion of, 29 Trypsin, absorption spectrum of, 330 difference spectra of, 348, 369 solubility of, 9 solution behavior of, 51, 52, 62 titration of, 161 Trypsinogen, difference spectra of, 348 spectrophotometric titrations of, 342 structure of, 170, 173 titration curve of, 160-161 Tryptophan, absorption spectra of, 310-311, 314, 317-319 difference spectra of, 352 poly-, absorption spectra of, 318 spectral analysis of in proteins, 378380 in spectroscopy of proteins, 80 Turnip yellow mosaic virus, structure of, 170
412
SUBJECT INDEX
Tyrosinase, in tyrosine spectrophotometric studies, 316-317 Tyrosine, absorption spectrum of, 310, 311, 314317, 352 of derivatives, 315-316 difference spectra of, 348 0-methyl-, difference spectra of, 353, 355 p K of, 73 role in protein structure, 32-34 spectral analysis of, in proteins, 378380 spectrophotometric titration of, 335337, 341 L-Tyrosine ethyl ester, HCl titration of, 23-25 UV spectral properties of, 45
U Ultraviolet spectra, of amino acids and proteins, 303-390 difference, use in protein titration, 81 Urea, as protein denaturant, 47
V Valine, absorption spectrum of, 325 Vasopressin, gel filtration of, 221 Veins, elastic tissue of, 233-236 Viscotoxin, gel filtration of, 218, 219 Vitamin Biz, in sea water, molecular sieves in study of, 214-215
z Zein, solubility of, 9 solution behavior of, 48 Zinc, binding site of, in proteins, 89-90
