Advances in Protein Chemistry: v. 20

ADVANCES I N PROTEIN CHEMISTRY Volume 20

CONTRIBUTORS TO THIS VOLUME M. Bettex-Galland W. G. Crewther

R. D.

B. Fraser

John J. Harding Robert 1. Hill

F. G. Lennox H. Lindley

E. F.

Luscher

ADVANCES IN PROTEIN CHEMISTRY EDITED BY

C. B. ANFINSEN, JR.

M. 1. ANSON

National Institute of Arthritis and Metabolic Diseases Bethesda, Marylond

New York, N e w York

J'OHN T. EDSALL

FREDERIC M. RICHARDS

Biological Laboratories

Deportment o f Molecular Biology and Biophysics

Horvord Univerrify

Yale University

Cambridge, Morrachureftr

N e w Hoven, Connecticut

VOLUME 20

1965

ACADEMIC PRESS

N e w York and london

COPYRICHTO 1965, B Y ACADEMIC PRESSINC. ALL RIGHTS RESERVED

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PRINTED IN THE UNITED STATES OF AMERICA

CONTRIBUTORS TO VOLUME 20 M. BETTEX-GALLAND, Theodor Kocher Institute, University of Berne, Berne, Switzerland W. G. CREWTHER, Division of Protein Chemistry, C.S.I.R.O. Wool Research Laboratories, Melbourne, Australia

R. D. B. FRASER, Division of Protein Chemistry, C.S.I.R.O. Wool Research Laboratories, Melbourne, Australia JOHNJ. HARDING, T h e Gelatine and Glue Research Association, Holloway, London, England

ROBERTL. HILL,Departm,ent of Biochemistry, D u k e University, Durham, ,Vorth Carolina F. G. LENNOX, Division of Protein Chemistry, C.S.I.R.O. Wool Research Laboratories, Melbourne, Australia

H. LINDLEY, Division of Protein Chemistry, C.S.I.R.O. Wool Research Laboratories, Melbourne, Australia

E. F. LUSCHER, Theodor Koche'r Institute, University of Berne, Berne, Switzerland

V

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PREFACE Kenneth Bailey, our former colleague, had many of the qualities of a Renaissance scholar transplanted to the machine age. His fruitful life of research and teaching was sparked by a true and selfless fascination with the understanding of natural phenomena and with the dissemination of knowledge to others. His contribution to the earlier volumes of this series was immense. Those of us who were privileged to know him will sorely miss his wry humor, his sound scholarship, and his great personal warmth. This volume of “Advances” opens with a tribute to him by one of his close friends, Professor S. V. Perry. The first article in this volume is concerned with the contractile protein from blood platelets known as thrombosthenin. This interesting niember of the actomyosin class of proteins appears to serve a central and critical role in the blood clotting process by forming networks of interlacing protein strands which can then undergo a n ATP-dependent contraction. The chapter written by M. Bettex-Galland and E. F. Luschcr presents a thorough sumiiiary of present-day knowledge of this system and points out arcas in which the study of thronibosthenin may be of unique value in the understanding of incchanisms involving contractile proteins in general. The hydrolysis of proteins by chemical and enzymatic methods is discussed by R. L. Hill in the second chapter. The chapter should serve as a standard source for information on the many operational techniques with which only the experienced specialist is likely to be familiar. Dr. Hill covers not only the partial hydrolysis of proteins by general and specific proccdures but also the total enzymatic hydrolysis of proteins for the purpose of amino acid analysis. In the tliircl article, .J. .J. Harding gives us a broad review of the unusual linkages and cross-linkages of collagen. Many of the kinds of bonds discussed are still of unknown importance in the structure of this protein and, indeed, the actual occurrence of some of them is still conjectural. The author has given a critical evaluation of the evidence in support of the various bonds that have been postulated, emphasizing the complexity of the problenis involved in unraveling the structure of collagen and its cross linkages. I n the final chapter W. G. Crcwtlicr, R. TI. B. Fraser, F. G. Lennox, :ind H. Lintllcy providc what w7‘c would consider very nearly a definitive vii

... Vlll

PREFACE

discussion of the cheinistry of the keratins, which should serve as a standard treatment of this field for many years. The authors describe the many chemical and physical studies that have recently begun to clarify the true chemical nature of this class of proteins. These studies reinforce the growing idea that, even for inaterials as complicated as hair and wool, macromolecular structure is generally describable in terms of structural subunits of moderate size. The editors are, as always, indebted to the staff of the Academic Prcss for their invaluable assistance in preparing the manuscripts for publication and for expediting the final production of the volume.

November, 1964

C. B. ANFINSEN,JR. M. L. ANSON J. T. EDSALL F. M. RICHARDS

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KENNETHBAILEY

KENNETH BAILEY 1 909-1 963

A Personal Tribute By

S. V. PERRY

Most of US can recall the single event which was to determine the pattern of our future career with sharpness and clarity. This was not so in my case when in 1946 I was first introduced to Kenneth Bailey, who at this time was rising to the height of his powers in what was a particularly favorable atiiiosphere for the developiiient of protcin chemistry in Cambridge. F. Sangcr, working a t the next bench to him in a sriiall basement laboratory in tlic Department of Biocheinistry, was laying the foundations of work eventually to merit the award of the Nobel Prize. The two junior occupants of the laboratory fortunate enough to share this environment were R. R. Porter and myself. Although at casual meeting Bailey’s personality and powers iriay not have been iiiiiiiediately evident to tlic undiscriminating, day-by-day contact quickly revealed his wide talents. These were clearly apprcciated by his colleagues who constantly sought his advice and treated i t with the greatest respcct. My strongest recollection of him in these early years of acquaintance was his skill a t cveryday procedures a t the bench-to see him determine the pH of a solution with an external indicator was almost an artistic experience in itself. Quite apart from matters of technique Bailey’s attitudc to research was more of that of the artist than is the case with most scientists. He was not interested in broad systematic investigations aided by cxtensive instrurncntation, characteristic of much modern work. His approach was intuitive, strengthened by an appreciation of the potential of the application of new techniques to an area of interest to him and vulnerable to his ideas. Essentially simple his cxperirncntation was aided little by the assistance of junior colleagues or large financial resources. Although practically all of his working life was spcnt in university departments he had rarely inorc than a single research student at any one time and in all only about half a dozen students werc fortunate enough to gain the P1i.D. degree under his supervision. Most of his best work was done without technical assistance and he only relied on it to any xi

xii

KENNETH BAILEY

extent during his last few years of research. So far as I am aware he rarely had a personal research grant and when he did it was never worth more than a hundred or so pounds a year. Thcse circumstances were largely of his own choosing, for Bailey clearly had the stature to attract substantial support. He was frequently offered senior posts which would have given him much greater personal resources if he had felt the need for them. Few biochemists can have made so much impact in the field of protein chemistry with so little resources. A son of the Potteries, Bailey was born in 1909 in a small village a few miles from the Five Towns immortalized in English literature by Arnold Bennett. I n this area which has had so much of its natural beauty seared by the excesses of the Industrial Revolution Bailey’s roots lay deep and here he returned to spend the last months of his life. As a schoolboy he was reserved but clearly of outstanding ability and won scholarships which took him to Birmingham University. I n 1927 very few university departments in Britain provided a degree course in biochemistry and to select such a course for university study in these early days of biochemical development required some initiative. Clearly during his school days Bailey had been attracted by a synthesis of the biological and chemical approach rather than the inore conventional courses of chemistry or biology then generally available. The curriculum a t Birmingham was then largcly orientcd toward the biochemistry of plants and fermentation and Bailey’s later introduction into research was in the field of carbohydrate chemistry under F. W. Norris and R. H. Hopkins. His early post-doctoral years were spent in London a t Imperial College with A. C. Chibnall and although for a while he continued with the work started in Birmingham his future interests were beginning to be molded by the active plant protein work going on around him a t Imperial College. Already Bailey’s talents wcre becoming evident from his early papers on carbohydrates and later on the plant proteins with Chibnall and collaborators. This was a world of protein purification and crystallization followed by careful chemical study and analysis by laborious chemical procedures. Now long outmoded by the post-war advances in techniques the habits and disciplines acquired a t this time made an iinportant contribution to Bailey’s later successes. Sensitive as he was to intellect and environment Bailey’s wider interests were nourished in London in a way that had never been possible in the Midlands. His interest in music was encouraged at Birniinghani and after teaching hirnsclf the piano without formal instruction he became a competent performer. Music became one of the passions of his life in which he was ablc to indulge deeply while at Imperial College.

S . V. PERRY

...

Xlll

At this time another important influence entered Bailey’s life. W. T. Astbury, then making his classical contributions to protein structure, frequently visited Chibnall’s group to discuss recent work on proteins. A life-long friendship born from mutual respect of ability, common origins in the Potteries, and a passion for music sprung up between these two scientists. Contact with Astbury not only stimulated Bailey’s thoughts but also influenced his day-to-day researches, and the foundation of much of the skill he acquired in handling proteins and inducing their crystallization was acquired at this time as a result of his efforts to grow large crystals of edestin for Astbury’s X-ray work. About this time he bccarne interested in denaturation, and in collaboration with A. C. Chibnall he confirmed experimentally the ideas of Astbury that seed globulins could be converted into fibrous denatured forms. This work was later taken up by Imperial Chemical Industries and led to the commercial production from arachin of a wool substitute known commercially as Ardil. This was an active period of experimental collaboration with Astbury which was renewed later just after World War I1 when Bailey stimulated Astbury’s interest in the properties of his then newly discovered tropomyosin B. Until a few years before Astbury’s death a fairly close association continued. It often took the form of meetings in Bailey’s rooms a t Trinity College, Cambridge, which developed into long discussions ranging from protein chemistry to music and lasted until the early hours of the morning. The talk was interspersed with duets on the piano and both activities were pursued with all the energy and enthusiasm characteristic of Astbury. Although he found them stimulating, I had the impression in later years that to Bailey these occasions were something of a physical ordeal and they became less frequent. Nevertheless the friendship of these two men was continued and was eloquently expressed in a tribute paid by Bailey at the Memorial Service held for Astbury in 1961, and later in his article on Astbury in Volume 17 of these “Advances.” It is probable that Bailey’s first interest in the muscle field, in which lies his greatest contribution, was aroused by the work of Astbury and Dickinson who showed that fibers of denatured myosin behaved in ways similar to keratin so far as their elastic properties were concerned and their structures wcre revcaled by X-ray analysis. At this time the Chibnall group was much interested in the amino acid composition of proteins. The obvious similarities in fibrous behavior between keratin and myosin despite their differences in amino acid composition, particularly in cystine content, stimulated Bailey to make a comparative study of the composition of some of the then recognized muscle proteins. This was Bailey’s first paper on muscle and extension of the

xiv

K E K N E T H BAILEY

ideas behind it led hiin to study the proteins of the electric organ of Torpedo wliicli has the sanie embryonic origins as iiiusclc itself. T o acquire the necessary experimental niatcrial lie had to work for soiiie tiiiie a t the Stazionc Zoologica, Naples, and thereby established a habit of regularly visiting Italy which was to stay with hiin for the rest of his life. He developed a great passion for that country and often thought of ultiniately retiring to live there. At the same timc contact with the world of marine biology opened up a biochemical treasure house from which, with his characteristic flair for appreciating what was worth doing, he was able to reap a rich reward. A period in the United States in 1939 as :L Rockefeller Fellow in tlic Harvard laboratory of E. .J. Colin widened his expcricncc of proteins and knowledge of their physical clicinistry and led to his first crystallization of a iiiuscle protein, a niyogen from rabbit niuscle which was later shown by other workers to be identical with triosephosphatc dchydrogenase. The war brought Bailey back to England and assignment to work of national importance a t Cambridge, wliere he was associatcd with the team directed by M. IXxon that was engaged in work on the enzymology of the action of war gases. However hc also found time for some further studies on muscle. Stiinulated by the work of Ljubimova and Engelliardt on the association of niyosin and adenosinctriphosphatase and by contact with the Needhams a t Cambridge he carried out a dctailcd cnzyrnological study of myosin. His investigations led hiin to suggest in 1942 that “the essential phase of excitation and contraction is the liheration of the Ca ion in the neighborhood of thc ATPase grouping.” This is an iiiiprcssivc anticipation of present day ideas although the precise ionic relationships are now considered to be soinewhat more subtle than could he anticipated from the knowledge available a t that time. After the war, Bailey returned to studies on muscle, and announced in 1946 the isolation from vcrtcbratc muscle of tropomyosin B, a new rnyofibrillar protein with unusual properties. This achievement and the later discovery of the fanlily of tropomyosins A are Bailey’s most important contributions to thc iiiusclc field. The latter protein was shown to be responsible for the paraniyosin X-ray diffraction pattern characteristic of molluscan adductor muscles and opened u p a new field of interest. The tropoiiiyosins possess a nurnber of remarkablc properties which are .just thosc which we would have expected to excite his int e r e s t o n c cannot help feeling that these proteins were almost custombuilt for him. Their function is still something of a mystcry but the

S. V. PERRY

XV

evidence is now accumulating to indicatc their fundamental role in inuscle activity. I n the collection of this evidence one of his former students tJ. C. Riiegg has played an important part. Naturally tlic interaction of actin and myosin was something to stir Bailey’s enquiring inind and as a raw research student suffering from dissociation from biochemistry by war service, I was put to investigate this problem. Here Bailey’s background and experience were of enormous help and it soon became apparent that the interaction was of a somewhat unique kind involving sulfhydryl groups. The idea that actin and adenosine triphosphatc might be alternative substrates for thc inyosin was a t that time a useful hypothesis which proimpted a search for a pyrophosphate group on the actin. A sinall amount was found but a t that time it was not thought to be large enough to be significant. Straub and collaborators were later to show t h a t this labile phosphate was due to the bound nucleotide of actin which was intimately associated with this protein and with the interconversion between its F and G forms. Although his contributions lie mainly in our knowledge of the inuscle proteins Bailey deserves much more credit for initiating the study of the relaxing factor than is generally appreciated, even by investigators with considerable experience in this subject. He had long becn struck by tlic fact that although it was very difficult to squeeze out juice froin freshly minced muscle, the texture of the mince changed on standing in some way, and the juice could readily be obtained. B. 8. Marsh, a New Zealandcr working a t the Low Temperature Research Station in Cambridge who had been assigned to Bailey as a research student, was given the problem of studying the underlying biochemistry of the changes taking place post rnortein in muscle minces. This work ultimately led to the discovery of a factor which controlled the voluine of cell fragments and inhibited tlic niyofibrillar adenosinctripliosphatasc. This was first known as the Marsh factor, but with a less generous supervisor of research than Bailey it could well have been the Bailey-Marsh factor. His normal rule was not to put his namc on a publication hy a research student unless he had made some direct contribution to the experimental work. The provision of ideas and dircction was in his view not enough to justify his name a t the head of a paper. Although from time to time Bailcy’s interests ranged outside the iiiuscle field he continued hiniself and with the aid of research students, such as T. C. Tsao and C. &I. Kay, to inake important physicochcmical studies on the niuscle proteins. His other major contribution lies in the mechanism of the foririation of fihrin from fihrinogcn. Hc was quick to appreciate the powrr of tlic

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KENNETH BAILEY

Sanger fluorodinitrobenzene method for dctermination of terminal amino groups, and naturally applied it to the muscle proteins and fibrinogen, which Astbury had previously classified together on the basis of the marked a-pattern that could be demonstrated by X-ray diffraction studies. Extension of the findings with fibrinogen led to his discovery with F. R. Jevons that fibrin formation involved the liberation of peptide material. This for the first time gave a clue to the mechanism of thrombin action. His interest in this field continued until his death and in collaboration with J. B. Clegg his last publication described a neat separation of the peptide chains of fibrin. At the same time his interests were ranging wider and he announced in 1961 with T. Weis-Fogh the isolation and properties of yet another new protein, resilin, which is responsible for the rubberlike properties of certain parts of the insect cuticle. A bare recital of scientific achievement does incomplete justice to Bailey. Undoubtedly one of the outstanding protein chemists of his time, he had personal qualities which endeared him to all who knew him and his talents reached far beyond the laboratory bench. A scnsitivc person, hc expressed himself during his leisure momcnts in sculpture, a t which he was rcinarkably able, and in painting. As a Director of Studies at Trinity Collcge he fired the interest of many students in biochemistry and his influence soon became felt in the number of able Trinity students who were entering the P a r t I1 Course in biochemistry. These students looked upon him not only as a teacher but as a counsellor and friend. He was acutely distressed by injustice in any form and indeed many students had reason to be grateful for his help and efforts on their behalf behind the scenes. The surest way for a student to arousc Bailey’s special interest was for him to have origins in the Potteries and show leanings to biochemistry. Despite frequent offers of professorships and positions of authority in various parts of the world Bailey preferred to stay in Cambridge and to maintain the freedom of action which he felt would he denicd to him in a post with greater administrative responsibility. H e remained an Assistant Director of Research in the Department of Biochemistry until 1961 when he was appointed University Reader in Biochemistry. His appointment carried no formal teaching deinands, but he organized and gave a tours(' of lectures and practical work on proteins to final year undergraduates. Few students have had as much good fortune as these to be introduced to protein chemistry with such sympathy and understanding. Bailey did, however, accept editorial responsibilities with the Biochemical Journal and there is no need to stress here that the protein literature is especially enriched by his later efforts. I well remember that

S . V. PERRY

xvii

in the 1950’s he rarely appeared without a inanuscript for editorial correction in liis hands. He had thc flair for doing this in odd moments betwen experinients in the laboratory and several doyens in the fields would hare visibly paled if they had a t these times heard his comments on their style. His own was impeccable, much admired and envied by all. Essentially somewhat introverted as a person he tended to retire from thc scientific world a t large and was not inclined to attend mcetings regularly. His reputation was solidly based on achievement as assessed by others; he never tried to draw public attention to what he had accomplished. H e did take, however, a normal pride in achievement and derived great pleasure froin his election as a Fellow of the Royal Society in 1953. Nevertheless, it was characteristic of the man that it was often the simple things that gave him greatest satisfaction. Although he never married or established a home of his own he loved gardening. This he practiced from time to time a t his father’s house near Newcastle under Lyme, but an appointment which gave him particular pleasure was nieiiibership of the Fellows’ Garden Coniinittee a t Trinity where his practical knowledge backed by early botanical training was especially valuable. Many a scientific visitor to Cambridge will reiiieniber wit11 pleasure an escorted tour of Trinity gardens and the pride he took in them. The last berm years of Bailey’s life were rnarrcd by rccurrent illness. After a complete breakdown in 1955 and five months absence from effcctive work he spent a period of convalescence working a t the Stazione Zoologica, Nal)lcs. I t wa5 then that he brilliantly isolated and crystallized troponiyosin A. This was his last inajor achieveiiient and the pattern of his life thence onwards consisted of troughs of depression and incapacitation interspersed with periods of scientific achievement. I n tlicsc working periods his nature lost some of its old spontaneity but his talents were still evident. Indccd his last literary effort was a brilliant tribute to W. T. Astbury in the “Advances” to which reference has already been made. I n the early suinnwr of 1962 Bailey’s Iicalth h(1gan to fail again antl he left Cambridge. After a long distressing period of illness which lasted a ycar he appeared to be regaining sonic of his health and confidcnce. He returned to Cambridge for a short stay in M a y 1963, but three days aftcr his arrival he took his own life. His tragic and baffling illness thus lirought liis brilliant career to an untimely end. His great achieveinents during the height of his activities, his broad learning, and his many-sided interests, and the inspiration antl encouragement that he gavc both to his contemporaries and his

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KENNETH BAILEY

junior collcagucs, will reiiiain as a living tribute to liis memory.

The ~)hotogr:q)liwhich faces the first page of this tribute was taken about 1954, when he was a visiting professor a t the University of Washington i n Seattle, and shows him as he was before his illness, a t the height of 111- ;~cliicvciiicntsin protein research.

CONTENTS 20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v

.....................................................

vii

KKNNICTH BAILEY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi

CoS'I'ItII$li'I'OHS TO VOLUME

PRW.\(T

Thrombosthenin, the Contractile Protein from Blood Platelets and Its Relation to Other Contractile Proteins

&I. BETTEX-GALLAND AND E. F. L ~ S C H E R 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . TI. The Blood Platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Contractile Proteins of Other Origin . . . . . . . . . . . . . . . . . . . . . . IT-. Rclation of Thrombosthenin to Other Contractile Mechanisms Rrfvwnccs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 22 28 32

Hydrolysis of Proteins

ROBERTI,. HILL 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . TI. nlethods for Measurement of Peptide 111. Acid Hydrolysis . . . . . . . . . . . . . . . . . . . . IJ-. Alkaline Hydrolysis . . . . . . . . . . . . . . . . ........... IT. Hydrolysis of Peptide Bonds by Catalysis with Metals and Metal Clielates . . . . . . . . . . . . . . . . . . . . . . . . . . . IT. Enzyniatic Hydroly,'qls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171. Total Enzymatic Hydrolysis . . . . . . . . . . . . . . . . . . VITI. Enzymatic Hydrolysis of Native Proteins . . . . . . . . . . . . . . . . . .......................... Reftwnces . .

37 38 39 61 62 63 89 94 99

The Unusual Links and Cross-Links of Collagen

*JOHN J. HARDING T. Introtluction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 11. The Cross-Links of Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 TIT. y-Gliitainyl and p-Aspartyl Peptide Linkages . . . . . . . . . . . . . 120 xix

XX

COS T E NT S

IY . t - h i i n o Peptidc I. inkages . . . . . . . . . . . . . . . . . . . . . . . . . . . Y . Ester-Like Linkages in Collagen . . . . . . . . . . . . . . . . . . . . . . . . . VI . Ciirbohydrate Linkages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Tlic Interrelationship between Carbohydrate and Ester Links arid the Cross-Links of Collagen . . . . . . . . . . . . . . . . . . . . . . . VIII . Otlicr Unusual Links and Cross-Links in Collagen . . . . . . . . . Rrfcrmces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

136 144 162 169 178 181

The Chemistry of Keratins

IT. (+. CREWTHER. K . D . B . FRASER, F . G . LENNOX, AND H . LINDLEY I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 I1. Isolation and Characterization of Proteins . . . . . . . . . . . . . . . 193 I11. IV . V. VI .

Cornposition of Keratins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 Clieiiiical Reactivity of Keratins . . . . . . . . . . . . . . . . . . . . . . . . . 247 3lolecular Structure of Keratins ......................... 287 Relationship between the Physical Properties and Chemical Structure of Keratin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

A r m o n IKDEX ..............................................

347

SVBJEPT INDEX ..............................................

364

THROMBOSTHENIN, THE CONTRACTILE PROTEIN FROM BLOOD PLATELETS AND ITS RELATION TO OTHER CONTRACTILE PROTEINS By M. BETTEX-GALLAND and E. F. LUSCHER Theodor Kocher Institute, University of Berne, Berne, Switzerland

I. Introduction . . . . . . . . . . . . . . 11. The Blood Platelet8 . . . . . . . . . . . . A. Origin and Morphology . . . . . . . . . . . B. Platelet Metabolism . . . . . . . . . . . . C. Function of Platelets in Blood Coagulation . . . . . . D. Role of Platelets in Hemostasis . . . . . . . . . E. Clot Retraction . . . . . . . . . . . . . F. Thrombosthenin, the Contractile Protein from Blood Platelets . . 111. Contractile Proteins of Other Origin . . . . . . . . . A. Contractile Proteins of the Actomyosin Type . . . . . . B. Other Contractile Systems . . . . . . . . . . . IV. Relation of Thrombosthenin to Other Contractile Mechanisms . . . .4. Quantitative and Morphological Criteria . . . . . . . B. A Comparison of the Enzymatic Activities of Different Actomyosin. . . . . . . . . . Like Proteins . . . C. Discussion . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . .

I 2 2 6 7 7 9 10 52 22 27

38 28 30 31 32

I. INTRODUCTIOT Movement according to a predetermined pattern is such a major characteristic of living things that unsophisticated observers often distinguish between living and nonliving structures on this basis. For the scientifically educated, a living organism is more specifically defined by its capacity for reproduction or growth. Further analysis, however, shows that the same basic mechanisms involved in the movement of entire cells, organs, or organisms play a capital role in reproduction and in cell division as well, and that, almost without exception, contractile proteins are intimately linked to all these processes. In view of this, it seems remarkable that only the contractile protein of the striated muscle has been the object of extensive studies. Our knowledge about the contractile components of smooth muscle is already much more limited, and only in 1951, based on indirect evidence, Lett& came to the conclusion that a relationship between the contractile system of muscle and the motility of other cells 1

2

M. BETTEX-GALLAND AND E. F. LUSCHER

might exist. In 1954 Hoffmann-Rerling was able to demonstrate that glycerol-extracted cells of nonmuscular origin show active contraction in the presence of ATP (adenosine triphosphate). In 1956 the same author confirmed and extended his previous findings by the isolation from tumor cells of a piirificd protein with properties comparable to the actomyosiri obtained from striated muscle. Unfortunately, further progress in the characterization of the contractile proteins of other than muscular origin has been greatly hampered by several technical difficdties. First of all, most cells contain these proteins in very low concentrations; furthermore, owing to the interference of the nucleoproteins with similar solubility characteristics, the classic method of extraction using media with high ionic strengths cannot be followed directly. It was therefore a most welcome discovery that blood platelets contain a contractile protein in relatively large amounts. Platelets, on the other hand, do not contain a cell nucleus and because of this are free of DNA(deoxyribonuc1eic. acid)-nucleoproteins. Finally, they can be isolated by simple methods from the blood, and studies even on material of human origin are therefore possible without difficulties. This review will deal primarily with this contractile protein from human blood platelets, which we have named “thrombosthenin” in view of its origin aiid function. A brief review on blood platelets in general and their role in hemostatis will be included. The role of thrombosthenin for platelet fuiictiori will be discussed arid its properties compared with those of other contractile proteins. 11. THEBLOOD PLATELETS

A . Origin and Morphology Platelets were first observed as structural elements of the blood by LIonn6 (1842), but it was only about 40 years later that more detailed accounts became available. A t that time Hayem (1878), Rizzozero (1882), and Eberth and Schimmelbusch (1886) not only gave excellent descriptions of the morphology of the blood platelet, but they also made remarkably accurate observations about their physiological arid pathological functions. For many years the origin of the blood platelets has remained a much disputed subjert. Wright (1910) was the first to see them derived from the megaliaryocyte ; this observation has since been confirmed many times, morc remitly by elec+xon mirroscopy (De Marsh et nl., 1955), by direct production of platelets by megakaryocytes in tissue culture (Izak ef nl., 1957) a i d by the demonstration of common antigens between the mcgakaryorytes arid thc platelets (Vasques aiid Lewis, 1960; Silbcr et uZ.,

T H E CONTRACTILE PROTEIN FROM BLOOD PLATELETS

3

1960). Kinosita et nl. and others have simessfully filmed the maturation cvycle of the megakaryocyte. These micrographs shorn that during the multiple divisions of the cell nucleus that accompany the transformation of the promegakaryocyte into the mature megakaryocyte, violent cytoplasmic movements occur; projections of variable morphology are formed and disappear again in a rapid sequence. This phenomenon might well indicate that contractile proteins are already present in the maturation phase of the megakaryocyte. The pIatelets are finally formed by fragmentation of the megakaryocyte cytoplasm. Platelets circulating in the bloodstream appear as colorless, often irregular discs with a diameter from 2 t o 4 p (Witte and Schricker, 1958). Their cytoplasm, which is devoid of a nucleus, nevertheless appears not as a homogeneous mass, but contains a variable number of granules, which can be observed even by light microscopy (Fonio and Schwendener, 1942). Further information about the internal structure of the platelets became available when suitable preparation techniques for electron microscopy were introduved (Bernhard and Leplus, 1955; Feissly et al., 1957). Ultrathin sections show that the cytoplasm is surrounded by a membrane and contains an impressive number of granules, vacuoles, but in general only a few, moderately developed mitochondria (Fig. 1) (concerning literature on the ultrastructure of the platelets see Marcovici et al., 1961). Apart from occasiorial deposits of glycogen (Jean and Gautier, 1961) the cytoplasm shows no structure. In particular there is no evidence for the presence of fiberlike structures which might remind one of the orientated arrangement of the contractile proteins within the muscle fiber. In extravasated blood, platelets rapidly undergo morphological changes. Provided blood coagulation is inhibited by the removal of ionized calcium, these changes, which include formation of more or less pronounced pseudopodes, swelling of the cell body, and rearrangement of the granules, may still be reversible. Even under these conditions wettable surfaces, such as glass, exert a typical influence on the platelets, which show a pronounced tendency to adhere. They thereby spread out, and in these spread forms evidence for a fibrous arrangement of the cytoplasm is often found (Bessis, 1950; Zucker and Borrelli, 1954; Hutter, 1957; Braunsteiner et al., 1960; Marx et al., 1960). Much more dramatic are the changes suffered by the platelets in the course of blood coagulation. The morphology of these changes was studied by the pioneers in this field during the last century. I n 1886 Eberth and Schimmelbusch proposed for the first time the name “viscous metamorphosis” (VM) which today includes the morphological as well as the biochemical changes which the platelets undergo under the influence of thrombin. This enzyme, as may be recalled, is formed by the activated

FIG.1. Sornial human blood platelets. Electron micrograph of an ultrathin secation of platelets. Magnification : x 30,000. The surrounding membrane, some initorliondri.t, as wrll as light and dmsc granulations, are clearly discernible. Courtesy of Dr. Gautier, Centre de Microscopie Electronique de l’Universit6 de Lausanne. 4

THE CONTRACTILE PROTEIN FROM BLOOD PLATELETS

5

FIG.2. Early phase of viscous metamorphosis. Addition of thrombin has induced aggrrgation and drgranulation of platelrts. Remaining dark bodies (arrows) are mitochondria (Parmegyiani, unpublished, 1962).

blood-clotting system (Luscher and Bettex-Galland, 1962). Isolated and washed platelets undergo typical VM if incubated in a buffered, isotonic medium containing adequate amounts of Ca++ or Mg++ ions and a small amount of thrombin (Liischer, 1956b; Setna and Rosenthal, 1958; Koppel, 1958; Zucker and Borrelli, 1959). The time sequence of the morphological changes, as observed by electron microscopy, is as follows (Parmeggiani, 1961): swelling of the cells and pronounced tendency towards mutual aggregation; rapid disappearance of the so-called a-granules and, a t a later stage, swelling and loss of the mitochondria (see Fig. 2); formation of large aggregates, in which the platelets are still separated by their mem-

6

M . BETTEX-GALLAND AND E. F. LUSCHER

branes, with extremely close packing of these aggregates, perhaps by interlinking pseudopodes (Hovig, 1962). Sokal (1960) has shown by a special mirroscopy technique that the formation of a dense aggregate is clearly linked to the manifestations of active contraction of barely visible protrusions emanating from the thrombin-activated platelet when surrounded by serum. The point of attack on the platelets of thrombin, an enzyme which is highly specific for fibrinogen, is still unknown; in particular it is still controversial whether fibrinogen in the form of a layer on the cell surface is involved (Schmid et al., 1962; Grette, 1962; Grigler et al., 1962). The situation is even more complex as shown by the finding that agents other than thrombin are equally capable of inducing VM. Platelets are known to adhere instantaneously to connective tissue particles, arid more recent studies have shown that, as a ronsequence of this first passive adhesion, they undergo morphological changes resembling, if not identical with, VM (Hugues and Lapierre, 1964; Hovig, 1963). Similarly, VM can be induced by adding to platelet suspensions such unrelated substances as certain heparinoids, antigen-antibody complexes, or aggregated y-globulins (Rettex-Galland et a2 , 1963b; Bettex-Galland and Luscher, 1964). The participation of the complement system has been suggested a t least in some of these reactions (Bettex-Galland arid Luscher, 1964). Since the secondary phenomena of VM, independent of the inducing agent, are always the same, it seems logical to postulate a common denominator for their appearance. A reasonable hypothesis would be that the inducing agent acts directly or indirectly on the membrane, thereby causing an increase in membraiie permeability. The ensuing phenomena would then be due to the influx of extracellular fluid, in particular, to a disturbance of the iritracellular ionic balance.

H. Platelet Metabolzsrn Blood platelets coiitaiii a csonsiderable number of different enzymes (see review by Rettex-Galland and Maupin, 1961). They show some respiratory activity (Maupin, l954a), which conforms with the presence of a limited number of mitochondria. It is important to note, however, that the energy metabolism of the platelets is based mainly on glycolysis arid that their enzyme pattern in this respect shows great resemblance to that of striated muscle (Gross, 1961). There exists a direct link between the morphological manifestations of VM and energy metabolism of the platelets; only viable (i.e., metabolically intact) platelets will undergo VM, and addition of glucose favors the manifestations of VM (Liischer, l956a; Rounameaux, 1956). The presence of this substrate leads to a n initial rise of ATP production in platelets in contact with thrombin (Bettex-

T11E CONTItACTILE P ROT E IN FltOM BLOOD PLA TELETS

7

Galland and Luscher, 1960; Luscahcr and Bettex-Galland, 1961). As first described by Born (l956), the net result of VM with respect to the ATP content of the platelets consists in a rapid loss of the nucleotide. In relation to the disappearance of ATP from the platelets during VM an observation by Hellem (1960) proved to be of particular importance. This author found evidence for the presence of a low molecular weight, thermostable compound, first isolated from red cells, that was capable of aggregating platelets suspended in plasma. Gaarder et al. (1961) identified this material as adenosine diphosphate (ADP). Kaser-Glanzmann and Luscher (1962) filially demonstrated that the A T P disappearing from the platelets during VM is mainly converted into ADP. Enough of this substance is released from the platelet to explain platelet aggregation by this niechanisrii without the participation of foreign cells. I n this respect the ATPase (adenosinetriphosphatase) activity of the platelets seems particularly important.

C , Function of Platelets in Blood Coagulation VM, which forms the basis of platelet function, is triggered under physiological circumstances by the blood-clotting enzyme thrombin, thus indicbating ail intimate linkage of the platelets to the plasmatic coagulation mechanism. In facat, this linkage goes so far that the platelets themselves contribute essential factors to the array of plasma factors involved in the conversion of the proenzyme prothrombin into active thrombin. This conversion is possible by two pathways, an “extrinsic” one based 0x1 the participation of injured tissue cells and an “intrinsic” one to which the platelets contribute essential components. A considerable number of such platelet factors are known today (Luseher, 1959, 1962); some of them are specifically adsorbed plasma factors, some of them are of cellular origin. Among the latter ones, platelet factor 3, a lipoprotein located in the a-granules (Srhulz and Hiepler, 1959) is essential for intrinsic prothrombin activation. By this means, intrinsic thrombin must necessarily form on the platelet surface in the first place. This may explain why platelets are very sensitive to even a limited activation of the clotting system; VM becomes discernible long before the plasma level of thrombin is high enough for the conversion of fibrinogen to fibrin.

D. liole of Platelets in Hemostasis The most important physiological function of the blood platelets consists in their participation in the mechanisms leading to the arrest of hemorrhage. Bleeding from smaller vessels, such as the arterioles and venules, is almost exclusively arrested by means of the formation of a “hemostatic plug” which is composed of densely packed blood platelets. In larger

8

M. RETTEX-GALLAND AND K. F. LUSCIIER

vessels the same mechanism is also, although not as exclusively, observed (Witte aud Schrivker, 1960), whereas capillary bleeding is often stopped by fibrin formation, again by the formation of a platelet plug or by some other means (JZrgensen and Borchgrevink, 1963). Although this cellular hemostatic mechanism was already described in some detail by early workers in the last century, much of our present-day knowledge about its importance is due to Roskam el al. (see review, 1961), Zucker (1947), and Chen and Tsai (1948). The contributioiis of these authors coiisisted in the application of modern methods of observation and experimentation and in the revival of intercst in the role played by the platelets in hemostasis. Hugues (1959), in particular, was able to show convincingly that the formation of a n efficient hemostatic plug consists of three phases: (1) adherence of the platelets to the injured endothelium; ( 2 ) growth of a loose aggregate of platelets by the addition of new platelets, (3) consolidation of this aggregate t o a stable arid impermeable hemostatic plug. It has since been found that phase 1 is explained by the fact that both connective tissue and isolated collagen particles specifically attract platelets (Bounameaux, 1959, 1961; Hugues, 1960, 1962); i n vitro mixed aggregates will result from the mutual adherence of the two components (Zucker and Borrelli, 1962). Connective tissue particles even seem capable of inducing VM in such adhering platelets (Hugues and Lapierre, 1964; Hovig, 1963), but since no tissue injury is possible without the activation of the bloodclotting mechanism through tissue thromboplastin, thrombin formation mill lead equally to the induction of VM in these first adhering platelets. The existence of a direct relationship of the clotting system to hemostasis must be concluded from the observations of Borchgrevink and Waaler (1958) on “primary” and “secondary” bleeding times in a variety of bloodcoagulation disorders. With the induction of VM we have already entered phase 2 of the formation of the hemostatic plug. Platelets in VM stick to each other, and the already mentioned ADP-mediated aggregation undoubtedly is an important component in the growth of a voluminous platelet plug. Simultaneously, the liberation of platelet factor 3 from the &-granules makes possible the thrombin formation by the intrinsic pathway. In order to become efficient, this aggregate must first solidify (phase 3); this most likely is achieved by an active contraction of the platelets. The result is a tightly packed mosaic of platelets with still discernible membranes, but showing evidence of multiple branching and interlinkage (Parmeggiani, 1961; Kjaerheim and Hovig, 1962). Direct proof for a n active contraction of loose platelet aggregates suspended in plasma comes from the microscopy work of Sokal (1960). The micrographs obtained leave no doubt that fine fibrous emailations draw together the different parts of a primarily loose platelet aggregate.

THE CONTRACTILE PROTEIS FROM BLOOD PLATELETS

9

The formation of the hemostatic plug is entirely independent of the conversion of plasmatic fibrinogen into fibrin (Zucker, 1947; Jglrgensen and Borchgrevinck, 1963). In primitive animals cellular aggregation is the only hemostatic mechanism (Lechler and Gross, 1962). In the course of evolution, then, plasmatic coagulation obviously is a later development which helps, but does not replace, the cellular system (see discussions by Heilbrunn, 1961 or Rudtz-Olsen, 1951). Finally, it should be mentioned that pathological deviations from the physiological process of the formation of a hemostatic plug are widespread and most important. Such deviations include the primary “white” thrombus in thrombosis and the formation of platelet microthrombi in the circulation in a variety of pathological states.

E. Clot Retraction The most spectacular example of a contractile process linked to the presence of the blood platelets is clot retraction. Freshly formed blood clots contract to a smaller volume, whereby serum is expressed. The extent of retraction is determined by a series of factors, the most essential being the number and the functional integrity of the blood platelets. An extensive literature on clot retraction has accumulated, and the reader is referred to the review by Budtz-Olsen (1951) for the earlier observations and theories. More recent work has shown that the prerequisites for clot retraction are the same as for thrombin-induced VM: thrombin a t the same time acts as a trigger for VM and supplies the substrate fibrin. Again, an intact metabolism and the presence of Ca++ or Mg++ ions in a buffered system are essential (Luscher, 1956b; Bettex-Galland and Luscher, 1960). Glucose, acting as a substrate for the energy metabolism, may improve clot retraction, particularly in aged platelets or in platelets of certain animal species (Luscher, 1956a; Bounameaux, 1956, 1957; Ballerini and Seegers, 1959; Corn el al., 1060; Zucker and Borrelli, 1961). The normally low speed of clot retraction is due to the braking action of the fibrin meshwork; clots containing very small amounts of fibrin contract in times that approach the speed of active contraction of pure platelet aggregates (Luscher, 1961). Many explariatioris have beeii aiid are still being offered to explain clot retraction. Platelets arc found to be completely incorporated, mostly in the form of small aggregates, in a newly formed fibrin clot. It seems that under these circvmstancw their memhranes-other than in a pure platelet, aggregate--arc easily disrupted, whereby a particularly close contact between the cytoplasmic constituents of the cell and its plasmatic surroundings can be established. As early as 1906 Le Sourd and Pagniez suggested that some material, capable of inducing the shrinkage of fibrin

10

M. BETTEX-GALLAND

A N D E. F. LUSCHER

fibers, was thereby liberated from the platelets. This hypothetical material was latrr termed “retractozymr” by Glanzmariri (1918). Although such an enzyme as a defined entity has never been isolated, the idea of the fibrin itself being the contractile material still persists (Kuhnke, 1958). On the other hand, evidence is accumulating which seems to indicate that fibrin plays a passive role in clot retraction and is simply carried along by actively contracting elements of platelet origin (Discombe, 1950; Bloom, 1955; Sokal, 1960; Castaldi et al., 1962; Rodman et nl., 1963). Discrepancies in opinion also exist with respect to the physiological significance of clot retraction. As shown by Rudtz-Olsen (1951), the force of clot retraction is so weak that its function as a “physiological ligature” seems unlikely. Hetraction of an intravascular clot would favor recanalizatiori of an obstructed blood vessel; but Quick (1950) has emphasized that this would lead t o the liberation of serum rich in thrombin, thus favoring further clot formation. Finally, the possibility that the orientation of the fibrin fibers under the influence of retraction might favor wound regeneration has been discussed (Lusc.her, 1956~).

F. Thromnboslhenin, the Contractde Protein f r o m Blood Platelets Many of the described properties of the blood platelets suggested thc presei1c.e of a coiitractile protein in these cells. This possibility seemed the more likely siiive Hoffmann-Eerling (1954, 1956) had already demonstrated that the morphological alterations of other cell types were due to the presence of actomyosin-like material. I n 1959 I3ette.x-Galland and Luscher succeeded in extracting from human blood platelets such a rontractile protein, which was subsequently named “thrombosthenin.” Its solubility properties, as well as its dependence for activity on the presenve of ATP and metal ions, soon led to its c+lassification as a member of the actomyosin group. Work on thrombost,heiiin has airwe coiitinued, both with respect t o its properties as a complex protein with enzymatic activity and to its biological significance. 1. The Isolation of Thromboslhenin

Thrombostheriiii is extracted from a concentrated suspension of washed blood platelets obtained by any of the described methods of isolation (Maupin, 195413). Bettex-Galland and Luscher (196l), starting with 50 liters of freshly rollected citrated human blood isolated by diff creiitial centrifugation in the (*old,from the buffy layers, 20 to 30 ml of a highly concentrated suspension of washed platelets. Since the isolatioii of thrombostheiiiii is bused on its solubility properties, spevial care must bc taken to elimiiiate the 1euroc.ytt.s;their content of deoxyriboiiiicleoproteids

T H E CONTRACTILE PROTEIN FROM BLOOD PLATELETS

11

with similar solubility characteristics could otherwise lead to impure products. a. Thrombosthenin from H u m a n Platelets. The first step in the isolation of thrombosthenin from concentrated human platelets consists in the extraction with a solution of high ionic strength. This is achieved by adding to the platelets enough concentrated “Weber-Edsall solution’’ composed of 3 M KC1, 0.05 M NarCOs, and 0.2 M NaHC03 to bring the final concentration of KC1 in the mixture to 0.5 M . I n their first experiments, Bettex-Galland and Luscher (1959, 1961) disrupted the cells by mechanical homogenization. Later it was found that addition of digitonin to a concentration of 0.1 yois a much more elegant means of breaking up the platelets without damaging the contractile material (Bettex-Galland et al., 1962). After standing overnight, the suspension of lysed platelets is centrifuged for 60 min at 20,000 g, in order to remove solid cellular debris. Thrombosthenin is then contained in the supernatant and can be precipitated by lowering the ionic strength to 0.05 p with distilled water. The precipitate is collected by mild centrifugation and is redissolved by adding concentrated KCl solution, buffered to pH 7.0 with imidazole-HC1. The final colleentration of KCl is brought to 0.4M. This process of precipitation and redissolution is repeated three times in order to remove the other, more soluble cytoplasmic proteins. All operations are carried out in the cold. Solutions of thrombosthenin are unstable on storage. Denaturation becomes discernible after about a week in samples kept a t 0°C a t ionic strength 0.6 p . Addition of the same volume of glycerol and storage a t - 5°C allows the conservation of unchanged samples for periods of 1month or longer. The glycerol is easily removed by dilution with cold water, whereby the thrombosthenin is again obtained as a precipitate. 6. Thrombosthenin from Pig Platelets. The method described by Grette (1962) for the extraction of the contractile protein from pig platelets uses butaiiol for the lysis of platelets, and the precipitation is effected a t an ionic strength of 0.2 p in the presence of Mg++ ions. It is noteworthy that, in spite of the differences in origin and in experimental procedure, the yield and the characteristic properties of the active material from pig platelets are the same as for thrombosthenin of human origin. c. Solubility of Thrombosthenin. The contractile proteins of both human and porcine origin are soluble a t a neutral pH only in aqueous media of ionic strength above 0.3 p. This applies to solutions a t a protein concentration below 1 yo;at higher concentrations stable gels form within a few hours. Such gels, most likely the result of intermolecular aggregation, cannot be reconverted to homogeneous solutions. On lowering the ionic strength to 0.2 p, thrombosthenin precipitates

12

M. BETTEX-GALLAND

AND E. F. LUSCHER

in coarse floccules, which are easily separated by low-speed centrifugation. Figure 3 presents a solubility curve for human thrombosthenin in KClimidazole buffer a t pH 7.0. The presence of ATP leads to an increased solubility a t lower ionic strength. Grette (1962) reports that solutions of porcine thrombostheiiin tend toward precipitation at an ionic strength of only 0.08 I.( provided 5 x M ATP is present. Perhaps it is not correct to speak of a solubilizing effect of ATP on thrombosthenin; more likely this seemingly increased

t

0

L

01

02 P

____

03

-

FIG. 3. Solubility of thrombosthenin as a function of ionic strength. From Bettex-Galhnd and Luscher (1961).

solubility is already the result of an ATP-induced dissociation of the complex molecule into its more soluble subunits. As will be shown later (cf. Section 11, F,3,d),this conclusion is supported by studies of the ATPase activity of thrombosthenin at low ionic strength and as a function of increasing amounts of ATP. Lowering the pH also leads to a decreased solubility of thrombosthenin. Grette (1962) obtained precipitation of porcine thrombosthenin a t pK 6.5. The addition of small amounts of Mg++ ions seems to enhance the precipitating effect. Care must be taken, however, because even a t p H 6 signs of progressive denaturation become discernible. It seems preferable for preparative purposes to work a t a neutral pH. 2. The Phenomenon of Superprecipitation

At an ionic strength of about 0.1 I.( with Mg++ ions and ATP present, thrombosthenin forms a flocculant precipitate which contracts rapidly to a smaller volume. Since the contracting units in the precipitate are randomly arranged, the magnitude of contraction is the same in every direction. The result is a small contracted pellet, still showing the con-

13

T H E CONTRACTILE PROTEIN FROM BLOOD PLATELETS

tours of the container (see Fig. 4) (Bettex-Galland and Luscher 1959, 1961). This phenomenon, termed “superprecipitatioii” by Szent-Gyorgyi (1951), is typical for the contractile proteins of the actomyosin group. It becomes even more spectacular if concentrated solutions of thrombosthenin are spun out to small fibers and allowed to shrink (Bettex-Galland and Luscher, 1961). Precipitates obtained from dilute solutions are not stable enough to show macroscopically visible contraction. In this case a fine granular

0.5

1.0

2.0

3

4.0 5.0

6.0

15.0

FIG.4. Superprecipitation of throtnbosthcnin a t 20°C. Upper series shows the contraction of precipitated thrombostlienin under the presence of 10.’ M ATP. Lower series: sedimentation of prcripitate without added ATP. From BettexGalland and Liischer (1961).

precipitate wiIl form which sediments much faster than the voluminous product obtained in the absence of ATP. Grette (1963) has published such pictures of porcine thrombosthenin. The speed of the contraction of a superprecipitate is greatly dependent upon temperature. For thrombostheniii a t room temperature (about 20°C) 15 to 20 min are required for complete contraction. At 37°C this process requires only 1 to 2 min. At 0°C superprecipitation does not take place. Accurate and comparable measurements of the contraction rates

14

M. BETTEX-GALLAND AND E. F. LUSCHER

of different preparations are di6cult to perform; nevertheless, it seems that the muscle actomyosins show still faster contraction. Magnesium ions are essential for siiperprecipitatiol~. This is borne out hy the fact that an excess of EDTA (ethylenediaminetetraacetic a d ) leads to complete inhibition. Another powerful inhibitor of superpreripitatioii is the sodium salt of o-[ (3-liydroxymercuri-2-methoxypropyl)~~arbamoyl]phenoxyac*eticacid ("Salyrgan"). 3. Ar(CnosznCir4phoepF,criase ( AI'l'ase) AcfzLity of Thrcmbcsthenin

The ATF-splitting activity is a typical property (1. Spectjicily of Acticn. of the vontracti e proteiiis of the actomyosiri type. Furthermore, VM of the platelets is characterized by maiiifestatioiis of the caoiitrartile system and a t the same time by the disappearance of ATP. The assumption that thrombostheriiri also has enzymatic activity therefore seemed reasonable. The following experiment verifies this assumption : A sample of the

t

Incubation: 15 min

KHCO,

3%

Incubation: 0 min

7%

4%

t

Fractions

FIG.5 . Enzymatic clcavagr of ATP by thrombosthcnin. Left half of picture shows chromatogram of nuclrotides after incubation of ATP with tlirombostlienin. ADP has been formed a t the expense of ATP. AMP has remained unchanged. Right half of pictures shows chromatogram obtained prior to incubation. Nnmhers on t,op of curves give KHCO,, concentrations used in elution of nuclcot,idos from column. From Rcttex-Galland and Liischer (1961).

protein is incubated with ATP for 15 min a t 20°C, arid the deproteiiiized supernatant aiialyzcd by chromatography for its niicleotide compositioii. As showii ill Vig. 5 , the sample prior to iiicubatioii contains only ATP, besides negligible ckoiitamiriat oils, whereas the active preparation has degraded part of the ATP, with ADP showing up as a distinct peak in the chromatogram. Thus thrombostheriin fulfills the criteria of a typical

THE- CONTRACTILE

PROTEIN

FIZOM BLOOD PLATELETS

15

ATPase ; the abseiiw of iirvly formed AMP (adenosine moiiophosphate) shows it to be free of contamiliatioris with apyrase or myokinase activities. In view of the finding that platelets contain both apyrase and myokinase, this latter observation seems particularly important. b. ATPase Actzuzty of Throinbcsthenzn in fielation to Ionic Strength. The ATPase activity of the actomyosins in general is highest at low ionic strength. For thrombosthenin a t an ionic strength of O.O8p, a value expressed as liberated inorganic phosphorus ( P J ,in the order of magnitude of lop3pmole Pt per minute per milligram of protein has been determined (Rettex-Galland and Iischer, 1961; 13ettex-Galland et al., 1963a). For the porcine material Grette (1962) has found an actixity in the same range. As will be discussed in detail later, these values are considerably lower than those obtained with muscle actomyosin. Figure 6 represents the influence of increasing ionic strength on the ATPase activity of the enzyme from human platelets. A significant,

t

. c_ 0004 E \ ._ U 0.003

ea

a, -

2

0.001 -

3.

o

.

0.2 o

o0.4

o 0.6

o

~

P

FIG.6. Influence of ionic strength on A4TPasc nct,ivit>y. Thrombosthenin reprecipitatcd three times (solid line) ; pH 7, IO-:'M ATP, lo-:' M Mg++,20°C. Values for contritctilc protein from undifferentiated cells (daslicd line) calculated according to Hoffman-Brrling (1956) assuming a ratio protein/N = 6.25. From BcttesCalland and Liisrher (1961).

though not very proiiounced, decrease in activity is evident; the good agreement with the findings of' Hoffmann-Perling (1!956) on the contractile protein from tumor cells is remarkable. c. Effects of Mg++ and Ca+f Ions on the A T P a s e Actzvity of Thrombosthenin. The influence of the two metal ions has been tested a t two different ionic strengths chosen to represent the insoluble (0.08 p), and the completely dissolved (0.6 p) states (Bettex-Galland and Luscher, 1961). At low ionic strength, Mg++ ions up to a concentration of lop3M increasingly afativate the thrombosthenin ATPase. Still higher csonventra-

1 (i

M . BETTEX-GALLAND AND E. F. LUSCHEIt

tions (10--LM), 011 the contrary, depress this optimal activity to a considerable extent. At higher ionic strength the presence of Mg++ ions exerts a predominaiitly inhibiting effect upon the thrombostheniii ATPase (Fig. 7). Calrium, on the other hand, independent of the ionic strength, always arts as a powerful activator, its potency increasing with inrreasing concentrations (highest conrentration tested, M). p = 0.60

+E&A

6

10-4 16-3 1 6 - 2

FIG.7. Influrnce of rnagncsium ion concentration on ATPase activity of thrombosthenin at low (left) and high ionic strength (right). ATP, 10T3M;temperature, 20°C. From Bcttex-Galland and Luscher (1961).

This difference in the effects of the two metal ions explains why the ATP loss suffered by the blood platelets in model systems containing only Mg++ ions is not as pronounced as the disappearance of the iiucleotide during normal blood coagulation (Born, 1958). It has been mentioned before that ADP production during VM of the platelets was of biological importanre. This production most likely is due mainly to the activation of the thrombosthenin ATPase by the influx of plasmatic Ca++ ions; their pronounced activator role therefore appears of particular significance. d. Enzyrnatic Activity of Thrombosthenin and A T P Concentration. As will be discussed later, ATP acts not only as a substrate for thrombosthenin, but it also exerts a profound influence on its structure. It is therefore not unexpected to find a pronounced dependence of the ATPase activity on the ATP concentration. On raising the ATP concentration from 0.5 to 1 mM a n increase in ATPase activity is observed; still higher concentrations, however, bring about a reduction in enzymatic activity and a t the same time a solubilization of the precipitate. This rorresponds to a dissociation of the thrombosthenin molecule, which makes it impossible to calculatb a Michaelis constant (Rettex-Galland and Luscher, 1961).

THE CONTRACTILE PROTEIN FROM BLOOD PLATELETS

17

e. Inhibition of ATPuse Activity. Salyrgaii is one of the most specific inhibitors for the contractile proteins of the actomyosin type. At a concentration of 0.5 m M and higher it was found to inhibit 80 yoof the thrombosthenin ATPase activity when measured a t a low ionic strength in the M Mg++ ions. Grette (1962) reports similar results for presence of thrombosthenin of porcine origin. Together with the inhibition of the ATPase activity, superprecipitation and contraction of filaments are also similarly affected . In view of the fact that the ATPase activity is strongly dependent on the presence of Mg++ or Ca++ ions, it is plausible that EDTA exerts a pronounced inhibitory effect, as shown in Fig. 7. Superprecipitation is totally inhibited by EDTA. Tephorine and Diparcol, both synthetic antihistamines known to inhibit clot retraction (Bounameaux, 1957; Hugues, 1959), only partially inhibit ATPase activity and superprecipitation of thrombosthenin. Finally, monoiodoacetate, a powerful inhibitor of clot retraction, inhibits neither phenomenon. The findings reported above may be summarized as follows: Inhibitors of the ATPase activity of thrombosthenin always are inhibitors of superprecipitation, whereas they do not necessarily affect manifestations of the contractile activity of the whole platelet, such as clot retraction, to the same extent.

4. Injuence of ATP on the Viscosity of Thrombosthenin in Solution (“ATP Sensitivity”) At low ionic strengths, ATP causes the superprecipitation of thrombosthenin. Applied a t higher ionic strength, i.e., under conditions where the protein is in solution, it exerts an entirely different effect-it causes the dissociation of the thrombosthenin complex into its two components. This dissociation is connected with a considerable decrease in viscosity that is easily determined quantitatively. With Zq values between 0.1 and 0.2, solutions of thrombosthenin appear less viscous than comparable preparations of actomyosin obtained from striated muscle. As shown in Fig. 8, the sudden decrease in viscosity which accompanies the addition of ATP is followed by a slow rise owing to the enzymatic breakdown of the nucleotide, until the original level is almost reached again. On repeated addition of ATP the same sequence of events is observed. Based on such quantitative determinations of the viscosity changes induced by adding ATP to soluble thrombosthenin, the so-called “ATP sensitivity” of thrombosthenin has been calculated according to the method

18

M. BETTEX-GALLAND AND E. F. LWSCHER

A 2.00

1 “J’

0

KCI ATP

cc

30

60

90

Min

FIG.8. Influence of ATP on viscosity of thrombosthenin. Extract reprecipitated once; protein concentration, 0.33 TO;ionic strength, 0.6 p ; p H 7; temperature, 25°C. ATP is added as a lo-’ M solution in 0.6 M KC1. All added solutions represent 2 % of volume of extract. From Bettex-Galland and Luscher (1961).

described by Portzehl et al. (1950). A value of about 90 % was found (Bettex-Galland and Luscher, 1961). 5. Thrombosthenin A and Thrombosthenin M

The changes in viscosity observed in solutions of muscle actomyosin on addition of ATP are explained by a dissociation of the actomyosin complex into its component parts, actin and myosin. Since thrombosthenin behaves similarly, it must be assumed that it too has dissociated into the corresponding components, which have been termed thrombosthenin A and thrombosthenin M, (A for actin-like, M for myosin-like) (RettcxGalland et al., 1962, 1963a). The dissociating effect of a given concentration of ATP is strongly dependent upon the ionic strength of the system. Thus, when the ionic strength is high, a lower concentration is sufficient to cause dissociation. At low ionic strengths much higher ATP concentrations are required, as is illustrated by the changes in ATPase activity in relation to the ATP coiicentration (cf. Section 11, F , 3,d). a. Preparation of Thrombosthenin M . The preparation of myosin from muscle is based mainly on the different rates of diffusion from the structured fibrils of myosin and actin, respectively. This method, as well as the KI extraction method described by Szent-Gyorgyi (1951), cannot be applied to thrombosthenin or to platelets. Thrombosthenin M was isolated from thrombosthenin by the use of polyethenesulfonate. This compound belonging to the group of “interaction inhibitors” (SArAny and Jaisle, 1960) potentiates the dissociation a t such a low ionic strength that thrombosthenin M precipitates, whereas

T H E CONTRACTILE PROTEIN FROM BLOOD PLATELETS

19

the actin-like component remains in solution (Bettex-Galland et al., 1962, 1963a). By this method, 10-20 yo of reprecipitated thrombosthenin M is obtained from thrombosthenin. b. Preparation of Thrombosthenin A . Thrombosthenin A was isolated from acetone-dried platelets, using a modification of the method described by BBr&nyet al. (1957). All procedures were carried out in the cold, and generally centrifugations were replaced by dialysis in order to prevent losses of material. Nevertheless, the yields are low. The preparation so obtained in a 1 yo solution is not sensitive to the addition of ATP, nor does it display ATPase activity. An alternative way of preparing thrombosthenin A uses the supernatant from the isolation of thrombosthenin M as a starting material. This solution is first concentrated and low molecular components are removed by gel filtration. This method has not yet been used to any great extent for preparative purposes (Bettex-Galland et al. , 1963a). c. Recombination Experiments with the A and M Fragments of Thrombosthenin. Tested alone, neither of the fragments of thrombosthenin shows ATP sensitivity, but sensitive preparations are obtained by simply mixing the two components. The observed viscosity effects are in the same order of magnitude as the ones from the original thrombosthenin. B y isolating actin and myosin from striated rabbit muscle and cross-reacting them with the thrombosthenin M and A, respectively, hybrids with pronounced ATP sensitivities are obtained (Table I) (Bettex-Galland et al., 1962). This TABLEI Sensitivities toward A T P of the Recombined Dissociation Products of Thrombosthenin and Actomyosin from Rabbit Muscle No.

Components of mixturea

1 2 3

Actin myosin Thrombosthenin A thrornbosthenin M Thrombosthenin A myosin Actin thrombosthenin M

4

+ +

+ +

Sensitivity toward ATP 136 120 117 147

a The components were present in the mixtures approximately in a 1:1 ratio. details see Bettex-Galland et al. (1962).

For

seems the more remarkable because the thrombosthenin components are of human origin. Of particular interest were the ATPase activities of the fragments and of the recombined complexes. Thrombosthenin M is a considerably weaker ATPase than thrombosthenin. Thrombosthenin A, as mentioned before, is completely inactive, but it is capable of potentiating the enzymatic

20

M. BEWEX-GALLAND AND E. F. LUSCHER

activity of thrombosthenin M. Here again, it was found that actin from rabbit muscle exerts the same enhancing effect toward the thrombosthenin M ATPase. Vice versa, thrombosthenin A acts as an activator for the TABLE I1 A T P a s e Activities of the Recombined Dissociation Product of Thrombosthenin and Muscle Actomyosin” Activity of recornhination product with Fragment

Activity

Thrombosthenin A

Actin

Thrombosthenin M Myosin

0.0032 0.0148

0.0050 0.0510

0.0068 0.0997

a Data represent mean values of inorganic phosphate (Pi) liberated by the action of the enzyme expressed in pmoles Pi per minute per milligram of protein. The conditions of the experiments were: p = 0.08, pH 7.0, 20°C, M Mg++, l O P M ATP. All values refer to the protein concentrations of the thrombosthenin M and myosin moieties, respectively. From Bettcx-Galland et al. (1963a).

muscle myosin ATPase (see Table I1 for quantitative relationships) (Bettex-Galland et al., 1963a). 6. Other Properties of Thrombosthenin

Studies on the double refraction of flow (von Muralt and Edsall, 1930) of thrombosthenin are being carried out. Preliminary results indicate the presence of a strongly asymmetrical molecule (von Muralt, unpublished observations, 1963). Ultracentrifugation of thrombosthenin as well as of thrombosthenin M led to conflicting results. As is known from the contractile proteins of muscular origin, poorly defined complexes of variable size may form very easily, making molecular weight determinations difficult. The same conditions were encountered with the contractile protein from human platelets (Bettex-Galland and von Tavel, unpublished observations, 1963). 7. The “Relaxing Factor” of the Blood Platelets-a of Thrombosthenin Activities

Natural Inhibitor

Recently, Grette (1963) has reported the extraction from pig platelets of a material with properties comparable to the “relaxing factor” from muscle (Marsh, l’~51). Solutions of this factor will inhibit superprecipitation as well as ATPase activity of thrombosthenin of porcine origin. Calcium ions, on the other hand, were found to inhibit this relaxing effect.

THE CONTRACTILE PROTEIN FROM BLOOD PLATELETS

21

8. The Physiological Signijicance of Thrombosthenin

Today thrombosthenin is the only contractile protein of nonmuscular origin available in larger quantities. This alone makes it a n interesting material from the point of view of comparative physiology. On the other hand, the high content of thrombosthenin in the blood platelets also suggests an important function of this material in physiological platelet activity. Two such possible functions are self-evident. The first is the contraction of platelet aggregates during viscous metamorphosis, a process of considerable importance in the formation of an efficient hemostatic plug. It may be recalled that the micrographs published by Sokal (1960) clearly show active contraction of primarily loose platelet clumps, obviously effected by fine contractile strands or very thin pseudopodes emanating from the platelets. It is important to note that these micrographs were taken under conditions which seem to exclude the interference of fibrin formation. Unfortunately, electron micrographs lend no support to the hypothesis that contractile material, as such, might leave the platelets, thereby forming contractile strands under the influence of the low ionic strength in the extracellular fluid. Also, it has not been possible to find thrombosthenin in a supernatant of elevated ionic strength of platelets after they have undergone VM, unless they are mechanically or chemically lysed. It is much more likely that the active contraction of packed platelet aggregates is the result of the contraction of pseudopodes, i.e., contractile structures which generally form part of the cell and are still covered with a membrane. The spontaneous contraction of pseudopodes has already been observed by Fonio and Schwendener (1942). In the hemostatic plug these pseudopodes are short and coarse; they seem to fill the interspaces between the platelets, thus forming a dense, interlinked cell mosaic. Owing to the relative incompressibility of the cell bodies, contraction under these conditions would not be expected to lead to a visible decrease in volume of the aggregate but to its solidification. Motion pictures of the formation of the hemostatic plug are in agreement with this prediction (Roskam et al., 1961). The evidence available seems to support the assumption that thrombosthenin does riot act in a free form but rather in the form of more or less pronounced pseudopodes. The formation of a loose platelet plug does not of itself lead to the arrest of hemorrhage; its solidification by means of the contractile mechanism therefore appears to be of vital importance. The second even more striking manifestation of contractile activity is clot retraction. Here again, the major problem consists in explaining how the contractile material can come in contact with the fibrin fibers. In the presence of fibrin, and different from VM in a fibrin-free system,

22

M. BETTEX-GALLAND AND E. F. LUSCHER

platelets often show torn membrane structures and evidence for shedding of the cytoplasm (Parmeggiani, 1961; Jorgensen and Borchgrevink, 1963). Thus, the assumption of a direct participation of thrombosthenin in retraction does not seem unlikely. Here again, additional experimental evidence speaks against this hypothesis. Salyrgan, a powerful inhibitor of thrombosthenin contractility, does not affect clot retraction. On the other hand, monoiodoacetate, known to inhibit retraction completely, does not interfere with the superprecipitation of isolated thrombosthenin. Equally divergent is the evidence from microscopy of retracting or retracted fibrin clots. Although contracting pseudopodes carrying along fibrin fibers have occasionally been observed (Sokal, 1960), the number and range of these structures seem inadequate to account for the observed retraction effect. Other micrographs, however (Bloom, 1955), very clearly show the platelet to be the contractile center of retraction. Kinetic data also support the idea of a close relationship between clot retraction and thrombosthenin contraction; retraction normally is a slow process, which is speeded up considerably with decreasing fibrin concentrations (Liischer, 1961). Extrapolation to a fibrin concentration of zero leads to rates of retraction which are in the same order of magnitude as those of contracting thrombosthenin. The situation may be summarized as follows: Most likely, thrombosthenin is the effector of clot retraction activity; there remain, however, a series of puzzling observations, which still await explanation. Until then, clot retraction is still as good a playground for speculation as it has been for the past 100 years. One last important function of thrombosthenin remains to be mentioned in relation to hemostasis, namely its ATPase activity. ADP arising from this activity must be considered of importance for platelet aggregation, and its production by the platelets during VM is perhaps an essential step in the arrest of hemorrhage as well as thrombus formation (Kaser-Glanzmann and Iiischer, 1962). 111. CONTRACTILE PROTEINS OF OTHER ORIGIN

A . Contractile Proteins of the Actomyosin T y p e A great variety of contractile proteins of the actomyosin type have been described. They are found in many different animal species and in different tissues. No attempt will be made here to give a complete list; this seems the more permissible as a detailed review on the subject has been published (Perry, 1960). Loewy (1952) has reported the extraction from the plasmodia of myxomycetes of a contractiie protein with the typical properties of an acto-

T H E CONTRACTILE PROTEIN FROM BLOOD PLATELETS

23

myosin. This observation, confirmed since then by Nakajima (1960), seems noteworthy because it shows that the occurrence of the actomyosins is not a t all restricted to the animal kingdom. The following discussion will deal mainly with the properties of the actomyosiiis from striated and smooth muscles, and those properties will be considered which appear pertinent to a comparison with thrombosthenin.

I . Actomyosin from Striated Muscle Most of our knowledge about contractile proteins comes from the extensive studies of the actomyosin from striated muscle. For more detailed accounts the reader is referred to one of the recent reviews on the subject (Weber, 1957; Perry, 1960; Szent-Gyorgyi, 1960). Actomyosin is generally extracted from fresh rabbit muscles by the use of buffered KC1 solutions of an ionic strength of 0.5-0.6 p (WeberEdsall solution). The solubility curve of the isolated actomyosin a t pH 7 shows an inflection a t 0.25 p ; above a value of 0.3 p the protein is completely soluble (Hasselbach et al., 1953). At low ionic strengths, actomyosin upon addition of ATP and provided Mg++ ions are present shows superprecipitation. By glycerol extraction, muscle fibers may be prepared t o contain essentially only the contractile system. Such fibers will contract normally under the conditions mentioned above for the isolated actomyosin (Weber and Portzehl, 1952). The muscle fibril contains the actomyosin in the insoluble state and in an optimal spatial arrangement (cf. Section IV, A,2). Actomyosin from striated muscle possesses ATPase activity (cf. Table IV), the extent of which is strongly dependent upon the ionic strength. At M ATP presents a n activity M Mg++ and 3 X pH 7.0, 1Y0C,with of 0.15 Fmole P , per minute per milligram of protein has been observed a t ionic strengths below 0.1 p. A rise in ionic strength to about 0.2 p under otherwise unchanged conditions brings about a decrease in activity to a value as low as 0.01 pmole Piper minute per milligram of protein. This in fact corresponds to the ATPase activity of the isolated myosin moiety, thus indicating complete dissociation of the actomyosin complex under these conditions (Hasselbach, 1952). At a low ionic strength, Mg++ ions i n concentrations up to lop3M exert an activating effect; still higher concentrations again depress the activity. On the other hand, Ca++ ions progressively activate up to concentrations of lo-' M (Hasselbach, 1952). Portzehl et al. (1950) have determined a value of 0.3 for the specific viscosity Zv; the ATP sensitivity was consistently higher than 100 yo. The dissociation of the actomyosin complex into its coiistituents is dependent upon the ATP concentration and the prevailing ionic strength ;

24

M. BETTEX-GALLAND AND E. F. LUSCHER

furthermore, it is favored by the presence of “interaction inhibitors,” such as the substances studied by R&r&nyand Jaisle (1960). Striated muscle contains a physiological interaction inhibitor, called the “relaxing factor” by its discoverer Marsh (1951). Porteehl (1957) found this activity linked to the “grana” fraction located, according to Muscatello et al. (1961), in the sarcotubular system. Perhaps this particulate fraction does not itself represent the relaxing factor, but only its site of production (Rriggs and Fuchs, 1960; Parker and Gergely, 1960). Myosin is easily extractable from homogenized muscle a t higher ionic strength. Since actin seems to be more solidly linked to the structural element, this offers a convenient means for the separation of the two components. Myosin has been studied in some detail, and a molecular weight of the order of 400,000 to 500,000 has been reported (cf. review by Perry, 1960). Actin is generally obtained from acetone-dried muscle powder by extraction with either water or, provided ATP is present, with KI solutions. Under these conditions the globular form of the protein is obtained, which polymerizes to the fibrillar form in 0.1 M KC1 containing traces of Mg++ or Ca++ ions. Fibrillar actin combines with myosin to give actomyosin. Nevertheless, the exact structure and mode of action of the actom yosin complex are still far from being fully understood. 2. Actomyosin from Smooth Muscle

Needham and Cawkell (1956) were the first to describe in some detail the properties of the contractile protein from smooth muscle isolated from the gravid uteri of different animal species. Later, Hasselbach and Ledermair (1958) compared the properties of the materials extracted from gravid and nongravid uteri, and finally Cretius and Jaisle (1960) and Jaisle (1961) discussed these results in relation to those obtained on material of human origin. Owing to the differences in the materials and techniques used, the results of these iiivestigations are not strictly comparable ; however, they show beyond doubt that the contractile protein of smooth muscle belongs to the actomyosin group. Fresh uterus contains from 1.1 to 3.5 yo contractile protein, which is extractable under the same conditions as the acstomyosin from striated muscle (Table 111). It is interesting to note that the solubility of this material changes in the course of gravidity, the actomyosin from nongravid uterus passing into solution at an ionic strength of 0.3-0.4 p, independent of the amount of ATP present. The material from gravid uterus is already soluble a t 0.2-0.3 p and its solubility is influenced by ATP (Ledermair, 1959). The specific viscosity 211 varies between 0.1 arid 0.2 (Needham and

T H E CONTRACTILE P R OT E I N FROM BLOOD PLATELETS

25

Cawkell, 1956; Hasselbach and Ledermair, 1(358),and the ATP sensitivity ranges from 60 to 80 %. The highest values are observed during gravidity, and this has led to specdations that under these conditions the ratio of actiii to myosin might be changed (Nasselbach and Ledermair, 1958). TABLE111 Quantitative Distribution of Contractile Proteins in Different Tissues Tissue and species Striated mnsrle Rabbit

Current denomina- Content (%) hasrd on tion of contractile material Total protein Wet weight Artomyosin

Uterus, nongravid Rat Actomyosin Bovine Actomyosin Human Act omyosin

50

I

-

-

References

10-1 2

Hassrlharh and Schnrider (1951)

3.5 1.53 1.11

Ledermair (1959) Ledermair (1959) Ledermair (1959)

Uterus, gravid Rat Bovine Human

Actom yosin Actomyosin Actomyosin

-

-

2.6 1.31 1.2

Ledermair (1959) Ledermair (1959) Ledermair (1959)

Platelets Human

Thrombosthenin

15

1-2

Contractile protein

20

-

Bettex-Galland and Luscher (1959) Grette (1962)

Contractile protein from undifferentiated rells

-

Porcine Sarcoma cells

0 . 1 - 0 . 2 Hoffmann-Berling (1956)

Upon additioii of ATP, precipitates of smooth muscle actomyosin a t low ionic strength show the phenomenon of superprecipitation (Jaisle, 1961). The ATPase activity of the actomyosins from smooth muscle is in the order of 0.1 pmolc P , per milligram of protein per minute (cf. Table IV). At an ionic strength of 0.6 p and in the presence of Mg++ ions the enzyme is less active than a t 0.1 p ; the opposite is found in the presence of calcium ions (Needham and Cawkell, 1956; Ledermair, 1959). Cretius arid Jaisle (1960) attribute a considerable proportion of the observed ATPase activity to the presence of granular particles in their preparations; this might then explain at least some of the observed descrepancies. Thc relaxing factor extracted from striated muscle is inactive toward the actomyosin from smooth muscle ; no comparable material has been found up to now in uterine muscle (Hasselbach and Ledermair, 1958).

26

M. BETTEX-GALLAND AND E. F. L ~ ~ S C H E I ~

TABLE IV li 1'Pase ilcticities o j the Contrattile Proteins of the iictottiyosin 'I'ype Isolated from Different Vertebrate Tissues

Tissue or cell

Enzymatic activity determined a t ionic I3ivalent ion strength' (concentration : Spcwirs M) 0.07-0. l p 0 . 5 4 . 6 p

References

Itahbit Ital h i t

Mg++ (:a++

0 . 253 0.257

0.005 0.078

Hasselbach (1'352) Hasselhach (1052)

Porcine Ihvine I'orrinc Ihvine

Mgf+ Mg+' Ca++ Ca++

0.016 0.010 0.025 0.011

0.015 0.040 0.092 0.025

Ledermair Ledermair Ledermair Ledermair

(1950) (1959) (1059) (195O)

Bovine Human Bovine Human

Mg++ Mg++

0.012 0 ,007

&++ Ca++

0.012

0.004 0.004 0.040 0.025

Ledermair Ledermair Ledermair Ledermair

(1959) (1959) (1959) (1959)

Tumor cells

Rat

Mg++

0.0027

0.0021 Hoffmann-Berling (1956)

IHood platclcts

Human

Mg+f

0 ,0038

Blood platelets

Human

Ca++

0.0056

0.0024 Bettex-Galland and Luscher (1961) 0.016 Bettex-Galland and Luscher (1961)

Striated musrlr Striated musde IJterus mnsclc Nongravid Nongravid Nongravid Nongravid Utrrus muscle Gravid Gravid Gravid Gravid

0.013

a Activity: pmoles Pi per milligram of protein per minutc. The conditions under which these activities were determined were approximately pH 7,20"C, and 10-3 M ATI'.

Most likely, proteins of the actomyosin type also exist in other smooth muscles. Iiiiegg and Strassiier (1963), for instance, have isolated from arterial walls a protein which is soluble a t high ionic strengths arid which exhibits ATPase activity. More detailed information about this material is not yet available, and therefore a comparison with the other contractile proteins is not possible a t the present time. 3. Contractile Protein from R a t Sarcoma Cells

As mentioned previously, Hoffmann-Berling (1954, 1956) was the first t o demonstrate the presence of a contractile material of the actomyosin type in cells of nonmuscular origin. He did SO by first proving the contractility of glycerol-extracted cells on addition of ATP, and later, in spite of the many difficulties involved, succeeded in extracting the contractile

T H E CONTRACTILE PROTEIN FROM BLOOD PLATELETS

27

protein. The properties of the actomyosin-like protein isolated from rat sarcoma cells will be discussed briefly here (Hoffmann-Berling, 1956, 1960, 1961). The starting material for the isolation were cells of the Jensen and Yoshida sarcomas, and the procedure used took into account the possible ’ based on the interference of the nucleoproteins. Yields of 0.1 to 0.2 % fresh weight of the cells were obtained (Table 111). Above an ionic strength of 0.3 p the protein is completely soluble; below this value, the solubility decreases steadily, but a sharp inflection point in the solubility curve is missing. Addition of ATP increases the solubility. With ATP and Mg++ ions, precipitates of these extracts will show superprecipitation, i.e., a comparatively slow contraction, which is abolished by the addition of Salyrgan. ATP sensitivity is about 70 yo and the ATPase activity amounts to 0.002-0.003 pmole P , per milligram of protein per minute (cf. Table IV). This activity varies with the ionic strength and with the magnesium concentration; it too is inhibited by Salyrgan. Most likely, a relaxing factor is present in the cells used because only exhaustive extraction with glycerol makes them susceptible to the action of ATP. The isolation of such a factor has not yet been attempted; it has been found, however, that the relaxing factor prepared from striated muscle remains without effect upon these contractile “cell models” (Hoffmann-Berling, 1961).

B. Other Contractile Systems The analogies in the contractile systems discussed above might easily lead to generalizations as to the reaction mechanisms of all contractile or even motile activities. There exists a considerable number of contractile systems which are based on different principles, in spite of the fact that they also depend for activity on the presence of ATP as well as the bivalent cations Mg++ and Ca++. Contractions of this type are observed in the stalks of Vorticella; the undulating rhythmical motions of flagella and cilia as well as the contractions of the tails of bacteriophages also belong to this alternative type of contractile mechanisms. Still other examples are certain cell movements restricted to the elongation of the cell body, the projection of the trichocystes of certain ciliated or flagellated protozoas, and finally the elongation of the inner portions of the mitotic spindle. Many of these systems have been studied in the form of cell models, and the results are summarized in two excellent reviews by Hoffmann-Berling (1960, 1961). Owing to the small dimensions of the structures involved, most of these materials are available only in very limited amounts and up to now this has prevented the preparation of the contractile proteins in a purified form.

28

M. BETTEX-GALLAND AND E. F. LUSCHER

IV. RELATION OF THROMBOSTHENIN TO OTHERCONTRACTILE MECHANISMS The discussion of the similarities and dissimilarities of thrombosthenin and other contractile proteins will be restricted to the group of actomyosinlike proteins. As already mentioned, contractile substances of other types have not yet been isolated in a purified form and most of our knowledge about them comes from studies of glycerol-extracted cell models, which have not yet been prepared from blood platelets. Lastly, there can be little doubt that the mode of action of these other systems must be quite different from thrombosthenin.

A . Quantitative and Morphological Criteria 1. Concentration of Contractile Mderials in Cells or Tissues

The concentrations of contractile proteins in different tissues are tabulated in Table 111. It is tempting to interpret these values as reflections of the different degrees of specialization of the tissues involved. The finding that the uterus a t the end of gravidity still seemingly contains the same amount of actomyosin is not in agreement with this view. It has been postulated, however, that in this case qualitative rather than quantitative changes brought about by a shift in the actin-myosin ratio are responsible for the adaptation to predominantly contractile activity (Hasselbach and Zedermair, 1958). Still appIying the same criterion of specialization, the blood platelets obviously must be considered a highly specialized cell, even when keeping in mind that the absence of a nucleus is partly responsible for the high ratio of contractile to other proteins. It might perhaps also be argued that contractile proteins are abundant in the thrombocyte because of its origin from the megakaryocyte, the latter cell displaying a particularly high degree of motility in the course of its evolution. The platelets themselves, however, show sufficient evidence for a high dependence of their functions on contractile activity to justify discarding such a one-sided view. 2. Stale or Structure of Contractile Materials

The structure of the striated muscle and in particular the distribution of actin and myosin within the myofibril is well established today. This concerns not only the topography but the functional state as well and is the result of a whole series of admirable observations involving more recently high-resolution electron microscopy on ultrathin sections (see review by Huxley and Hanson, 1960). The striated muscle is composed of fibers consisting of giant multinurleatcd cells. These fibers contain the myofibrils in a longitudinal arrangement which is characterized by a transversal

THE CONTRACTILE PROTEIN FROM BLOOD PLATELETS

29

periodicity with isotropic and anisotropic bands, i.e., the striation typical for this type of muscle. The fibrils consist of the contractile elements, the actin and the myosin being arranged in filaments parallel to the longitudinal axis. The anisotropic band of the fibril is constituted mainly of myosin filaments, whereas the finer arid isotropic strands of the actin are in close relation to the Z-baiid which forms the boundaries of the sarcomeres. In a very regular, hexagonal pattern, the actin fibers enter the interspaces between the ends of the strands of the A-band, composed mainly of the myosin molecules. Contraction and elongation of this structure is then explained in terms of the more or less pronounced interdigitation of the actin and myosin moieties. This implies that even exteiisive contraction of the muscle is possible without a corresponding shortening of the myosin or actin fibers, respectively. On a molecular level the actin strands slide along the myosin chains (Huxley and Niedergerke, 1954; Huxley and Hanson, 1954; Huxley, 1963). This process must be due to the cyclic formation and breaking of weak bonds between the two constituents of the contractile complex. It might well be that ATP is directly or indirectly involved in the latter process, but it remains a fact that, in spite of many hypotheses, a better understanding of the essential role of the nucleotide still has not been achieved. It seems suggestive to assume that the very strict spatial arrangement of the constituents of the contractile system, as realized in striated muscle, is capable of mediating an optimal energy transformation. It is remarkable though that precipitates of extracted actomyosiri are equally capable of contracting to 20 yoof their original dimensions. Most likely, the basic mechanism, i.e., the sliding of the two component molecules along each other is the same, and it must be coricluded that the spacial arrangement which is a prerequisite for this mechanism is a property inherent to the molecules themselves. The orderly array within the muscle fibril is then perhaps a structural necessity for the synchronization of the contractions of the many units involved. The smooth muscles of the vertebrates are composed of mononucleated cells surrounded by a membrane. On excitation these cells may nevertheless behave as a syncytium. Electron microscopy studies have not led to newer findings with respect to the internal organization of these cells; they have mainly confirmed that there exists only one type of filament orientated parallel to the longitudinal axis of the myofibril. Striations are completely missing. These results have been obtained on a variety of different organs, such as the gall bladder, the urinary bladder, and the uterus (Caesar et al., 1957; Hanson and Lowy, 1957; Schoenberg, 1958; Csapo, 1960). Although many micrographs of thin sections from blood platelets have been published, no evidence for the presence of orientated or filamentous

30

M. BETTEX-GALLAND

AND E. F. L ~ S C H E R

strurtures which can be ascribed to thrombosthenin has ever been found. This may be due to inadequate techniques for detecting such structures; equally well it could mean that thrombostheniii is not present in the platelet in aiiy organized form. Taking into consideration that thrombosthenin is present in ten times higher concentration than the contractile proteins in undifferentiated cells, such as those described by Hoffmann-Berling (1956), it is not astonishing that, in the latter case also, no evidence for any such structures has been detected. This of course leads to the questions as to the state in which these last mentioned proteins, as well as thrornbosthenin, are present within the cell. Let us recall that thrombosthenin is distinctly more soluble than muscle actomyosin, although it is still insoluble within the so-called physiological range of ionic strength. It should be kept in mind, however, that it might be possible that the ionic concentration is not constant throughout the different compartments of a cell. Furthermore, the ATP concentration is equally if not more important for the solubility of the contractile substances, and again, adequate methods for estimating local concentrations of the nucleotide in different regions of the cytoplasm are not yet available. Thus, the possibility remains that the contractile proteins described by Hoff mami-Berling, as well as thrombosthenin, are present within the cell in a soluble form.

B. A Comparison of the Enzymatic Activities of Diflerent Actomyosin-Like Proteins Among the different properties of the actomyosins which can be quantitatively determined, the ATPase activities give the most reproducible results. With thrombosthenin, as well as with the actomyosin from striated rabbit muscle, minor variations in the method of isolation or storage effects may already give rise to inconsistent viscosity values or to changed properties with respect to superprecipitation. Nevertheless, it should always be kept in mind that a strict standardization of the ATPase test remains a necessity; thus, the enzyme of the actomyosin from striated muscle is very sensitive to changes in ionic. strength, and the thrombostheniri ATPase is greatly influenced by very small vhanges in the Ca++ concentration. In comparing the different enzymatic activities, the pronounced species dependence of the actomyosins from smooth muscle ought to be taken into consideration. In Tablc I V an attempt has been made to summarize the available data, as far as they appear comparable, on the enzymatic activities of the different actomyosiiis. In the presence of Mg++ ions and a t a low ionic strength, the actomyosin from striated muscle is by far the most powerful ATPase, in fact about ten times as active as the corresporiding activity of

T H E CONTRACTILE PROTEIN FROM BLOOD PLATELETS

31

smooth muscle. Still weaker are the activities of thrombostheniii and the contractile protein from tumor cells. The two last mentioned enzymes appear very similar, if not identical; they even show the same dependence of the ATPase activities on changes in the ionic strength (cf. Fig. 7).

C. Dzscussion

A comparison of the properties of the actomyosin-like contractile proteins shows qualitative similarities as well as quantitative differences. Throughout, these proteins are soluble only a t an elevated ionic strength, they show ATP sensitivity as well as the phenomenon of superprecipitation, and they are all active as ATPases. This last mentioned property offers the most convenient means for a differentiation of the actomyosins; in fact, the enzymatic activities of the contractile proteins from striated muscle, from smooth musck, and from tumor cells or blood platelets differ markedly from each other. Further work will have to be done in order to permit the conclusion that the ATPase activities always decrease in the sequence mentioned above. Both thrombostheriiri and the proteins from undifferentiated cells are soluble a t a somewhat lower ionic strength than the corresponding muscle proteins. Finally, it is of particular interest to note that the relaxing factor obtained from striated muscle does not affect contractile models of smooth muscle and most likely is also ineffective against tumor-cell models. It should be mentioned that only the actomyosins of muscular origin are arranged in visible and well-organized structures within the cell. All these differences proved very valuable in establishing beyond doubt that the diff went members of the actomyosin family are distinct entities. This seems important in view of the overwhelming evidence which speaks for their very close similarity. The most impressive observation undoubtedly is the iiiterchangeability of the actiii arid myosin moieties obtained from striated muscle with the thrombosthenins A and M. It may be recalled that iii the cited experiments even the species were different, and nevertheless, active products were obtained on cross-reacting the different components of the system. As would be expected, this cross-reactivity is not a special feature of the actomyosiii-thrombosthenin system. Csapo (1960) mentions unpublished experiments by Nagy and Csapo which showed that actin from striated muscle restores ATP sensitivity in a preparation of myosin isolated from smooth muscle. This could mean that relatively nonspecific complexing is already sufficient for restoring ATPase activity and for the manifestation of ATP sensitivity. It would be interesting t o know whether the more stringent conditions for re-establishing contractility are also fulfilled. Such experiments have not yet been performed with respect to the actomyosin-thrombosthenin system.

32

M. BETTEX-GALLAND AND E. F. LUSCHER

Another intercsting question relates to the problem of cellular differentiation. The fact that undifferentiated cells contain their contractile protein in a nonstructured form might lead to the speculation that this represents the state of a precursor of the structured actomyosins in the course of cellular devclopment. Actually, this does not seem to be true. Holtzer et al. (1957) working with fluorescent antimyosin antibodies were able to show that, whenever myosin appears in the cellular protoplasm of the chicken embryo myoblasts, it does so already incorporated into a fibrillar structure. It has generally been assumed that the energy required for muscular contraction was derived directly from the ATP split in the course of the reaction. The establishment of accurate relationships, however, has been hampered by the fact that the actomyosin ATPasc continues to degrade the nucleotide long aftcr contraction has reached an optimum. The availability of thrombosthenin with its much Iower ATPase activity might be an interesting material for a comparative investigation of this basically important mechanism. Thus, thrombosthenin may prove not only interesting with respect to the functions of the blood platelets but also with respect to the general importance of the elucidation of the many remaining problems in the field of the contractile proteins.

REFERENCES Ballcrini, G., and Secgcrs, W. H. (1959). Thromb. Diath. Haemorrhag. 3, 147. Bhriny, M., and Jaislc, F. (1960). Biochim. Biophys. Acta 41, 192. BkLny, M., Bgrhy, K., and Guba, F. (1957). Nature 179, 818. Llernhard, W., and Leplus, R. (1955). Schweiz. M e d . Wochschr. 85, 897. Bessis, M. (1950). Blood 5, 1083. Rcttex-Galland, M., and Liischer, E. F. (1959). Nature 184, 276. Bcttex-Galland, M., and Liischcr, E. F. (1960). Thromb. Diath. Haemorrhag. 4, 178. Lkttex-Gslland, M., and Liischer, E. F. (1961). Biochim. Biophys. Acla 49, 536. Bettex-Galland, M., and Liischer, E. F. (1963). Proc. Intern. Congr. European SOC. Hematol., Sth, Lisbon, Vol. 2, p. 1227. Karger, Basel. Bettex-Galland, M., and Liischer, E. F. (1964). Pathol. Microbiol. 27, 533. Bettex-Galland, M., and Maupin, B. (1961). ZZe'mostase 1, 375. Bettex-Galland, M., Portzehl, H., and Liischer, E. F. (1962). Nature 193, 777. Bcttex-Galland, M., Portzehl, H., and Liischcr, E. F. (1963a). Helv. Chim. A d a 46, 1595. Bettex-Galland, M., Liisi~hcr,E. F., Simon, G., and Vassalli, P. (196313). Nature 200, 1109. Bizzozero, J. (1882). Arch. Pafhol. Anat. Physiol. 90, 261. Bloom, G. (1955). Z. Zellforsch. Mikroslcop. Anat. 42, 365. Borchgrevink, C. F., ant1 Waaler, B. A. (1958). Acta M e d . Scand. 162, 361. Born, G. V. R. (1956). J. Physiol. (London) 133, 61P. Born, G. V. R. (1958). Biochem. J. 68, 695.

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Bounameaux, Y. (1956). Experientia 12, 355. Bounameaux, Y. (1957). Rev. Hematol. 12, 16. Bounameaux, Y. (1959). Compt. Rend. SOC. Biol. 153, 865. Bounameaux, Y. (1961). Thromb. Diath. Haemorrhag. 6, 504. Braunsteiner, H., Pakesch, F., and Sailer, S. (1960). Acta Haematol. 24, 311. Briggs, F. N., and Fuchs, F. (1960). Biochim. Biophys. Acta 42, 519. Budtz-Olsen, 0. E. (1951). “Clot Retraction.” Blackwell, Oxford. Caesar, R., Edwards, G. A., and Ruska, H. (1957). J . Biophys. Biochem. Cytol. 3, 867. Castaldi, P. A., Firkin, B. G., Blackwell, P. M., and Clifford, K. J. (1962). Blood 20, 566. Chen, T. L., and Tsai, L. (1948). J . Physiol. (London) 107, 280. Corn, M., Jackson, D. P., and Conley, C. L. (1960). Bull. Johns Hopkins Hosp. 107, 90. Cretius, K., and Jaisle, F. (19601. Arch. Gyneakol. 194, 266. Csapo, A. (1960). I n “The Structure and Function of Muscle” (G. H. Bourne, ed.), Vol. I, p. 229. Academic Press, New York. De Marsh, G. B., Kantz, J., and Motulsky, A. G. (1955). J. Clin. Invest. p. 929. Discombe, G. (1950). Acta Haematol. 3, 151. Donnk, A. (1842). Compt. Rend. Acad. Sci. 14, 366. Eberth, J. C., and Schimmelbusch, C. (1886). Arch. Pathol. Anat. Physiol. 103, 39. Feissly, R., Gautier, A., and Marcovici, J. (1957). Schweiz. Med. Wochschr. 87, 1218. Fonio, A,, and Schwendener, J. (1942). “Die Thrombocyten des menschlichen Blutes.” Huber, Bern, Switzerland. Gaarder, A., Jonsen, J. V., Laland, S., Hellem, A., and Owren, P. A. (1961). Nature 192, 531. Glanzmann, E. (1918). Jahrb. Kinderheilk. 88, 113. Grette, K. (1962). Acta Physiol. Scand. 56, Suppl. 195. Grette, K. (1963). Nature 198,488. Gross, R. (1961). I n “Blood Platelets” (Henry Ford Hosp. Intern. Symp.), p. 407. Little, Brown, Boston, Massachusetts. Gugler, E., Stillhardt, H., Kaser-Glanzmann, R., and Liischer, E. F. (1962). Proc. Congr. Intern. SOC.Pediatrics, loth, Lisbon, p. 37. Hanson, J., and Lowy, J. (1957). Nature 180, 906. Hasselbach, W.(1952). Z. Naturforsch. 7b, 163. Hasselbach, W., Hofschneider, H. P., Kasper, E., and Lutz, R. (1953). 2. Naturforsch. 8b, 204. Hasselbach, W., and Ledermair, 0. (1958). Fortschr. Geburtshilfe Gynaekol. 7, 15. Hasselbach, W.,and Schneider, G. (1951). Biochem. Z. 321, 462. Hayem, G. (1878). Arch. Physiol. Norm. 5, 692. Heilbrunn, L. V. (1961). In “The Functions of the Blood” (R. G. Macfarlane and A. H. T. Robb-Smith, eds.), p. 283. Blackwell, Oxford. Hellem, A. J. (1960). Scand. J . Lab. Clin. Invest. 12, Suppl. Hoffmann-Berlmg, H. (1954). Biochim. Biophys. Acta 14, 182. Hoffmann-Berling, H. (1956). Biochim. Biophys. Acta 19, 453. Hoffmann-Berling, H. (1960). I n “Comparative Biochemistry” (M. Florkin and H. S. Mason, eds.), Vol. 11, p. 341. Academic Press, New York. Hoffmann-Berling, H. (1961). Ergeb. Physiol. Biol. Chem. Exptl. Pharmakol. 51, 98. Holtzer, H., Marshall, J. M., and Finck, H. (1957). J . Biophys. Biochem. Cytol. 3, 705.

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Hovig, T. (1962). Thromb. Diath. Haemorrhag. 8, 455. Hovig, T. (1963). T h r o m b . Diath. Haemorrhag. 9, 264. Hugues, J. (1959). Thromb. Diath. Haemorrhag. 3, 35. Hugues, J. (1960). C o m p t . Rend. SOC.Biol. 154, 866. Hugues, J. (1962). Thromb. Diath. Haemorrhag. 8, 241. Hugues, J., and Lapierre, Ch. M. (1964). T h r o m b . Diath. Haemorrhag. 11, 327. Hutter, R. V. P. (1957). Am. 1. Clin. Pathol. 28, 447. Huxley, H., and Hanson, J. (1954). Nature 173, 973. Huxley, H. E., and Hanson, J. (1960). I n “The Structure and Function of Muscle” (G. H. Bourne, ed.), Vol. I, p. 183. Academic Press, New York. Huxley, H. E. (1963). J . Mol. Biol. 7, 281. Huxley, A. F., and Niedergerke, R. (1954). Nature 173, 971. Izak, G., Nelken, D., and Gurevitch, J. (1957). Blood 12, 507. Jaisle, F. (1961). Klin. Wochschr. 39, 1044. Jean, G., and Gautier, A. (1961). C o m p t . R e n d . Acad. Sci. 253, 2274. JGrgensen, L., and Borchgrevink, C. F. (1963). Acta Pathol. Microbial. Scand. 57, 40. Kaser-Glanzmann, R., and Liischer, E. F. (1962). Thromb. Diuth. Haemorrhag. 7, 480.

Kinosita, R., Ohno, S., and Bierman, H. R. Motion picture : “Thrombopoiesis.” City of Hope Medical Center, Du:irte, California. Kjaerheim, A., and Hovig, T. (1962). Thromb. Diath. Haemorrhag. 7, 1. Kiippel, G. (1958). Z. Zelljorsch. 47, 401. Kuhnke, E. (1958). Arch. Ges. Physiol. 268, 87. Lechlcr, E., and Gross, R. (1962). Thromb. Diuth. Haemorrhag. 8, 355. Ledermair, 0. (1959). Arch. Gynaekol. 192, 109. Le Sourd, L., and Pagniez, P. (1906). C o m p t . Rend. Soc. Biol. 61, 109. I,ctt.rP, H. (1951). Naturwissenschaften 38, 490. Loewy, A. G. (1952). J . Cellular C o m p . Physiol. 40, 127. I,iischer, E. F. (1956a). Experientia 12, 294. Liischer, E. F. (1956b). V o x Sanguinis 1, 133. Luscher, E. F. ( 1 9 5 6 ~ ) Schweiz. . M e d . Wochschr. 86, 345. Liischer, E. F. (1959). Ergeb. Physiol. B i d . C h e m . Exptl. Pharmakol. 50, 1. Liischer, E. F. (1961). Jn “Blood Platclets” (Henry Ford Hosp., Intern. Symp.), p. 445. Little, Brown, Boston, Massachusrtts. Liischer, E. F. (1962). In “Erbliche Stoffwerhselkranlrheiten” (F. Linneweh, ed.), p. 480. Urban Schwarzenberg, Munich, Germany. Liischer, E. F., and Bettex-Galland, M. (1961). J . Physiol. (Paris) 53, 145. Liischer, E. F., and Bettex-Galland, M. (1962). Proc. Intern. Congr. Union Physiol. Sci., 22nd, Leyden, Vol. I, p. 229. Marcovici, I., Gautier, A., and Jean, G. (1961). Waemalologica (Pavia) 46, 921. Marsh, B. B. (1951). Nature 167, 1065. Martonosi, A. (1960). Biochem. Biophys. Res. Com,mun. 2, 12. Marx, R., Ibrom, H., and Stanislawski. F. (1960). Blut 6, 335. Maupin, B. (1954a). C o m p t . Rend. SOC.Biol. 148, 439. Maupin, B. (1954b). “Les plaquettcs sanguincs de l’liommc.” Masson, Paris. Muscatello, U., Anderson-Cedergren, E., and Azzonc, G. F. (1961). Biochim. Biophys. Acta 51, 426. Nakajima, H. (1960). Protoplasma 52, 413.

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Needham, D. M., and Cawkwell, J . M. (1956). Biochem. J. 63, 337. Parker, C. J., Jr., and Gergely, J. (1960). J . Biol. Chem. 235, 3449. Parmeggiani, A. (1961). Thromb. Diath. Hacmorrhag. 6, 517. Perry, S. V. (1960). Zia ‘Comparative Biochemistry” (M. Florkin and H. S. Mason, eds.), Vol. 11, p. 245. Academic Press, New York. Portzehl, H. (1957). Biochim. Biophys. Acta 24, 474. Portzehl, H., Schramm, G., and Weber, H. H. (1950). 2. Naturforsch. 5b, 61. Quick, A. J . (1950). Surg. Gynecol. Obstet. 91, 296. Rodman, N. F., Jr., Painter, J. C., and McDevitt, N. B. (1963). J . Cellular Biol. 16, 225. Roskam, J., Hugues, J., and Bounameaux, Y. (1961). J. Physiol. (Paris) 53, 175. Riiegg, J . C., and Strassner, E. (1963). Helv. Physiol. Phamacol. Acta 21, C57. Schmid, H. J., Jackson, D. P., and Conley, C. L. (1962). J. Clin. Invest. 41, 543. Schoenberg, C. F. (1958). J. Biophys. Biochem. Cytol. 4, 609. Schulz, H., and Hiepler, E. (1959). Klin. Wochschr. 37, 273. Setna, S. S., and Rosenthal, R. L. (1958). Acta Haematol. 19, 209. Silber, R., Benitez, R., Eveland, W. C., Akeroytl, J. H., and Dunne, C. J . (1960). Blood 16, 943. Sokal, G. (1960). “Plaquettes sanguines et st,rncturc tlu caillot. Etude morphologique et thrombClastographique.” Arscia, Brussels. Szent-Gybrgyi, A. (1951). “Chemistry of Muscular Contraction.” Academic Press, New York. Szent-Gyorgyi, A. G. (1960). In “Structure and Func.t,ion of Muscle” (G. H. Bourne, ed.), Vol. 11, p. 1. Academic Press, New York. Vasquez, J. J., and Lewis, J. H. (1960). Blood 15, 968. von Muralt, A. L., and Edsall, J. T. (1930). J. B i d . Chem. 89, 315, 351. Weber, H. H. (1957). Ann. Rev.Biochem. 26, 667. Weber, H. H., and Portzehl, H. (1952). Advan. Protein Chem. 7, 161. Witte, S., and Schricker, K. T. (1958). KZin. Wochschr. 36, 1119. Witte, S., and Schricker, K. T. (1960). 2. Ges. Exptl. Med. 133, 361. Wright, J . H. (1910). J . Morphol. 21, 263. Zucker, M. B. (1947). A m . J . Physiol. 148, 275. Zucker, M. B., and Borrelli, J. (1954). Blood 9, 602. Zucker, M. B., and Borrelli, J. (1959). J. Appl. Physiol. 14, 575. Zucker, M. B., and Borrelli, J. (1961). In “Blood Platelets” (Henry Ford Hosp., Intern. Symp.) . Little, Brown, Boston, Massachusetts. Zurker, M. B., and Borrelli, J. (1962). Proc. Sac. Exptl. Biol. Med. 109, 779.

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HYDROLYSIS OF PROTEINS By ROBERT 1. HILL Deportment of Biochemistry, Duke University, Durham, North Carolina

I. Introduction . . . . . . . . . . . . 11. Methods for Measurement of Peptide Bond Hydrolysis . . 111. Acid Hydrolysis . . . . . . . . . . . A. Partial Acid Hydrolysis of Proteins: The Specificity of Acid Hydrolysis . . . . . . . . . . . . B. Partial Acid Hydrolysis: Structural Analpsis of Proteins . C. Complete Acid Hydrolysis . . . . . . . . D. Hydrolysis of Protein Derivatives . . . . . . IV. Alkaline Hydrolysis . . . . . . . . . . V. Hydrolysis of Peptide Bonds by Catalysis with Metals and Metal Chelates . . . . . . . . . . . VI. Enzymatic Hydrolysis . . . . . . . . . A. Trypsin . . . . . . . . . . . . . B. Chymotrypsin . . . . . . . . . . C. Pepsin . . . . . . . . . . . . . D. Bacterial Proteinases . . . . . . . . . E. Papain . . . . . . . . . . . . F. Carboxypeptidase A and B . . . . . . . . G. Leucine Aminopeptidase . . . . . . . . . . . VII. Total Enzymatic Hydrolysis . . . . . . VIII. Enzymatic Hydrolysis of Native Proteins . . . . . References . . . . . . . . . . . . .

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I. INTRODUCTION The first experiments on the acid hydrolysis of proteins were performed by Braconnot in 1820. Over the next 100 years studies by other workers with a variety of hydrolytic agents led to major advances in our knowledge of proteins, including the identification of the amino acid constituents of proteins and the development of the polypeptide concept of protein structure.' These studies are now, for the most part, of historical interest, and a detailed insight into protein hydrolysis has come only in the past 20 to 30 years. This is largely the result of three significant developments: Excellent reviews of these pioneering studies are given by Vickery (19221, Vickery and Osborne (1928), Mann (1906), and Greenstein and Winitz (1961) and provide exciting reading for those interested in historical aspects of protein chemistry.

37

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ROBERT L. HILL

(1) the discovery of chromatographic and electrophoretic methods for the examinatioii of the hydrolytic products of pure proteins; (2) the use of acids and enzymes as reagents for the systematic degradation of proteins into small peptides which are amenable to sequence analysis; and (3) the preparation of highly purified proteolytic enzymes which can be used for selective hydrolysis. The present review will deal with recent developments in the use of acids and enzymes for the hydrolysis of proteins, with particular emphasis on the hydrolytic methods which are employed in amino acid sequence studies. Particular attention will be given to those factors which must be considered in selection of a suitable hydrolytic reagent. These factors have been listed by Sariger (1952): 1. The reagent should produce a minimum of side reactions which lead to drstruction of the constituent amino acids, or if such reactions occur they should give stoichiometric amounts of known products. 2. The reagent should have a known specificity. Methods which lead to a limited specific degradation are needed in addition to methods which produce more extensive degradation with the production of small peptides. 3. The reagent should not result in synthesis or rearrangement of peptide bonds. Enzymatic hydrolysis will be discussed only as a method for degradation of proteins and peptides and no attempt will be made to discuss the vast literature on the physical and chemical properties of each of the proteolytic enzymes. A discussion of much of the older literature on hydrolysis can be obtained from the reviews of Vickery and Osborne (1928), Synge (1943), and Greenstein and Winitz (1961). Reviews of more recent aspects of hydrolytic degradation are those of Sanger (1952), Leach (1953), Desnuelle (1953), Thompson (1960), and Light and Smith (1963). Methods for nonenzymatic degradation have been discussed by Witkop (1961).

11. R~ETHODSFOB MEASUREMENT OF PEPTIDE BONDHYDROLYSIS Several physical and chemical methods have been used to follow the course of hydrolysis of proteins. Each method depends upon measurement of the disappearance of protein or the appearance of hydrolytic products during the course of the reaction. The choice of a method depends upon specific factors which are unique to each experimental situation, and one must choose a method in light of the information desired. Several methods can be employed that measure the amount of protein which remains after hydrolysis (Layne, 1957; Kirk, 1947). These depend upon differences in the solubility, optical rotation, absorption below 230 mp, or precipitability of unhydrolyzed protein arid hydrolytic products. Such

HYDROLYSIS O F PROTEINS

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methods, reviewed by Leach (1953), give little iiidicatioii of the nature of the bonds that are broken during the course of hydrolysis. Other methods which are based upon estimation of the appearance of hydrolytic products (peptides or amino acids) are widely used. Many of these methods have been reviewed by Davis and Smith (1955). The ninhydrin reagent can be used as a spectrophotometric (Moore and Stein, 1954; Spies, 1957) or a gasometric (Van Slyke et al., 1941) method. Other spectrophotometric methods are based on the biuret (Layne, 1957) and the Folin (Spies, 1957) reactions. Acidimetric titration in acetone, alcohol, and formaldehyde (Levy, 1957) is often convenient, especially with acid hydrolysis. Titrimetric methods in aqueous solution with aid of a pH-stat (Jacobsen and LBonis, 1951) are rapid and quite reproducible. The preceding techniques are applicable only for the measurement of the rate of hydrolysis of peptides and proteins, and the methods employed in the sequence analysis of polypeptides are required for identification of the residues which form the susceptible bonds. These methods have been reviewed in detail elsewhere (Moore and Stein, 1956; Anfinsen and Redfield, 1956; Greenstein and Winitz, 1961; Canfield and Anfinsen, 1963) and do not require comment here.

111. ACID HYDROLYSIS Despite the extensive use of acids for the hydrolysis of proteins, few of the parameters which control the extent and specificity of acid hydrolysis have been evaluated completely. It would be desirable to know in quantitative terms how hydrolysis varies as a function of temperature, pressure, acid concentration, kind of acid employed, and presence of nonprotein substances, but at this time such information is fragmentary. From the data on partial hydrolysis that are recorded, it is evident that each protein with its specific, unique sequence of amino acids presents, for purposes of hydrolysis, a wide variety of peptide bonds of unequal strength. Thus, partial hydrolysis of a protein involves parallel and consecutive cleavage of a variety of bonds, leading to a mixture of products so complex that kinetic analysis of the process often involves insurmountable experimental problems. The complex nature of partial acid hydrolysis and the lack of detailed knowledge of the kinetics of the process have not prevented its application to many problems in the determination of protein structure. Partial hydrolysis of peptides in strong acids may produce smaller peptides containing the overlapping sequences necessary for determination of the order of amino acids in the parent peptide. Dilute acids, under appropriate conditions, serve to cleave those bonds formed only by aspartic acid, and thus can be used to specifically degrade high molecular weight polypeptides into smaller peptides which are amenable to isolation and further charac-

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ROBERT L. HILL

terization. Total acid hydrolysis of proteins has been studied somewhat more extensively, because of its use in quantitative methods for analysis of amino acids (Moore and Stein, 1963). It also is employed routinely to liberate the amino acid derivatives which are formed in many determinations of amino-terminal end groups.

A . Partial Acid Hydrolysis of Proteins: T h e Specijicity of Acid Hydrolysis Gordon and associates (1941) were the first to examine partial acid hydrolysis in a semiquantitative manner, largely because of the development of chromatographic methods for the microdetermination of amino acids and peptides (Martin and Synge, 1941). In these studies wool, edestin, and gelatin were hydrolyzed at 37°C. in an excess of 10 N HC1 for several days. Throughout the course of hydrolysis, aliquots of the reaction mixtures were analyzed by methods which allowed estimation of total free amino acids, total number of peptides, average length of the peptide chains, and the rates of liberation of cysteine, ammonia (amide nitrogen), and the amino groups of hydroxyamino acids. From these data a number of conclusions could be made concerning the character of partial hydrolysis. Free amino acids were liberated from the outset of hydrolysis, and after about 1week approximately one-third of the total residues were free amino acids. The remainder of the total amino acids were smalI peptides, with dipeptides in the greatest proportion. Ammonia was liberated rapidly and was almost completely released within 48 hr. Cysteine was liberated a t a rate not significantly different from the average rate of other amino acids. The bonds involving the amino groups of serine and threonine were more labile to acid hydrolysis than bonds formed by other amino acids. Several workers subsequently confirmed these observations. Stein et al. (1944) examined the hydrolytic products with the manometric methods developed by Van Slyke and co-workers (1941) and demonstrated that dipeptides represented the overwhelming proportion of the products formed when silk fibroin was hydrolyzed for 96 h r a t 40°C in concentrated HCl. After 40 hr, the hydrolyzate contained about 25% free amino acids with the remainder of the residues existing as di- and tripeptides. Bull and Hahn (1948), using a spread monolayer technique for estimating molecular weights, examined partial acid hydrolysis of egg albumin. By this method, immediate cleavage of about fifty bonds was observed when egg albumin was dissolved in 7.6N HC1 a t 60°C. The remainder of the bonds were hydrolyzed a t much slower rates. These results show that the course of partial acid hydrolysis is not a random process but that it exhibits a certain degree of specificity. For purposes of discussion it is convenient to consider separately each aspect of this specificity.

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1. Hydrolysis of Amides

The rapid and complete release of ammonia during the early stages of hydrolysis is the result of cleavage of the amide groups of glutamine and asparagine. Studies with glutamine and asparagine (Gilbert et al., 1949; Vickery and Pucker, 1943), as well as with proteins (Chibnall et al., 1958), demonstrate the ease of hydrolysis of the amide bonds. Estimation of the ammonia in acid hydrolyzates prepared under conditions which result in only limited hydrolysis of peptide bonds (e.g., hydrolysis in vacuo of a protein for two hr at 110°C in 2 N HC1) serves as an excellent means for determination of the total amide content of a protein (Chibnall et al., 1958). Under conditions of hydrolysis where all peptide bonds are broken (see Section C . ) the yield of ammonia is somewhat greater than that expected on the basis of the amide content. This presumably results from liberation of the a-amino nitrogen of serine and threonine a s ammonia (Smyth et al., 1962). 2. Electrostatic gffects

The resistance of dipeptides to acid hydrolysis appears to be the result of inhibitory effects of the positively charged ammonium group gdjacent to the susceptible bond. The positive charge tends to repel Hf ions, and thus the dipeptide bond is more resistant to hydrolysis than a similar type of bond a t a greater distance from the amino terminus. Bonds which resist hydrolysis, as demonstrated by isolation of dipeptides from partial hydrolyzates, need not possess a greater thermodynamic stability than bonds which are hydrolyzed. Once a dipeptide is formed, its peptide bond is somewhat more kinetically stable than an analogous bond in a polypeptide. This type of electrostatic effect has been demonstrated in a number of studies on the kinetics of hydrolysis of simple peptides. Hamme1 and Glasstone (1954) determined the rates of hydrolysis of di, -tri-, tetra-, penta- and hexaglyciiie in 2 N HCl a t 65"-75°C and found that the specific rates of hydrolysis of the tetraglycirie and higher polyglycines increase almost linearly with the number of glycine residues in the chain. Lawrence and Moore (1951) showed that the rate of acid hydrolysis of the first peptide bond of triglycine is about eight times that of the bond in diglycine. Furthermore, the peptide bond in benzoylvalylvaline is much more labile to acid hydrolysis than the bond in valylvaline (Christensen, 1943, 1944). The influence of elcctrostatic effects on thc rate of hydrolysis of peptides is demonstrated vividly by the studies of Long and co-workers (1963) who made quantitative kinetic studies of the parallel and consecutive reactions which occur on hydrolysis of tripeptides. These workers employed an

42

ROBERT L. HILL

k

=

k = 15.5

2.17

A.

k = 1.45 B.

@ HI O I il Hl H,N- C-C-NI

0 II C Hz-COH

7%

H,c

+\ H

cH,

k=3.0

C.

k = 3.50

D.

k = 6.42

0 O I1 I HI HI OI1 I HI H,N-CH,-C-N-C-C-N-CH,-C-OH I

0 II

FIG.1. The mean first-order rate constants ( X lo3 min-') for the hydrolysis of leucylglycylleucine (A), leucylglycine (B), glycylleucine (C), and glycylleucylglycine (D) in 2 N HCI a t 94.15"C.

TABLEI Heat and Entropy Values for the Hydrolysis of Peptide Bonds i n Leucylglycylleucine and Glycylleucylglycinea Peptide Bond hydrolyzed AH: (kcal) AS' (cal/mole deg) a

Leu-Gly

Leu-Gly-Leu

Gly-Leu-Gly

Gly-Leu

Leu-Gly-Leu

Gly-Leu-Gly

Leu-Gly 21.6 -23.4

Leu-Gly 22.3 -20.1

Leu-Gly 20.2 -23.2

Gly-Leu 19.0 -27.1

Gly-Leu 20.2 -21.8

Gly-Leu 19.9 -25.4

From Long and Truscott (1963).

m

+4

tr

r +4

$ 0 r

w

e

E

3

44

ROBERT L. HILL

automatic amino acid analyzer (Spackman et al., 1958) to examine quantitatively partial acid hydrolyzates of leucylglycylleucine, glycylleucylglyciiie, and related dipeptides. The compounds and the rate constants for hydrolysis of the peptide bonds are given in Fig. 1. It is evident th a t the bonds in the tripeptides which are farthest removed from the positive charge on the a-amino group are hydrolyzed most rapidly. The leucylglycyl bond in glycylleurylglycine is hydrolyzed about three times as fast as the same type of bond in leucylglycylleucine. Similarly, the glycylleucyl bond in leucylglycylleucine is hydrolyzed about five times as fast as the glycylleucyl bond in glycylleucylglycine. Determination of the rate constants a t different temperatures permitted calculation of the quantities AHt and AS' for each bond. The heat and entropy values given in Table I show the influence of electrostatic effects when a mechanism for the hydrolysis of a peptide bond is considered. A possible mechanism is represented schematically as follows : O H 0 11 I H,N-CHR-C-N-CHR..

-

. . .COOH + Hf

O H 0 II I H,N-CHR--C-N-CHR.. 6tl

. . .COOH

O H 0 II I H.,N-CHR-C-N-CHR.

go

. ..

,

COOH

*Products

)6+

HO H H

Compound (I) is the iiiiprotoiiated form of the peptide, (11) the conjugated acid of (I), and (111) the activated complex.2 Recause of the repulsion of the H+ ion by the positive charge on the a-ammonium group, AHt should be somewhat larger for hydrolysis of the adjacent peptide bond than for hydrolysis of a bond further along the peptide chain. This is observed to be the case for the leucylglycyl bond, although steric effects (see below) also influence the values of AH'. The positive charges in (11) should also allow strong solvation in the region of the peptide bond, therefore ASi would be more negative for a terminal bond than for a bond farther removed This is the mechanism proposed by Long and co-workers (1963). It is generally agreed that protonation of the carbonyl oxygen rather than the amide nitrogen is predominmt (Martin, 1962) (luring acid hydrolysis of amides, but the nature of the protonatcd species does not alter the conclusions givcii by Long and co-workers.

HYDROLYSIS OF PROTEINS

45

from the terminal a-ammonium group. Thus, the electrostatic effect decreases the rate of hydrolysis of a bond adjacent to the a-ammonium group on both energy and entropy grounds. The positive charge on eammonium, imidazolium, or guanidinium groups does not decrease the rate of hydrolysis of peptide bonds significantly. On analysis of acid hydrolyzates, Gordon et al. (1941) found little difference between the ratio of the free basic amino acids to the total basic residues in a protein and the corresponding ratios for neutral amino acids. This suggests that the charge on the side chains of lysine, histidine, and arginine is too far removed from the peptide bonds formed by these amino acids t o play a n important stabilizing role. 3. Steric Eflects

Although the studies of Gordon et al. (1941) did not reveal which peptide bonds were most resistant to acid hydrolysis, later studies by Synge (1944) and Christensen (1943, 1944) showed that dipeptides containing aminoterminal valine were not cleaved completely on prolonged hydrolysis. Later, Synge (1945) compared the susceptibility of various synthetic dipeptides to hydrolysis by an equal mixture of 10 N HCl and glacial acetic acid a t 37°C. Table I1 lists the relative rates of hydrolysis of the peptides examined in this study. Of the compounds studied, glycylglycine was hydrolyzed most rapidly and valylglycine most slowly. Glycylglycine was hydrolyzed a t a rate about 70 times that of valylglycine and some 25 times that of leucylleucine and leucyltryptophan. The resistance to acid hydrolysis of peptides with amino-terminal valine and leucine has been confirmed by others (Table I). Synge attributed the stability of these peptides to the steric limitation imposed by the isopropyl and isobutyl side chains of valine and leucine on the approach of H+ ions to the peptide bond. Leucine stabilizes peptides less effectively than valine because the branched portion of its side chain is a t a greater distance from the susceptible bond than the isopropyl side chain of valine. Dipeptides with aminoterminal isoleucine are also resistant to acid hydrolysis as judged b y studies with insulin (Harfenist, 1953) and model peptides (Hirohata et aE., 1953; Muramatu et al., 1963a,b). The rate constants for the hydrolysis of a series of dipeptides of the type glycyl-X and X-glycine were determined a t three different concentrations of H+ ion by Muramatu et aE. (1963a,b). Table I11 lists the rate constants reported by these workers. It is noteworthy that dipeptides which contain carboxyl-terminal valine, leucine, or isoleucine are not as resistant to hydrolysis as those containing these amino acids as amino-terminal residues. Hirohata et al. (1953) and Muramatu et al. (1963a,b) have attempted to estimate the magnitude of steric effects on the basis of the known con-

cp

TABLE I1 Hvdrolysis of Peptides i n Acid Solution

Q,

Relative rate of hydrolysis (Gly-Gly = 1)

Peptidea DL-Aa-Gly Ala-Leu# DL-.kla-DL-Asp Ala-Ser Gly-~-Ala Gly-D-Ala Gly-DL-Ala Gly-ilsp Gly-Gly Gly-Leu Gly-Ser Gly-Tyr Gly-Try Gly-DL-Val Gly-Leu-Gly Gly-Leu-Gly ~U-ASP Leu-Glu Leu-Gly DL-L~u-G~~ Leu-Gly-Leu Leu-Gly-Leu Leu-D-Leu

2 N HC1, 99"cb

10 iV HCl-glacial acetic acid (50:50), 37"CC

0.69 0.32

0.62 -

-

2 N HCl, 100"Cd

1 A- HCI, 104"Ce

Dowex-50e

0.62

0.61

2.15

0.60 -

-

-

-

1.1 0.37

-

0.8 A; HCl, 54.5"CJ 0.56 -

-

0.40

0.62 1.94 1.0 0.34 1.83 0.43

-

-

1 .o 0.40

1.0 0.48

-

0.52

0.35 0.31

0.86 0.23 0.23

-

-

0.23 -

0.06

-

0.35 (Gly, Leu) 0.65 (Leu, Gly) 0.23 0.22 (Leu, Gly) 1.55 (Gly, Leu)

0.34 -

0.38

-

-

-

0.22

0.22

-

0.44

0.18 -

7

B

F

DL-hU-DL-LeU Leu-Try Pro-Phe Pro-Tyr Ser-Ala Ser-Gly Ser-Ser DL-Val-Gly DL-Val-DL-Ileu

-

-

0.045 0.041

0.29 0.74 0.40 0.40

I

0.015

-

All optically active amino acids are L isomers unless otherwise stated. Harris et al. (1956a). Synge (1945). Long and Lillycrop (1963). 6 Whitaker and Deatherage (1955). f Lawrence and Moore (1951). 0 Optical configuration undetermined.

a

0.0091

48

ROBERT L. HILL

Rate

TABLE I11 Constants for the Hydrolysis of L)ipeptidcsIL

_______Pcptide

Gly-uL-Tlir Gly-L-Me t (GIY-L-CYS-)? Gly-L-Leu Gly-L-His Gly-L-Pro GIy-L-Lys Gly-L-Phe Gly-L-Lyr G~Y-DL-V~ Gly-DL-Ileu L-Pro-Gly or,-Ala-Gly (1rCyS-Gly)z L-LYs-GIY L-Met-Gly ~-Leii-Gly L-Phe-Gly L-Tyr-GI y DL-Val-Gly m-Ileu-Gly a

1 . 5 N HCI 0 216 0 IT9 0 157 0.150 0 130 0.115 0. 115 0 . 104 0.103 0 .09!) 0.087 0.189 0,130 0.087 0.066 0.064 0.058 0.044 0.037 0.031 0.025

Rate constants (100°C) ________6 N HC1 Cone. HC1 2.08 1.63 1.15 1.27 1.48 0.57 1 .ti4 I .I(; 1 15 1.12

I .07 1.195 0 850 0.322 0.322 0.542 0.322 0.270 0.255 0.104 0.086

-

-

1.96 I , O!) __ -

-

2.265 -

0.716 0.615 1.359 0.703 0.659 0.476 0.231 0.207

From Muramata et al. (1963a,b).

figuration of the amino acid side chains. Their analysis permits calculation of a steric hindranre factor for each side chain. Figure 2 shows the relationship between this factor F and the observed rate constants for hydrolysis of a series of dipeptides of the type X-glyc*ine. The data of Long and co-workers (1963) also serve to illustrate the influenre of steric effects on the kinetic stability of peptide bonds. From the rate constants for the peptides listed in Fig. 1 it is clear that the leucylglycyl bond in glycylleucylglycine is hydrolyzed a t about one-half the rate of the glycylleucyl bond in leurylglycylleucine. Because the large isobutyl side chain of leucine would tend to break up the solvation sheath in the strongly solvated species (11),shown in the mechanism of hydrolysis g’ riven in Section III,A,2, a decrease in the value of AS’ is expected, and the rate of hydrolysis of the hindered bond is reduced. The data in Table I are in good agreement with the expected effects. Recently, Whitfield (1963) has applied the well-known “Rule of Six,” which is used t o explain steric factors in organic chemistry, to assess the

HYDROLYSIS OF PROTEINS

49

0.8

s

0.6

I-

2

a 0.4 u W

5 a

0.2

0

-

STERIC FACTOR F

FIG.2. The relationship between the first-order rate constant ( k ) for hydrolysis and the steric hindrance factor ( F ) for dipeptides of the type X-glycine. From Muramatu et al. (1963a,b).

stability of peptide bonds toward hydrolysis. He has shown that a peptide bond is most stable when atoms occupy position 6 in a dipeptide that is numbered arbitrarily as follows: 5c-C6

.“T’:

I

“ ,,w 5

I: ’i I:

I

N-C-C-N-C-C 3

2

3

4

-A-BThe “six number” for a peptide bond formed by residues A and B, is obtained by adding the number of atoms in A and 3. Thus, in alanylglycine, the “six number” is zero since the atoms in the side chains of alanine and glycine do not occupy position 6, whereas the “six number” for valylglycine is 6 since atoms in the side chain of valine occupy position 6 of the dipeptide. Peptides with a “six number” of 6, such as valylglycine or leucylleucine, are hydrolyzed slowly, whereas those with numbers of 3 are hydrolyzed a t intermediate rates and those with numbers of zero, are hydrolyzed most rapidly. The six numbers of the dipeptides examined by Synge (1945) as well as those for some synthetic polyamino acids (Heyns et al., 1958) correlate fairly well with the experimentally determined relative

50

ROBERT L . HILL

rates of hydrolysis. This method (mi be rxpected to give orily a rough indication of the kiiietir stability of peptide bonds, but might prove useful iii the absence of experimrntal data.

4. Hydrolysis of BorLds Forrtted by the Amino Group of

SerirLe and Threonine

The extreme lability of the peptide bonds formed by the amino groups of seriiie and threoriine has been studied in some detail since the origiiial observations of Gordon et aZ. (1941). Desriiielle arid Casal (1948) employed the dinitrofluoroberiaciie method to examiiie the liberation of amino groups during hydrolysis of proteins in 10 N HCl at 30°C. Desiiuelle atid Roiijour (1951), employiiig similar methods, determined the appearance of thrconine and seriiie amino erid groups 011 hydrolysis of globiii in 1 2 N HC1 a t 20°C. Both studies revealed a rapid liberation of amino groups of the hydroxy amino acids without extensive liberation of other erid groups. Harris et aZ. (1956a) determined the rates of hydrolysis of several synthetic dipeptides aiid found that those which contain the amino group of seriiie iu peptide linkage are more labile than other dipeptides (see Table I). Desriuelle arid Casal (1948) suggested that the lability of seryl and threoiiyl bonds could be explained by a mechanism similar to the N-0 acyl migration, a reaction which was shown first with N-acyl-pOH

H I -N-

I 0 H CH, H I I I II CHR-C-N-C-C-N-CHR’II

H O-FH2 I I -N- CHR-C=N-

H

I

O-YH2 I

-N-CHR-C=O

H I -N-CHR-

II

C -OH

7

C-C-N-CHR’II

B

C -C-N-CHR’I II H,N 0

+

H,N-

I YHz CH-C-N-CHH‘I1 0

HYDROLYSIS O F P R OT E I N S

51

hydroxy amino acids (Bergmann et al., 1923). Under the action of concentrated HCl, serine (or threonine) residues would form oxazoline rings followed by formation of an O-peptide structure in which the amino group of serine is liberated and an ester band is formed between the hydroxy group of serine (or threonine) and the carboxyl group formerly in peptide linkage. To test this hypothesis, edestin was treated with anhydrous sulfuric acid a t -20°C for several days and then hydrolyzed for 6 hr a t 18°C in 6 N HC1. Amino end-group analysis of the hydrolyzate revealed that the majority of the bonds broken by this treatment were those formed by the amino groups of serine and threonine. Treatment with sulfuric acid was believed t o promote N-0 acyl migration, and the ester bonds formed would be hydrolyzed specifically by HC1 a t 18°C. Subsequent studies of a similar kind (Elliott, 1951, 1953; Wiseblatt et al., 1955; Ramachandran and McConnell, 1955; Chibnall and Rees, 1953) have been interpreted as additional support for this hypothesis. At this time, however, some reservation must be given to the mechanism of Desnuelle and Casal (1948). If the N-0 acyl migration induced by sulfuric acid occurs, it should be reversed in alkaline solution. Lysozyme, after treatment with sulfuric acid and then dilute alkali, difiers considerably in its properties when compared to untreated enzyme, and extensive destruction of tryptophan occurs (Elliott, 1953). Furthermore, a considerable number of the amino groups of serine and threonine, which should be available after the sulfuric acid treatment, do not react with acylating agents. And finally, bonds formed by seryl and threonyl residues are very labile in dilute aqueous acids (Sanger and Tuppy, 1951) in contrast to the anhydrous conditions which are employed to obtain N-0 acyl migration in model compounds. It is also noteworthy that reaction of anhydrous formic acid with trypsin (Smillie and Neurath, 1959) might not involve N-0 acyl migration as suggested by earlier studies (Josefsson, 1958; Josefsson and Edman, 1957) with lysozyme and ribonuclease. 5. Hydrolysis of Bonds Formed by Aspartyl Residues

The bonds formed by aspartyl residues are very susceptible to hydrolysis in dilute acid. Sanger (1949) found high yields of peptides containing amino-terminal glycine in hydrolyzates of insulin prepared with 0.1 N HC1, whereas hydrolyzates prepared with more concentrated solutions of acid contained poor yields of the same kinds of peptides. He suggested that the negatively charged carboxyl groups of aspartic acid would attract hydrogen ions in dilute acid solutions and thereby increase the lability of neighboring peptide bonds. Subsequently, specific hydrolysis at aspartyl residues was demonstrated by Partridge and Davis (1950) who found

52

ROBERT L. HILL

preferential release of aspartic acid from several proteins on hydrolysis with dilute oxalic and acetic acids at 100°C. Thus the major products formed on hydrolysis of a peptide under these conditions are aspartic acid and peptides which represent the sequences between aspartyl residues. This is represented schematically as follows :

. . . A-B-C-~)-AS~-E--F-AS~-G-H-I-J-K 1 . . . A-B-C-11 + E-I! + GH-1-J-K

. .

. . + 2Asp

Other free amino acids, notably glutamic acid, appear only after most of the aspartic acid is liberated. Subsequent studies (Blackburn, 1950; Biserte and Pigache, 1951, 1952; Blackburn and Lee, 1954; Sanger and Tuppy, 1951) confirmed these observations, and Blackburn (1950) showed that the liberation of aspartic acid is a function of the H+ ion concentration and iiot a property of the weak acid. In light of these results, the high relative rate of hydrolysis of synthetic peptides which contain aspartic acid is not surprising (see Table 11). On the other hand, certain linkages, such as that of valylaspartic acid, may be sufficiently resistant to prevent complete liberation of aspartic. acid. A number of recent studies have examined in detail the preferential cleavage of proteins a t aspartyl residues (Schultz et al., 1954; Grannis, 1960; Schultz, 1961; Schultz et al., 1962). Schultz et al. (1962) determined quantitatively the release of aspartic acid from insulin, ribonuclease, and glucagon by 0.03N HC1 at 105°C. Asparagine is released at a slower rate than aspartic acid, although the absolute number of residues of aspartic acid released is related to the content of aspartic acid and asparagine in the molecule. Other amino acids are not liberated as rapidly. Schultz et al. (1962) calculated that one equivalent of the following residues is liberated from ribonuclease in the given times: aspartic acid < 1 hr; lysine, 30 hr; alanine, 40 hr; threonine, 44 hr; serine, 48 hr; glutamic acid, 54 hr; and glycine, 56 hr. The order of release of these residues is not in the same order as their abundance in ribonuclease (aspartic acid > seriiie > glutamic acid > alanine > threonine > lysirie > glycine). The application of this method to specific peptides is given in Section III,B,2.

B. Partial Acid Hydrolysis: Structural Analysis of Proteins 1. A pplicutions to Ihe Sequence Anmlysis o j Proteins and Polypeptides

The studies of Sanger and co-workers (1945-1955) on the amino acid sequence of insulin provide the best example of the use of partial acid hydrolysis for determination of the covalent structure of poIypeptides. When oxidized A- or B-chains were submitted to hydrolysis in 11-12 N HCl

HYDROLYSIS OF PROTEINS

53

for 3 4 days at 37"C, at least 48 peptides were obtained from the hydrolyzates of B-chain and 34 from hydrolyzates of A-chain. This large number of peptides was derived from polypeptides which contain only 30 (B-chain) and 21 residues (A-chain). The majority of the products were dipeptides with about half as many tripeptides and few higher peptides. Despite evidence for acid splits at almost all of the peptide bonds in each chain, the yields of certain peptides were very low, and complete sequence analysis of each chain required examination of fragments produced by enzymatic means. In accord with the earlier studies of Gordon et al. (1941) and Synge (1945), extensive cleavage of peptide bonds formed by the amino groups of serine and threonine was noted, whereas bonds formed by the carboxyl groups of valine and isoleucine were most stable. Bonds formed by glycine also proved to be unusually labile. From these observations it can be concluded that as a technique for producing fragments for sequence analysis partial acid hydrolysis has several limitations. The nature of hydrolyzates is very complex, the yields of many peptides are low, and fractionation of the products often is extremely difficult. Because of the small size of the peptides and fragments, reconstruction of the sequence of the parent peptide from overlapping sequences often cannot be achieved. When partial acid hydrolyzates of proteins which are larger than insulin were examined, the limitations of this technique became particularly apparent. Schroeder and co-workers (1953, 1954, 1957) and Kay et al. (1956) were unable to reconstruct sequences of any length from the peptides obtained from partial acid hydrolyzates of gelatin, silk fibroin, or feather calamus of turkey. Thompson (1955a,b) encountered similar difficulties when partial acid hydrolyzates of lysozyrne were examined. Despite the limitations of partial acid hydrolysis it has been employed by several investigators for analysis of small peptides which were produced by enzymatic hydrolysis of large polypeptides or proteins (e.g., Harris and ROOS,1959a,b; Margoliash, 1962; Dus et al., 1962). When used properly partial acid hydrolysis should remain a valuable technique in sequence analysis. 2. Hydrolysis in Dilute Acid

Hydrolysis in dilute acid under conditions which lead to the preferential rupture of aspartyl bonds may provide an excellent means for specific cleavage of polypeptides and proteins. Of all acid degradative techniques, hydrolysis in dilute acid appears to be the most specific and closely approaches the specificity of certain proteolytic enzymes. This method, however, has not been applied widely to problems on sequence analysis. Ingram and Stretton (1962) hydrolyzed a tridecapeptide from the &chain of human hemoglobin A2 for 12 hr a t 105°C in 0.25 M acetic acid. Prefer-

54

ROBERT L. HILL

ential cleavage of the two aspartyl bonds in the peptide oacurred without extensive hydrolysis of other bonds. More quantitative studies were performed by Schroeder and co-workers (1'363) who employed 0.25 M acetic arid a t refluxing temperatures for hydrolysis of peptides derived from 7-chains of human fetal hemoglobin. As shown in Table IV, extensive TABLEIV Hydrolysis of Peptides with Dilute A c i d a

Yield after hydrolysis Sequence*

Peptide

(%)

T-3

Val-Asn-Val-Glu-Asp-Ala-Gly-Gly-Glu-Thr-Leu-Gly-Arg

A.4-1 AA-2 AA-3 AA-4 T-9 AA-5 T-11 AA-6 Ah-7 AA-8 AA-9 AA-10

Val-Asn-Val-Glu Val-Asn-Val-Glu-Asp ASP

Ala-Gly-Gly-Glu-Thr-Len-Gly- Arg

Val-Leu-Thr-Ser-Leu-Gly-Asp-Ala-Ileu-Lys Ala-Ileu-Lys

Gly-Thr-Phe-Sla-Glu-Leu-Ser-Glu-Le ti-His-CMC-Asp-Lys LYE

ASP

Gly-'l'hr-Phe-Ala-Glu-Leu-Ser-Glu-Len-His-CM C Ser Gly

93 61 32 26 17

a From Schrocder et al. (1963). T-3,T-9, and T-11 are tryptic peptides which on hydrolysis gave the pcptitles designated by the symbol AA. * CMC, S-carboxymethylcystine.

hydrolysis occurred a t aspartyl bonds with little effect on bonds formed by asparaginyl, glutamyl, or glutaminyl residues. In one instance (T-11) random hydrolysis occurred as judged by isolation of significant amounts of serine and glycine in addition to the expected products. The low yields of some of the peptides also suggest that random hydrolysis occurred. 3. Partial Acid Hydrolysis of Peptides and Proteins Containing DisulJide

Bonds

Under special conditions partial acid hydrolysis can be used to obtain peptides which contain disulfide linkages in the form in which they occur in the intact protein. When acids are used for this purpose, disulfide interchange reactions, which allow random cleavage and reformation among the different disulfide bonds, must be avoided. Ityle and Sanger (1955) were the first to examine in detail the extent of disulfide interchange in acid solutions. Under the usual conditions of partial acid hydrolysis (12 N HCl, 37"C), extensive interchange occurred within a few hours. The

HYDROLYSIS O F PROTEINS

55

reartion was diminished in more dilute acid solution or on addition of thiol compounds a t the outset of hydrolysis. Hydrolysis in sulfuric acid resulted in less interchange, and mixtures of equal amounts of 2 0 N sulfuric acid and glacial acetic acid a t 37°C in the presence of thiol compounds gave little or no interchange. When insulin was treated in this manner, it was possible t o isolate peptides associated with two of the three unique disulfide bonds (Ryle et al., 1955). Similar methods were employed by Ryle and Aiifiriscii (1957) for loc3ationof the four unique disulfides in ribonuclease, but extensive interchange occurred and the results were inconclusive. Subsequently, enzymatic methods of hydrolysis were found to be necessary to obtain peptides in which the disulfide linkages of ribonuclease remained intact (Spackman et al., 1960). It is noteworthy that partial acid hydrolysis of proteins under conditions favoring the disulfide interchange reaction forms the basis of a method for determination of the cysteine and cystine content of proteins (Glazer and Smith, 1961).

4. Side Reactions during Partial Acid Hydrolysis I n their studies on insulin, Sanger and co-workers (1955) were concerned particularly with the question of whether the peptides in acid hydrolyzates truly reflect sequences in an intact peptide. For the most part, it would appear that they do, but in two instances interconversion of primary bonds has been detected. Sanger and Thompson (1953) noted that diketopiperazine formation during partial hydrolysis with dilute (0.1 N) HC1 could produce inversion of glycylvaline. When this dipeptide was heated with dilute acid, amino-terminal valine was detected, suggesting valylglycine formation. The ease of formation of diketopiperaziiies in dilute acid a t high temperatures has long been known (Greenstein and Winitz, 1961), but on partial hydrolysis with 12 N HC1 at 37"C, little of this type of cyclic anhydride is formed. The probability of inversions of this kind is increased with dipeptides which contain carboxyl-terminal isoleucine or valine. The strength of the two peptide bonds in the cyclic anhydrides which are formed with these residues will be quite different. The bond formed by the carboxyl group of valine is stabilized through steric limitations imposed by the branched aliphatic side chains (see Section III,A,3). Muramatu and co-workers (1963) have examined the relationship between hydrolysis and diketopiperazine formation with a variety of synthetic peptides at three different acid concentrations. The ease of formation of diketopipcrazines of proline dipeptides has been demonstrated (Smith and Rergmann, 1944), but does riot appear to have been observed in structural studies. A second type of acid-catalyzed process which might interfere with sequence studies is the interconversion of the a- and P-carboxyl groups of

56

ROBEKT L. HILL

HzN-CHz-C

O H H O II I I II -N- C -C- OH I CH / \ H3C CH,

H O H I It I HzN-CC-NI CH /I H,C CH,

0 I1 (2%- C OH

aspartic acid. This reaction was first noted by Swallow and Abraham (1958) with aspartyllysine derived from hydrolyzates of bacitracin. Similar reactions have been studied by Bernhard (1958). The results of Naughton et al. (1960) demonstrate how this reaction leads to difficulties in peptide-sequence studies. On acid hydrolysis of diisopropylphosphoryl proteins with 12 N HCl a t 37°C for 2 days, at least three different forms of peptides with the sequences, aspartylphosphoserine and aspartylphosphoserylglycine, were found. These forms represented the a-, p-, or a#-structures produced by interconversions of the aspartyl residues. H,C-CO,H I HC NH-HN/

\c’

II

Hzo + H,C-C=O I I HC Nf H ~ O -HN/ \c/

+-

It

0

0

a-Aspartyl f o r m

a,p-Aspartyl form

+

--HzO

HzC-C=O I I HC NH-HN/ ‘CO,H

+

P-Aspartyl f o r m

The three forms are interconvertible and were found in greatest yields when 12 N HC1 a t 100°C was employed. Short periods of hydrolysis a t 100°C with 5.7 N HC1 gave lower yields of the a,p- and p-forms. Longer periods of hydrolysis a t 37°C with 5.7 N HC1 did not produce detectable amounts of either the a,& or &form. The latter method seems to be preferred for partial hydrolysis if the interconversions of aspartyl residues are t o be avoided. Hydrolysis of the a,p-imide a t alkaline pH values leads predominantly to the p-form (Swallow and Abraham, 1958; Bernhard, 1958). Naughton et ul. (1960) noted that this type of reaction occurred with the peptide aspartyltyrosine on partial hydrolysis of A-chain of insulin (Sanger

HYDROLYSIS O F PROTEINS

57

and Thompson, 1953), but was not recognized as aspartyl interconversioii a t the time. Cyclization reactions of glutamic avid analogous to those of aspartic acid have not been found in sequence studies with peptides, but a n a! - y interconversion in model peptides of glutamic acid has been observed (Kornguth ei al., 1963). Another reaction which occurs readily at acid pH values is transformation of amino-terminal glutamine into amino-terminal pyrrolidorie carboxylic acid. 0

H,"

0

It C

\

H

This was first found by Sanger et al. (1955) in a peptide from insulin and was observed with other peptides by Hirs et al. (1956) and Smyth et 02. (1962). The reaction appears to occur when acidic buffers or dilute acids are employed for isolation of peptides. Conversion of the cyclic pyrrolidone carboxyl residue to a glutamyl residue is obtained on mild hydrolysis in dilute acids or alkalies. The cyclization reaction leads to difficulties when sequence methods are used which proceed from the amino-terminal end of a peptide. I n addition, this reaction can occur when a n internal glutamine residue becomes amino-terminal in the course of stepwise sequence analysis under acidic conditions, as in the Edman methods. An incorrect sequence for a peptide from ribonuclease was deduced a s the result of cyclization of amino-terminal glutamine and acidic destruction of serine and threonine in the same peptide (Smyth et al., 1962).

C. Complete Acid Hydrolysis The present chromatographic methods for the quantitative analysis of amino acids in protein hydrolyzates are sufficiently precise that the methods employed for preparation of hydrolyzates often limit the accuracy of the analytical methods (Hill et al., 1959). The experience gained from several studies indicates that total acid hydrolysis can best be achieved by treatment of a protein or peptide for 24 h r with 6 N HC1 a t 1lO"C, under conditions that rigidly exclude oxygen, nonprotein substances, and metals. Conditions which meet these requirements have been described by Moore and Stein (1963). Present evidence suggests that the composition of acid hydrolyzates prepared in this manner correctly reflects the amounts of most

58

HOBERT t. HILL

amino acids in the protein before hydrolysis. In order to determine the amino acid composition of a protein by analysis of acid hydrolyzates, two l&ctors must be taken into account. (1) Those amino acids which are destroyed almost caompletely by acid must be determined by other means. (2) Amino acids, which are destroyed to only a moderate degree or are liberated from peptide linkage very slowly, must be estimated by measuring the kinetics of destruction or release, as the case may be. Glutamine, asparagine, and tryptophan are destroyed extensively on acid hydrolysis. Glutamine is converted quantitatively to glutamic acid and asparagine to aspartic acid with the concomitant release of stoichiometric amounts of ammonia as the result of hydrolysis of the amide groups. It is evident that the ammonia which is produced within the first few minutes of hydrolysis is derived solely from these amides. Quantitative methods based on determination of the ammonia in short-term hydrolyzates provide accurate estimates of the total asparagine and giutamine content in proteins (Rees, 1946). Estimation of the total number of amide groups in this manner is required if a precise determination of glutamic and aspartic acids is sought, since the total aspartic and glutamic acid in complete acid hydrolyzates represents the sum of the two amino acids plus their amides. Methods for differential analysis of glutamic acid and glutamine, and aspartic acid and asparagine have been sought by selective modification of glutamic acid and aspartic acid side chains (Chibnall et al., 1958) or complete enzymatic hydrolysis (see Section VII) . Tryptophan in proteins is destroyed by acid, although considerable amounts often can be detected if oxygen and reduving substances are rigidly excluded. Olcott and Fraenkel-Conrat (1947) demonstrated that tryptophan is not destroyed extensively when heated a t 100"-125°C in 6-7 N HC1 (or sulfuric acid) an vacuo. When tryptophan is heated under similar conditions in the presence of air, edestin, zinc, serine, pyruvic acid, cysteine (or cystine), and a number of other substances, large losses are observed with formation of humin. The exact nature of the degradation products is unknown, although disruption of the indole ring seems to occur. For these reasons, tryptophan is most often estimated by direct analysis of the intact protein (Spies, 1950; Bencze and Schmid, 1957) or by analysis of alkaline (Dreze, 1960) or enzymatic hydrolyzates (Hill and Schmidt, 1962; Tower et al., 1962). Other amino acids are destroyed to a lesser extent as demonstrated by decreasing yields as a function of time of hydrolysis. Almost invariably 5-10 % of the serine and threonine is destroyed (Rees, 1946), the exact amount depending on the time of hydrolysis. Other amino acids, such as cysteine, aspartic acid, glutamic acid, lysine, arginine, tyrosine, and proline, have also been reported to be destroyed. Inasmuch as destruction varies

HYDROLYSIS OF PROTEINS

59

as a function of time of hydrolysis and differs from one protein to another, it is necessary for accurate measurements to determine the kinetics of destruction of each amino acid in the presence of the protein under examination and thereby correct analytical values to the appropriate extent. Hydrolysis of each protein appears to be an individual problem, and fixed destruction factors, which can be used to correct for losses, cannot be employed generally. A linear extrapolation to zero-time of hydrolysis based on zero-order kinetics has been used to estimate the destruction of each amino acid (Smith and Stockell, 1954; Wilcox et al. 1957). In contrast, Hirs et al. (1954) measured the destruction of amino acids in a mixture of serine, threonine, aspartic acid, and glutamic acid and showed that first-order kinetics are applicable to the destruction observed. Although not a direct consequence of acid hydrolysis, low yields of serine and glutamic acid often occur when HC1 is removed from acid hydrolyzates (Ikawa and Snell, 1961) by desiccation. Under these conditions a carboxyl group of glutamic acid is esterified with the hydroxyl group of serine. Low yields of cysteine and cystine are often encountered in acid hydrolyzates, especially when carbohydrates are present. For this reason determination of these amino acids as cysteic acid in acid hydrolyzates of oxidized proteins is required for accurate results (Schram et al., 1954; Moore, 1963). Often small losses of cysteic acid are encountered, but these can be estimated by time studies as described above. The products formed on destruction of the moderately labile amino acids have not been fully identified, although in two studies the increase in ammonia that is observed on prolonged hydrolysis is proportional to the serine and threonine lost (Smith et al., 1954; Smith and Stockell, 1954). Sanger and Thompson (1963) have shown that losses of tyrosine often may result from formation of 3-bromo- and 3,5-dibromotyrosine or the analogous chloro derivatives (Thompson, 1954a). Formation of these substances occurs owing to the presence of traces of chlorine or bromine in commercially available HC1. Minor amounts of oxidizing agents, such as air, increase the yields of these derivatives. Losses could be reduced if small amounts of substances which can be readily oxidized or halogenated are added a t the outset of hydrolysis. The stability of peptide bonds formed by valine, isoleucine, and leucine (see Section 1111A,3)often leads to low yields of these substances in total acid hydrolyzates. The optimal time of hydrolysis for complete liberation of these amino acids will depend upon the nature of the linkage in each particular protein, but generally, maximal yields are obtained after 70 hr (Mahowald el al., 1962; Noltmann et al., 1962; Smith el al., 1955; Wilcox

60

ROBERT L. HILL

et al., 1957). The isoleucylvalyl bond in insulin proved especially resistant to acid hydrolysis (Harfenist, 1953), and the maximal yields of these two residues could be obtained only after 96 hr hydrolysis. D. Hydrolysis of Protein Derivatives In view of the large amount of literature which has accumulated on the preparation, properties, and analysis of derivatives of proteins, a few comments on the problems encountered on acid hydrolysis of these substances are warranted. Complete acid hydrolysis of dinitrophenyl proteins is employed in the end-group methods of Sariger (1945). Under the usual conditions (6 N HC1, llO"C, 4-24 hr) almost all of the dinitrophenyl amino acids are destroyed to some extent, and large losses are noted using the derivatives of glycine, proline, hydroxyproline, serine, and threoniiie (Porter and Sanger, 1948; Fraenkel-Coiirat and Singer, 1956; Steven and Tristram, 1962). The destructive effect of tryptophan, cysteine, arid carbohydrates during hydrolysis has been indicated (Thompson, 1951; Desnuelle et al., 1951). For quantitative studies it is necessary to determine the destruction factors for breakdown of each dinitrophenylamino acid (Thompson, 1954b; Levy, 1954). Special conditions for hydrolysis often increase the yields of the most labile derivatives. Thus, dinitrophenylglycine is obtained in high yield on hydrolysis in mixtures of mineral and organic acids (Hanes et al., 1952). Steven (1962) has reported good yields of some dinitrophenyl derivatives from dinitrophenylgelatin after hydrolysis with Dowex-50. Special problems in the hydrolysis of the dinitrophenyl derivatives of human globin were noted by Hhinesmith et al. (1957a, 195713). In accord with the results of earlier studies, nonstoichiometric amounts of aminoterminal valyl residues were found in acid hydrolyzates of this derivative when prepared in the usual manner. Further studies revealed that hydrolysis for 15 min in refluxing 6 N HC1 yielded 90 yoof two valyl end groups as dinitrophenylvalylleucine, whereas on continued hydrolysis dinitropheiiylvaline appeared a t a rate higher than could be accounted for by hydrolysis of dinitrophenylvalylleucine. Measurement of the kinetics of formation of diriitrophenylvaline led these workers to conclude that hemoglobin coiitairis four valyl end groups per mole, but two are in the sequenw valylleucine, a particularly acid-resistant dipeptide, aiid two are in a sequence which gives dinitropheriylvaline in good yields during normal periods of hydrolysis. The inability to obtain the four elid groups in stoichiometric amounts resulted from the resistance to hydrolysis of the valylleucine peptide bonds. Phosphoserine and phosphothreoriine ester bonds (Perlmann, 1955)

HYDROLYSIS OF PROTEINS

61

are labile to acid hydrolysis, and quantitative determination of these derivatives cannot be made by direct chromatographic analysis of acid hydrolyzates prepared in the usual manner. Phosphoserine- or phosphothreoninc-containing peptides can be isolated from partial acid hydrolyzates (Hipp et al., 1957), although hydrolysis of the labile phosphoesters probably occurs to some extent. Some protein derivatives, in which specific amino acid side chains are modified, yield acid-stable amino acids. Thus, homoarginine produced by guanidination of proteins with 0-methylisourea is not destroyed extensively by acid and can be quantitatively estimated on analysis of acid hydrolyzates (Chervenka and Wilcox, 1956; Klee and Richards, 1957). Carboxymethylation of proteins with iodo- or bromoacetic acid yields S-carboxymethylmethionine, 1- or 3-~arboxymethylhistidine, 1,3-dicarboxymethylhistidine, ecarboxymethyllysine, and S-carboxymethylcysteine (Gundlach et al., 1959). The lysine and histidine derivatives are stable to acid hydrolysis, but extensive degradation of the methionine derivative occurs, and special methods are required for quantitative analysis of this substance in acid hydrolyzates (Neumann et al., 1962). Acid hydrolysis of proteins treated with nitrous acid results in an apparent destruction of t-hydroxy-a-amino-n-caproic acid (Shields et al. , 1959) , the deaminated product of lysine. S-(N-ethylsuccinimido)cysteine,which results when the thiol group of cysteine reacts with N-ethylmaleimide (Smyth et al., 1960) gives equal amounts of Ssuccinylcysteine and ethylamine in high yields (Smyth et al., 1961). Reaction of proteins with cyanate results in extensive conversion of lysine to homocitrulline (Stark et al., 1960). This derivative is decomposed to lysine to an extent of about 24 % per 24 hr under conditions of complete acid hydrolysis.

IV. ALKALINEHYDROLYSIS Alkalies have not been used extensively for degradation of proteins and polypeptides. Their limitations have been recognized for some time, and few attempts have been made in recent years to further evaluate this medium for hydrolysis of proteins. Much of the available information on alkaline hydrolysis has been reviewed elsewhere (Sanger, 1952; Leach, 1953; Desnulle, 1953), and only a few comments are necessary here. A number of amino acids are destroyed on alkaline hydrolysis; serine decomposes to give glycine and alanine ; threonine yields glycine, alanine, and a-aminobutyric acid; arginine gives ornithine, citrulline, and ammonia; cysteine and cystine yield alanine, hydrogen sulfide, ammonia, and pyruvic acid. Those amino acids which are stable in alkali are racemized to a considerable degree. Because alkalies promote such extensive changes, they have not been applied widely t o problems in protein hydrolysis.

62

ROBERT L. HILL

I n a few cases, alkaline hydrolysis has proved applicable to special problems. Tryptophan is not destroyed in alkali, and analysis of alkaline hydrolyzates forms the basis of one method for quantitative determination of this amino acid (e.g., Dreze, 1960). Despite the fact that tryptophancontaining peptides should be more stable in alkali than acid, partial alkaline hydrolysis has not been employed for identification of this type of peptide. Amino acids often can be regenerated by alkaline hydrolysis from derivatives obtained by the amino-terminal end-group methods. Dinitrophenyl amino acids and phenylthiohydantoin (Fraenkel-Conrat et al., 1955) as well as hydantoin (Stark and Smyth, 1963) derivatives of amino acids can be treated in this manner.

V. HYDROLYSIS OF PEPTIDE BONDSBY CATALYSIS WITH METALSAND METALCHELATES

A few examples are known of the effects of metal ions on hydrolysis of peptide bonds. Lawrence and Moore (1951) found that cobaltous chloride almost doubled the rate of acid hydrolysis of glycylglycine. The effect of the cobalt ion was reflected by a significant decrease in the activation energy and the activation entropy of the hydrolysis. Meriwether and Westheimer (1956) examined the effects of copper, cobalt, and nickel ions on the hydrolysis of glycinamide and phenylalanylglycinamide. Cupric ions were the most effective catalysts, and between p H 7.9-9.25 a t 75"C, they increased the rate of hydrolysis of glycinamide by a factor of thirty over the uncatalyzed hydrolysis. Considerable diketopiperasine formation occurred on hydrolysis of phenylalanylglycinamide under the same conditions, but cupric ion a t pH 5 directed the reaction to formation of glycine, phenylalanine, and ammonia, rather than formation of the anhydride. Although the mechanism of catalysis by metal ions is unknown, it is suggested that the ions are acting as Lewis acids in aqueous solution and may facilitate the attack of hydroxide ion or water on the peptide bond. Collman and Buckingham (1963) have reported preliminary results of studies on the hydrolytic cleavage of amino-terminal peptide bonds by cis-hydroxyaquotriethylenetetraaminecobalt(II1) ions. The amino-terminal residues of di- and tripeptides are selectively hydrolyzed by one equivalent of metal chelate and are converted to an inert metal complex. The reaction proceeds as shown on p. 63. The reaction is stoichiometric rather than catalytic and goes to completion within a few minutes at pH 7-8 at 6Oo-65"C. When four equivalents of chelate react with tetraglycine, four equivalents of the glycine-metal complex are formed. In view of the specificity as well as the rapidity of the reaction under rather mild conditions, it is evident that metal chelatcs of this type may prove useful for stepwise degradation of peptides.

63

HYDROLYSIS OF PROTEINS

1

pH 7-8 60”-65°C

+

O H 0 I1 I H,N-CHR-C-N-CHR”.

* *

-

VI. ENZYMATIC HYDROLYSIS The major recent advances in techniques for the hydrolysis of proteins have come from studies with proteolytic enzymes. Partial hydrolysis of proteins, as required for amino acid sequence studies, can best be obtained by enzymatic means, and complete enzymatic hydrolysis has been used on a few occasions. The advantages of enzymatic as compared to acid hydrolytic methods have been indicated by many workers (Sanger, 1952; Smith, 1950; Thompson, 1960; Anfinson and Redfield, 1956), but require additional emphasis here Possibly the most useful property of the proteolytic enzymes is their specificity. The ability of proteolytic enzymes to hydrolyze peptide bonds formed by specific amino acids give these enzymes several advantages over acids as hydrolytic agents. Among these are the following. High yields of the peptides (or amino acids) are obtained; less complex and more easily fractionated mixtures of peptides are produced; nonhydrolytic alteration of the products is rare; only catalytic amounts of enzyme are required; and finally, enzymes can be used to specifically modify biologically active substances in such a way that valuable information relating structure to function is obtained. The one limitation of enzymatic hydrolysis, which has been discussed repeatedly, is the possibility that artifacts are produced through transpeptidation (Katchalski and Sela, 1959; Waley and Watson, 1954; Blau and Waley, 1954). Although transpeptidation should be considered a major problem with proteolysis, it has not been demonstrated to occur to any extent in amino acid sequence studies reported a t the present time. Indeed, the best indication that transpeptidation has not allowed deduction of wrong sequences, has come

64

ROBERT L. HILL

from the successful synthesis of biologically active polypeptides whose structures were proved in great part with the aid of partial enzymatic hydrolysis (Hofmann et al., 1962; Hofmann et al., 1963; Li et al., 1963). The history of the development of proteolytic enzymes as agents for the specific degradation of proteins and polypeptides encompasses the work of numerous investigators over the past century, and no attempt will be made to describe this aspect of such an extensive subject. The following sections are devoted in great part to consideration of the specificity of proteolytic enzymes as revealed by recent studies on proteins and polypeptides whose primary structures are now almost completely established. The specificity of many proteolytic enzymes has been described in detail with the aid of synthetic substrates, but certain features of the specificity have come to light only when larger, more complex substrates have been examined.

A . Trypsin The limited specificity of trypsiri for hydrolysis of peptide bonds formed by the carboxyl groups of lysirie and arginine was first demonstrated by Bergmaim (1942) and co-workers and subsequently confirmed by others with synthetic substrates and proteins. Hesidues adjacent to the susceptible bond influence the rate of hydrolysis. This is demonstrated by the data in Table V which lists the relative rate of hydrolysis of various lysine and arginine peptides. It is evident that bonds adjacent to a free a-amino group are hydrolyzed very slowly. The observations that the amino-terminal lysine in riboriuclease (Hirs et al., 1960) and egg-white lysozyme (Canfield, 1963) is only partially removed on tryptic hydrolysis suggest that this specificity also applies t o polypeptide substrates. It would appear that the proximity of polar groups to the susceptible bond generally results in a decrease in the rate of hydrolysis. Thus, in the y-chain of human hemoglobin, the susceptible bonds in the sequences -aspartyllysylleucyl- and -aspartyllysylalaiiy1- are riot hydrolyzed completely on 3 to 4 hr digestion under conditions which show almost complete cleavage of other bonds (Schroeder el al., 1963). Similarly, the lysyl bond adjacent to the carboxyl-terminal aspartyl residue in P-MSH (nielanocytestimulating hormone) is somewhat resistant to hydrolysis. The only type of lysyl or arginyl bond which appears to be completely resistant, to trypsin is that formed with proline. I,ysylprolyl bonds in oxidized riboiniclease (Hirs et al., 1960), cortit.otropin (Leonis et al., 195Y), p-MSH (Harris and Roos, 1959b), and whale myoglobin (l
65

HYDROLYSIS O F PROTEINS

TABLE V SpeciJcity of T r y p s i n Toward Synthetic Substrates Substrates4 Benzoyl-L-argininamideb Glycyl-L-1ysinamideb L-Alanyl-L-1ysinamideb L-a-Aminobutyr yl-L-lysinamideb L-Norleucyl-L-lysinamideb @-Alanyl-L-lysinttmideb L-Valyl-L-1ysinamideb L-Leucyl-L-lysinamideb L-Phen ylalan yl-L-1ysinamideb L-Arginyl-L-leucinec L-Arginylgly cinec L-Arginylphenylalaninec L-Arginylglutamic aci& Cba-nitro-L-arginyl-L-leucinec Cbz-nitro-L-arginyl-L-phenylalaninec Cba-nitro-L-argininamidcc Benzoyl-L-homoargininamided a

At 0.05 M substrate, 25"-40"C, pH 7.5-7.8. Iaumiya et al. (1960). Van Orden and Smith (1954). Shields et al. (1959).

Relative rate of hydrolysis 100 91 200 406 424 82 324 155 124 0.036 0.029 0.029 0.017 0 0 0 0

Cbz, carbobenzoxy.

strates and numerous polypeptides and proteins of known structure, several workers have observed tryptic hydrolysis of ester substrates which do not possess the usual specificity features (Schwert et al., 1948; CastezedaAgull6 and del Castillo, 1959). Inagami and Sturtevant (1960) have shown that acetyl-L-tyrosine ethyl ester, a specific substrate for chymotrypsin, is hydrolyzed by trypsin under optimal conditions a t a rate onetwelfth that given by chymotrypsin. Hydrolysis of this substrate could not be accounted for by chymotrypsin contamination of the trypsin. This type of hydrolysis, as well as tryptic hydrolysis of numerous "virtual" substrates such as p-nitrophenylacetate and N-trans-cinnamoyl imidazole (Bender and Kaiser, 1962), has indicated the possibility of nonspecific cleavage an tryptic hydrolysis of proteins. The first suggestion that this might occur came from studies with glucagon. Thus, in a 50 hr tryptic hydrolyzate of glucagon (25"C, pH 7.8) cleavage occurred not only a t Iysyl and arginyl bonds but a t bonds carboxyl-terminal to phenyllanine and tryptophan (Bromer et al., 1957a,b). Hydrolysis for 2.25 hr did not result in these nonspecific splits. The amounts of chymctrypsin that contaminated the trypsiri preparations used in this study are unknown, but it is possible that the nonspecific splits occurred as the result of tryptic

66

ROBERT L. HILL

action. In several recent studies similar results were obtained, whether as the result of chymotrypsiii or trypsin. Prolonged digestion of oxidized papain resulted in three peptides with cerboxyl-terminal tyrosine and one with carboxyl-tcrminal asparagine (Kimmel et at., 1962), whereas four peptides, which could be obtained only as the result of “chymotrypticlike” action, were observed in tryptic digests of human cytochrome c (Matsubara and Smith, 1963). Walsh et at. (1962) observed hydrolysis at the asparagiiiylthreonyl and tyrosylthreonyl bonds on tryptic hydrolysis of bovine trypsinogen. Similar results were obtained by Tomitbek et al. (1963). Several attempts have been made to purify trypsin to the extent that it is inactive toward substrates of chymotrypsin. Trypsinogen, purified chromatographically by the methods of Keller et al. (1958) and activated just prior to use, is reported to produce a small amount of cleavage a t bonds in a-chains of human hemoglobin other than those formed by arginine and lysine (Guidotti et at., 1962). Chromatography of crystalline trypsin in 8 M urea on carboxylic acid ion-exchange resins (Cole aiid Kincaid, 1961) or on carboxymethylcellulose a t pH 3.2 (Liener, 1960) gives highly purified trypsin which possesses chymotryptic activity. Thus, it appears that the specificity of trypsiri is not as narrow as suggested by many earlier studies. On the other hand extensive hydrolysis a t bonds other than those formed by the carboxyl groups of lysine and arginine can be expected only on prolonged hydrolysis or when high concentrations of trypsin are employed. Despite the observations of hydrolysis a t bonds other than those formed by lysyl or arginyl residues, trypsin remains the most specific endopeptidase available at this time. When it can be employed for moderate periods of digestion, it is an ideal enzyme for producing one of the two sets of overlapping peptide fragments which must be obtained for proof of the sequence of amino arids in long polypeptides. Since it was iiitroduced for this purpose by Sanger (1952) aiid co-workers (Sanger and Tuppy, 1951; Sanger and Thompson, 1953; Sanger et al., 1955) in proof of the structure of insulin, it has been employed in every subsequent study on sequence analysis of large protein molecules. The peptides which result in high yield from tryptic action can be expected to possess carboxyl-terminal lysine or argiriinc with the exception of the single peptide formed from the varboxyl-terminal sequence of the substrate. Thus, with knowledge of the lysine and argiiiiiic rontent and the molecular weight of a protein, dctermiriatiori of the number of tryptic peptides derived from the protein provides valuable information about the number and kind of submiits in the substrate. Peptide maps of the tryptic hydrolyzates of human hemoglobin allowed Ingram (1956) to conclude that this molecule possessed two kinds of polypeptide chains and that the tctrameric molecule (molecular

HYDROLYSIS O F PROTEINS

67

weight 64,000) must contain two of each kind of chain. A similar type of analysis has been made with several other proteins including hemerythrin (Margoliash et al. , 1962) glyceraldehyde 3-phosphate dehydrogenase (Harris et al., 1963; DBvBnyi et al., 1963), and myosin (Kielley and Barnett, 1961). Although alternative methods for obtaining such information are available, analysis of tryptic digests in this manner is often useful. The action of trypsin on proteins and peptides can be limited to the bonds formed by arginine if the €-ammonium groups of lysine are modified. Reaction of proteins with fluorodinitrobenzene (Anfinsen and Redfield, 1956)) carbobenzoxychloride (Anfinsen et al., 1956), O-methylisourea (Weil and Talka, 1957), succinic anhydride (Li and Bertsch, 1960), potassium cyanate (Stark and Smyth, 1963), carbon disulfide (Merigan et al., 1962)) or ethylthiotrifluoroacetate (Goldberger and Anfinsen, 1962) results in derivatives which contain almost no free 6-ammonium groups, and tryptic hydrolysis proceeds only a t arginyl bonds. Dinitrophenyl proteins are insoluble, difficult to hydrolyze, and the dinitrophenyl peptides obtained on tryptic hydrolysis are often difficult to fractionate. These disadvantages, along with the difficulty of removing the dinitrophenyl groups from purified peptides, limit the usefulness of this method. Although carbobeiizoxy groups are readily removed from peptides by catalytic hydrogenation or cleavage in HBr-acetic acid, this type of protein derivative has not been examined extensively. The succinylated or guanidiriated proteins and their tryptic digestion products are more soluble than the dinitrophenyl or carhobenzoxy derivatives. However, removal of these masking groups is difficult. Extensive study has not been made of the tryptic digestion of carbamyl proteins obtained by reaction with isocyanate. The dithiocarbamoyl or trifluoroacetyl derivatives have few of the disadvantages of the derivatives made with other reagents. They are easily formed, they yield tryptic peptides which are water soluble, and the masking groups are removed easily under. mild conditions. Fractionation of the peptides by chromatographic means proceeds smoothly after removal of the masking groups. The general utility of these derivatives for limiting the action of trypsin cannot be ascertained without additional studies, but many of the methods may prove to be particularly helpful in amino acid sequence studies. Bonds formed by cysteinyl residues can be made susceptible to tryptic action when the thiol group of the cysteine side chains is reacted with p-bromoethylamine (Lindley, 1956). The resulting 8-(p-aminoethy1)cysteinyl side chains provide the specificity requirements necessary for tryptic action. One methylene carbon of the lysyl side chain is replaced by a sulfur atom in thioether linkage. Bonds formed by the carboxyl group of this derivative are susceptible to tryptic action. Reduction of

68

ROBERT L. HILL

the disulfide linkages in proteins with agents such as mercaptoethanol or thioglyvolate, followed by coupling with p bromoethylaminc, can hc used to render bonds formed by cystinyl linkages susceptible to trypsin. Application of this technique to problems in struetural analysis are few a t this time (Tietze el nl., 1057), possibly because of the sluggish reactivity of YHz

H,N-CH,-CH,-CH,-CH,-CC-COOH

I H

Lysine

H,N-CH,-

CH,-

S- CH,-

YHz C-COOH I H

S - (P-Aminoethy1)cysteine

bromoethylamine. Formation of the derivative occurs more rapidly on reaction of thiol groups of cysteirie with ethyleneiniine (Raftery and Cole, 1963). Use of this reagent in lieu of 0-bromoethylaniine might increase the ease of preparation of S-aniinoethyl derivatives of proteins and thereby allow wide application of this method to problems in structural analysis of proteins.

B. Chymotrypsin The specificity of chymotrypsin for hydrolysis of peptide bonds formed by the carbo,xyl groups of tyrosine, phenylalanine, and tryptophan has been recognized for some time (Green and Neurath, 1954; Desnuelle, 1960). Action on synthetic substrates of leucine (Goldenberg et al., 1951) and methionine (Kaufman and Neurath, 1949) also has been noted although a t much slower rates than observed with the aromatic amino acid derivatives. When protein substrates or synthetic ester substrates are examined, it is evident that a variety of bonds can be hydrolyzed by chymotrypsin. Inagami and Sturtevant (1960) observed that chymotryptic hydrolysis of a-benzoyl-L-argiiiine ethyl ester, a “typical” trypsin substrate, occurred a t a maximum rate which was 20% of that observed with trypsin. Several ester substrates, such as p-nitrophenylacetate (Hartley and Kilby, 1954), are also hydrolyzed. In the course of sequence analysis studies, chymotryptic digests of several proteins and peptides have been examined in detail. These studies have revealed significant chymotryptic cleavage at many bonds often thought to be resistant to chymotrypsin. Table V I lists the types of bonds in nine

TABLEVI Specificity of Chymotrypsin for Hydrolysis of Pepti& Bonds i n Proteins and Polypeptidesa

Type of bond

Type of bond

. . .

Type of bond

Type of bond

Type of bond

-Glu-Gln . . Ala-ArgJ -Lys-His . . . Ileu-Ileu-e -Leu-Met . . . Glu-Tyr-c -Ala-Thr . . , Asn-Arg-d -Val-Gln . . . Ala-Ser-J -Val-Asn . . . CMC-Ala-d -Ala-Gln . . Lys-His-" -Lys-His . . Lys-Thr-cJ -Lys-Met . . . Ileu-Phw -Gly-Thr . . . Glu-GlnJ -Try-Gln . . Lys-Met-] -Asp-Ileu . , . Asn-Lewh -hla-Met . . . Ly~--4rg-~-Lys-Thr . . . Gly-Gln-f -Ser-Gln . . . Val-Thr-h -Ala-Asn , . . Lys-Bsn-f -1leu-Thr . . . Ser-Leu-] -Val-Lys . . . Ala-His-' -Lys-Asn . . . Lys-Glyc -Lys-Gln . . .h -Glu-Lys . . . Gly-Gly-C -Thr-Met . . . Se+ -Arg-Thr . . . Val-Glu-h -Lys-Met . . . Val-Thr-1 -Ala-Phe . . . Pro-Leu-k -Val-Thr . . . Val-ArgJ -Val-Gly . . Lys-Lys-k -Val-Lys . . Gly-Hkb -Lys-Asn . . . Val-Ala-" -Thr-Asn . . . Val-Lysi -Leu-His . . Ma-His-* -Gly-Lys . . . Lys-Arg-& -Phe-Ser . . a -Glu-Val . . . Arg-Gln-h -Lys-Asn . . .* -Val-His . . . Ala-Ser-b -1leu-Lys . . . Lys-LysJ -Thr-Ser . . Ala-Ala-e -Pro-Val . . . Lys-Val3 -Ala-His . . . Gly-Lys-J -Tyr-Thr . . . illa-Ala-f -Val-CySOaH . . Ser-Leu-8 -Gly-His . . Gly-LysJ -Lys-Lys . . . Lys-f -Leu-His . . . Gly-Leu-c.f -Gly-Met . . Asn-Ala-d -Val-Thr . . . Ala-Lewh -Thr-Glu . . . Gln-dla-h -1leu-Met , . . Gly-Asn-1

-Thr-Asn . . . Arg-Aswd

. .

.

.

.

.

. .

.

5tY

fd 0

2

3 0

1

8el M

In the same substrates a total of 32 phenylalanyl bonds, 36 leucyl bonds, 24 tyrosyl bonds, and 6 tryptophyl bonds were also hydrolyzed. * Human hemoglobin, a-chain (Hill and Konigsberg, 1962). Horse cytochrome c (Margoliash, 1962). Egg-white lysozyme (Canfield, 1963). Ribonuclease (Hirs et al., 1956, 1960; Smyth et al., 1962). f Human cytochrome c (Matsubara and Smith, 1963). 0 Insulin (Sanger and Tuppy, 1951; Sanger and Thompson, 1953). Tobacco mosaic virus (Anderer et al., 1960). * Papain (Light and Smith, 1962). 1 Human hemoglobin, ychain (Schroeder et al., 1963). Ovine corticotropin (Leonis et al., 1959). a

z

70

ROBERT I,. HILL

proteins (or peptides) that have been found susceptible to chymotryptic hydrolysis. Only those proteins whose amino acid sequences are known in detail are listed here, inasmuch as lack of knowledge of the sequeiice around the susceptible bond limits the interpretation of chymotryptic specificity. In addition to hydrolysis of a large number of bonds formed by the aromatic amino acids and leucine, examples of splitting of bonds formed by the carboxyl groups of asparagine, cysteic acid, glutamine, glycine, histidine, isoleucine, lysine, serine, threonine, and valine also are found. Indeed, only bonds formed by alanine, aspartic acid, arginine, glutamic acid, proline, and cysteine or cystine have not been found to be hydrolyzed by chymotrypsin. Although this suggests that chymotrypsin has a rather broad specificity, it is evident that hydrolysis of many of the bonds listed in Table VI is iiot extensive. I n order to find an explanation for the observed hydrolysis of bonds which are usually difficult to split, it is helpful to examine the nature of the amino acid sequence around the unusual bonds and consider the conditions employed for hydrolysis when these bonds were split. From the data in Table VI it appears that after hydrolysis of bonds formed by the aromatic amino acids and leucine, bonds of methionine, glutamine, asparagine, histidine, lysine, and threonine are most susceptible t o chymotrypsin. Certainly, maiiy of these bonds are split slowly, and within the same substrates several bonds formed by the carboxyl groups of the same amino acids were not hydrolyzed t o a detectable extent. Thus, in the a-chain of human hemoglobin only one of the four ssparaginyl bonds, three of the eight histidyl bonds, and one of the eleven lysyl bonds were hydrolyzed. Similar results are obtained with the other substrates. It can be expected that the residues adjacent to the amino acids which form the susceptible bond influence chymotryptic action. The only residues which frequently are adjacent to the susceptible bonds are lysine, arginine, and histidiiie. Of the six asparagine splits, five occur where lysyl or arginyl residues are present on either side of the susceptible bond. Four of the six histidyl splits and four of the five lysyl splits are also in sequences rich in basic amino acids. In addition, few of the asparaginyl, lysyl, or histidyl bonds which were not hydrolyzed in these substrates occur in sequences which contain basic amino acids. Without additional data, the significance of such neighboring group effects remains to be determined. The few examples of chymotryptic hydrolysis of bonds formed by cysteic acid, glycine, isoleucine, serine, and valine suggest that cleavage a t these residues is possible. There are not enough data available a t this time to suggest a correlation between susceptibility of these bonds and the influence of neighboring basic residues. On the other hand, chymotrypsin would

HYDROLYSIS O F PROTEINS

71

not be expected to hydrolyze these bonds frequently, especially under coilditioris of limited digestion. Chymotrypsin does not hydrolyze extensively bonds formed by the imiiio group of proline. Bonds of this type were not cleaved in ribonuclease, cytochrome c, and the a-chain of human hemoglobin. The -Phe-Pro- bond in oviiie corticotropin has been reported to be hydrolyzed by chymotrypsin. The extent of hydrolysis was low as judged by the 5 % yield of the peptide which contained the carboxyl-terminal phenylalanine of the -Phe-Pro- bond. Table VII summarizes the conditions for chymotryptic hs drolysis of the proteins and peptides listed in Table VI. The parameters which would be expected t o determine the rate of hydrolysis (apart from the nature of the bonds in the particdar substrates) are temperature, pH, time of hydrolysis, and the molar ratio of chymotrypsin to substrate. All these factors often differ considerably for the substrates listed. Hydrolyses have been performed under conditions which vary from 2 to 24 hr, from pH 7.0 t o 9.0, from 22" to 40°C, and a t enzyme to substrate molar ratios between 1/360 to 1/21. It is not obvious how variations in pH and temperature affect the apparent specificity of chymotrypsin, but a t low molar ratios of enzyme to substrate only the most susceptible bonds would be expected to be hydrolyzed. The lowest molar ratio was employed in the studies with ribonuclease. The only bonds of a n unusual nature which were split were those formed by serine and histidine in the following sequences, -Thr-Ser . . . Ala-Ala- and -Lys-His . . . Ileu-Ileu-. Many of the unusual splits listed in Table VI were observed in equine or human cytochrome c and in oxidized papain. Each of these substrates was digested for long periods of time and at high ratios of enzyme to substrate under coiiditioris which would be expected to split bonds that are usually resistant to hydrolysis. One of the best examples of the differences in susceptibility of peptide bonds in a single substrate is provided by the studies on a-chains of human hemoglobin. Chymotryptic peptides with carboxyl-terminal tyrosine, tryptophan, or phenylalanine were isolated from 6-hr digests in high yield. but on 24-hr hydrolysis the same peptides were found in lower yields as the result of secondary splitting a t less susceptible bonds. This is demonstrated in Table VIII, which shows the yields of chymotryptic peptides from two separate sequences in the a-chain. The two largest peptides (Ca 14 and C a 9) were isolated in high yield after 6 hr hydrolysis, but were obtained in much lower yield (20 %) after 24 hr hydrolysis. These low yields were the result of extensive hydrolysis of less susceptible bonds within the larger peptides. Additional data comparable to those for a-chains are not available for the other substrates given in Table VI.

TABLEVII Conditions f o r Ghymotryptic Hydrolysis of Certain Proteins ~

Substrate Performate oxidized ribonucleasea Oxidized A-chain insulinb Oxidized B-chain insulin= Ovine corticotropind Human hemoglobin, a-chaine Human hemoglobin a-chain6 Carboxymethyl lysozymef Equine cytochrome c@ Human cytochrome ch Oxidized papain' Hirs et al. (1960). Sanger and Thompson (1953). Sanger and Tuppy (1951). Leonis et al. (1959). "Hill and Konigsberg (1962). a

Substrate (pmoles)

Chymotrypsinj (moles)

72 22 17 7.5 121 60 70 100 70 10

0.20 0.1 0.1 0.046 0.80 0.40 0.8 2.4 2.3 0.48

~

~

Volume of reaction (ml)

Enzyme substrate molar ratio

100 5 5 5 200

1:360 1:220 1:170 1:160 1:150 1:150 1:87 1:42 1:33 1:21

90 100 120 90 15

~

Temperature ("C) 25 37 25

40 30 25 37 22

Room temp. 39

Canfield (1963). Margoliash (1962). * Matsubara and Smith (1963). Light and Smith (1962). i -4ssumed molecular weight 25,000. f @

PH 7.0 7.5 7.5-8.0 9.0 9.0 8.0 8.0 i.8 7.85 i.6

Time of hydrolysis (hr) 24 24 24 24 24 6 2 29 26 6

2

0 W M

z

?

zr P

TABLEVIII Products Formed on Chymtryptic Hydrolysis of a-Chains of Human Globin under Two Different Conditions of Hydrolysisa Peptide substrate, a-chain, human hemoglobin. Bonds hydrolyzed are indicated by arrows

1

-

1

110

5

115

Val-LeuSer-Pro-Ala-Asp-Lys-Thr-Asn-Val-Lys-Ala-Ala-Try-Gly.. .. . .. . ..

Products on hydrolysis for 6 hr at pH 8,25"C, Ca14 Val-Leu-Ser-Pro-Ala-Asp-Lys-Thr-Asn-Val-Lys-Ala-Ala-Try (7597, yield) CalO Ser-Pro-Ala-Asp-Lys-Thr-Asn-Val-Lys-Ala-Ala-Try (20'7' yield) with a chymotrypsin:substrate molar ratio of 1 : 150

Ca14 CalO Ca15

Products on hydrolysis for 24 hr a t pH 9, 30°C, with a chymotrypsin: substrate molar ratio of 1 :150 ~~

Val-Leu-Ser-Pro-Ala-Asp-Lys-Thr-Asn-Val-Lys-Ala-Ala-Try (15% yield) Ser-Pro-Ala-Asp-Lys-Thr-Asn-Val-LysAla-Ala-Try ( 10Yo yield) Val-Lys-Ala-Ala-Try

(15% yield)

2

E 0

~

Peptide substrate, a-chain, human hemoglobin. Bonds hydrolyzed are indicated by arrows .

...

Products on hydrolysis for 6 hr a t pH 8, 2 5 T , with a chymotrypsin: substrate molar ratio of 1 : 150

Ca9

Products on hydrolysis for 24 hr a t pH 9, 3O"C, with a chymotrypsin: substrate molar ratio of 1 :150

Ca9 Ca16 Ca29

a

2 E 0

i 25

i 30

I

1

35

. Tyr-Gly-Ala-Glu-Ala-Leu-Glu-Arg-Met-Phe-Leu-Ser. . . . . . . . . . . . . . . . . . Gly-Ah-Glu-Ala-Leu-Glu-Arg-Met-Phe

(80% yield)

'd

s 0 M

3 Gly-Ma-Glu-Ala-Leu-Glu-Arg-Met-Phe Glu- Arg-Met-Phe G1y-Ala-Glu-Ala-Leu

Only portions of the a-chain (substrate) are shown. From Hill and Konigsberg (1962).

(32% yield) (50% yield) (15% yield)

74

ROBERT L . HILL

At present, it would seem that evaluation of the substrate specificity of chymotrypsin with the aid of synthetic prptides or appropriate amino acid derivatives has indicated which bonds will be split in the protein Substrates. Much useful information which bears on the problem of chymotryptic hydrolysis in sequence studies could be obtained if other protein substrates were examined so that all parameters affecting hydrolysis could be evaluated more clearly.

C. Pepszn Pepsin has often been used to obtain one of the sets of peptides required for determination of the amino acid sequence of a protein or polypeptide. For this reason a large amount of information is available concerning the specificity of pepsin for protein substrates. Sel era1 earlier studies with synthetic substrates (reviewed by Hovey and Yanari, 1960; Grccn and Neurath, 1954) demonstrated that pepsin could hydrolyze bonds formed by the amino or carboxyl groups of phenylalanine, tyrosine, glutamic acid, eystine, and cysteine. Other types of synthetic substrates have not been examined in detail, and the best picture of the specificity of pepsin has come from recent studies with protein substrates. Table I X presents a list of the bonds split by pepsin in seven proteins or peptides whose amino acid sequence have been established. In addition to the bonds which were shown t o be susceptible in synthetic substrates, examples have been found of hydrolysis of bonds formed by the carboxyl groups of all of the other natural amino acids with the exception of proline and isoIeucine. In order t o determine which bonds are most susceptible to pepsin, it is neressary t o consider how many bonds of a particular type occur in the substrate and how many of this type arr hydrolyzed. Analysis of the available data shows that the majority of the bonds formed by the aromatic residues and lcuvyl residues are hydrolyzed. Data of this type for the a- and &chains of human hemoglobin arr presented in Tables X and XI. Hydrolysis was observed a t the carboxyl or amino side of ten of the rleven aromatic residues in a-chains. Four bonds formed by the amino groups of these residues and sewn bonds formed by the rarboxyl groups of the aromatic residues were hydrolyzed. Hydrolysis was found a t both the carboxyl and amino bond of only one phenylalanine residue. In @-chains only two of the thirteen aromatic residues formed a carboxyl or amino group bond which was not susceptible to pepsin, despite the rather mild conditions of hydrolysis Similar results were observed with leurinc, as shown in Table XI. Bonds formed by the other amino acids do not appear to be hydrolyzrd as readily as those of the aromatic amino acids and lencine. The large number of alariyl boiids whirh were hydrolyzed in the proteins given in Table I X suggest that this residne also forms bonds that are split easily.

TABLE IX Specificity of P e p s i n for Hydrolysis of Peptide B o n d s in Proteins a n d Polypeptidesa Type of bond -&4sn-Brg. .

. Ileiic

-Val-Asn . . . P h e s -Phe-Asn . . . Thr-d -Gly-Asn . . . Try-d -Val-Asp . . . Glu-' -Ser-Asp . . . Phe-c -Ma-Asp . . . Pro-c -Val-Asp . . . Pro-fZc -Leu-CMC . . . -Try-CMC . . . Asn-d -Val-Glu . , . Alas -Leu-Glu . ilsn-0

. .

-Val-Glu .

. . Gln-0

Type of bond

. . . His-%

-Leu-Gly . . . Arg-COO-/ -Leu-Gly . . . -Tleii-G!u . . . Leu-c -Glj-G!y . . . fulu-, ll.. I -Glu-Glu . . . Lys-c -Asp-Gly . . . Leu-' -Ma-Glu . . . Phe-* -Arg-Gly . . . Phe-g -Lys-Glu . . . Phe-fbh -Arg-Gly . . . Tyr-d -Gly-Glu . . . Tyr4 -Val-His . . . Alas -Bsp-Glu . . . Val-f -Glu-His . . . Phe-* -Val-Gln . . . Ala-"Sh -His-His . . . PheJ -Am-Gln . . . His-0 -His-Lys . . . Lewb -Tyr-Gln . . . Leu-0 -Phe-Lys . . . LeuS -Ssn-Gln . . . Phe-c -Arg-Met . . . Phe-b -Brg-Cln . . . Phe-c -Ser-Ser . . . Asp-d -Leu-Ser . . . Ser-h -Ser-Gln . . . Val-c -Val-Ser . . . Thr-b -Met-Glu

Type of bond

Type of bond

-Gly-Ser . . . Tyr-. 1 -vai-inr -7

-7

-Val-Thr -Am-Thr -Ala-Thr -Arg-Thr -Val-Thr -Ah-Try -Arg-Try -Ma-Try -Am-Try -Am-Tyr

. . . Aia-c

-Leu-Tyr

. . . Leu-g

-Ser-'ryr . . . Ser-c -Pro-Tyr . . Val-e -1leu-Val . . . -&la-@ -Ala-Val . . . Asp-c tl -Am-Val . . . Asp-' -Ser-Val . . . C ~ S O ~ H - ~ r -CySH-Val . . . Leu-' 4 -Am-Val . . . Lys-b -Ma-Val . . . ThrJ 0 -Gln-Val . . . Try-C CySOaH-0 -Val-Val . . . TyrJ

. . . Leu-b . . . Phe-e . . . Val-. . . . Val-. . . . Val-c . . . Gly-b . . . Try-d . . . Val-d . . . Val-d

...

Type of bond

.

5

z

0

-Leu-Tyr

. . . Gln-g

In the same substrates a total of 22 alanyl bonds, 34 leucyl bonds, and 23 phenylalanyl bonds were also hydrolyzed. Asn, Asparagine; Gln, glutamine; CMC, S-carboxymethylcystine. b Human hemoglobin, a-chain (Hill and Konigsberg, 1962). c Tobacco mosaic virus (Anderer et al., 1960). d Egg-white lysozyme (Canfield, 1963). a Ribonuclease (Him et al., 1960). f Human hemoglobin, p-chain (Konigsberg and Hill, 1962). 0 Insulin (Sanger and Tuppy, 1951; Sanger and Thompson, 1953). h Human hemoglobin, 7-chain (Schroeder et al., 1963). p-MSH (Harris and Roos, 1959b).

e

E

Z m

TABLE x Peptic Hydrolysis of Bonds Formed by the Aromatic Amino Acids i n Human Substrate a-Chaina

@-Chain*

Hydrolysis amino-terminal to aromatic residue

Hydrolysis carboxyl-terminal to aromatic residue

-Glu-Glu . . . Tp-Gly-Arg-Met . . . Phe-Leu-Val-Asn . . . Phe-Lys-Ala-Glu . . . Phe-Thr-

-Ah-Try . . Gly-Lys-Met-Phe . . . Leu-Ser-His-Phe . . . Asp-Leu-Tyr-Phe . . . Pro-His-Ser-Phe . . . Pro-Thr-Am-Phe . . . Val-Lys-Lys-Phe . . . Leu-Ala-

-Ala-Leu . . . Try-Gly-Val-Val . . . Tyr-Pro-Arg-Phe . . . Phe-Glu-Gly-Ala . . . Phe-Ser-Glu-Asn . , . Phe-Arg-His-His . . . Phe-Gly-Lys-Glu . , . Phe-Thr-Ala-Ala . . . Tyr-Gln-

.

.

-Arg-Phe . . Phe-Glu-Ser-Phe . . . Gly-Asp-Thr-Phe . . . Ala-Thr-

a-

and &Chains Bonds not hydrolyzed

-Thr-Tyr

. . . Phe-Pro-c

TI W

M

s -Pro-Try . . . Thr-Gln-His-Lys . . . Tyr-His-COO-Lys-Tyr . . . His-COO-

Conditions of hydrolysis: 2 gm a-chain in 200 ml a t pH 2.8, 20 mg of pepsin, 16 hr hydrolysis at room temperature (Konigsberg and Hill, 1962). * Conditions of hydrolysis: 0.8 gm @-chainin 80 ml at pH 2, 8 mg of pepsin, 1 hr hydrolysis at 25°C (Konigsberg et uZ., 1963). Hydrolysis at -Phe-Pro- observed.

r

B

r

TABLEXI Peptic Hydrolysis of Bonds Formed by Leucine in Human a- and 8-Chains

Substrate

Hydrolysis amino-terminal t o leucine

Hydrolysis carboxylterminal to leucine

Bond not hydrolyzed ~

a-Chaina

&Chainb

-Glu-Ala , . . Leu-Glu-c -Met-Phe . . . Leu-Ser-His-Lys . . . Leu-Arg-Phe-T,ys . . . Leu-Leu-V?:-rhr . . . Leu-Ala-e -Lys-Phe . . . Leu-Ala-c

-Ma-Leu =41a-Leu -Ala-Leu -Ala-Leu -Asp-Leu -Leu-Leu -Thr-Leu -Ser-Leu -Phe-Leu -Val-Leu

. . . Ser-Ala. . . Ser-Asp. . . His-Ala. . . Val-Thr. . . Ala-Ala-c . . . Asp-Lys. . , Ala-Ser-c . . . Thr-Ser-

-hrg-Leu . . . Leu-Val-Arg-Leu . . . Leu-Gly-CySH-Val . . . Leu-Ala-Am-Ala . . . Leu-Ala-

-Ala-Leu -Ala-Leu -Arg-Leu -Asp-Leu -His-Leu -Am-Leu -Thr-Leu -Glu-Ser -Lys-Leu -Arg-Leu

. , . Try-Gly. . . Gly-Arg. . . Leu-Val. . . Ser-Thr. . . Asp-Asn. . . Lys-Gly. . . Ser-Glu. . . His-CMC. . . His-Val. . . Leu-Gly-

Konigsberg and Hill (1962). Konigsberg et al. (1963). c Hydrolysis at bonds on either side of leucine waa observed. * Hydrolysis at -Lys-Leu- and -Leu-Ser- bonds was observed. Hydrolysis at -Leu-Val- bond waa observed.

. . . Glu-Arg-c . . . Thr-Asn-

~~

HZN-Val-Leu . . . Ser-Pro-Asp-Leu . . . Ser-His-Lys-Leu . . . Leu-Serd -Cyso~H-Leu. . . Leu-Vale -Ala-His . . . Leu-Pro-

-His-Leu . . . Thr-Pro-Val-Leu . . . Gly-Ala-Gly-Leu . . . Ala-His-Val-Leu . . . Val-CySH

0

r

Ee I2

3

-4

78

ROBERT L . HILL

Only a small number of the alanyl bonds, however, are hydrolyzed. In a-chains only three of the twenty bonds formed by the carboxyl groiips of alaniiie are cleaved, whereas in P-chains only four of the fifteen alanyl bonds are cleaved. Thus, the bonds formed by carboxyl groups of amino acids other than leucine and the aromatic amino acids may be split because they form the peptide linkage with amino groups of the aromatic residues or leucine. An explanation is lacking for the hydrolysis of bonds such as -Asp-Pro-, -Arg-Ileu-, -Gly-Arg-, and -Ser-Asp-. Cleavage of bonds of this type has not been observed frequently and caiinot be expected to be encountered often. It has been suggested that the specificity of pepsin can be accounted for if it is assumed that pepsin requires a “hydrophobic binding site” near the peptic susceptible bond (Tang, 1963). This suggestion is based upon calculation of the frequency of appearance of amino acids with hydrophobic side chains a t positions on either side of the bond which is hydrolyzed. Although it is evident that susceptible bonds are often found in a hydrophobic environment, several instances of cleavage of bonds in portions of peptide chains which contain hydrophilic residues make it difficult to evaluate the importance of the proposed binding site. Specificity must be explained ultimately on the basis of the events which occur during coinbination of those structures which form the susceptible bond and structures in the active site of pepsin. Until a more detailed knowledge of the mechanism of the action of pepsin is known, it will be difficult to determine whether structures somewhat far removed from the susceptible bond contribute significantly to peptic specificity. Comparison of limited and extensive hydrolysis of the same substrate by pepsin demonstrates not only its specificity, but shows how it can be used advantageously to degrade peptides into fragments of varying size. The studies of Goldstein et aE. (1963) on peptic hydrolysis of pure tryptic peptides of the P-chain serve as the best examples for comparison. Figure 3 shows the products obtained on limited and extensive hydrolysis of two tryptic peptides. Clearly limited digestion a t p H 2 for a few minutes with low pepsin concentrations results in hydrolysis of only a few bonds, whereas hydrolysis for several hours a t higher concentrations of pepsin results in more extensive cleavage. An additional factor that could limit peptic hydrolysis is the inhibitory effect of a free a-amino group adjacent t o a potentially susceptible bond. It has been found that acylated dipeptides are split more readily by pepsin than corresponding unprotected dipeptides. Thus, amino-terminal aromatic residues or leucine would not be released readily by pepsin. The lack of significant hydrolysis of the amiiio-terminal leucyl bond in the peptide shown in Fig. 3 might be the result of the inhibitory effect of the a-amino group.

A.

105 110 115 120 Leu-Leu-Gly-Asn-Val-Leu-Val-CyS0,H-Val-Leu-Ala-His-His-Phe-Gly-Lys

1

0.00% pepsin, 25"C, 4 min, pH 2

Leu-Leu-Gly-Asn-Val-Leu ( 8 0 % yield)

1

+

0.01% pepsin,

25"C, 18 hr, pH

Leu-Leu-Gly (90% yield)

+

Val-CyS03H-Val-Leu-A-His-His-Phe-Gly-Lys (53% yield)

p

Asn-Val-Leu (yield not given)

i

o.ooz%

pepsin,

2 5 T , 4 min, pH 2

o.ooz% pepsin, 2 5 T , 21 hr, pH 2

Ala-Thr-Leu-Ser-Glu-Leu-His-CySO~~-A~p-~~~-Leu-His-Val-Asp-Pro-Glu-Asn-Phe-Ar~ (69% yield)

+

Gly-Thr-Phe (27% yield)

80

ROBERT L. HILL

D. Bacterial Proteinases Three bacterial endopeptidases have been used recently for partial enzymatic hydrolysis of proteins and polypeptides. One, from a culture filtrate of Bacillus subtilis, was crystallized by Guntelberg and Ottesen (1952) and has been named subtilisin. A second crystalline enzyme from a different strain of the same organism was isolated by Hagihara et al. (1958a,b) and has been marketed commercially under the trivial name Nagarse. The third enzyme was obtained from a strain of Streptomyces griseus in highly pure form by Nomoto and Narahashi (1959) and is often cited in the literature as “Pronase.” The physical and chemical properties of these enzymes has been reviewed recently by Hagihara (1960). The two B. subtilis proteiiiases appear to have very similar substrate specificities. Table XI1 lists the types of bonds in a number of peptides which have been recorded as susceptible t o hydrolysis. Because of the wide variety of bonds that are split by this type of enzyme, it is difficult to detect a pattern of specificity. Since both enzymes degrade proteins more extensively than trypsin, chymotrypsin, or pepsin (Hagihara et al., 1958a,b; Nomoto et al., 1960a,b), they provide an excellent means for degrading small polypeptides into smaller peptides and amino acids. Such extensive degradation is analogous to partial acid hydrolysis, but unlike acid hydrolysis, destruction of amino acids does not occur and high yields of the hydrolytic products are obtained. The proteinase from S. griseus seems to be the least specific of all proteolytic enzymes known a t this time. Its action on protein (Nomoto et al., 1960a) and peptide (Nomoto et al., 1960b) substrates reveals few types of peptide bonds which are totally resistant to hydrolysis under optimal conditions. The action of S. griseus proteinase, trypsin, chymotrypsin, and subtilisiri on hydrolysis of casein is given in Fig. 4. When the two pancreatic enzymes and subtilisin have digested casein exhaustively, several additional bonds are susceptible to the proteinase. Conversely, few bonds which remain intact after hydrolysis of casein with the proteinase are susceptible to trypsin, chymotrypsin, or pepsin. It was shown that about 75 % of the bonds in casein, 87 % of the bonds in ovalbumin, and 80 % of the bonds in wheat gluten were hydrolyzed after digestion for 72 hr (pH 7.4, 40°C) at enzyme concentrations which were 0.5 % (by weight) that of the substrate concentration. In addition, many free amino acids were present in these hydrolyzates. For example, 45 % of the total aspartic acid, 94 % of the total methionine, 61 % of the valine and leucine, and 74 yo of the total lysirie in casein were found as free amino acids after 72-hr hydrolysis. The studies with synthetic substrates show that almost all di- or tripeptides, or amino acid amide or ester derivatives are hydrolyzed, a t least partially, by prolonged action of the enzyme. Initial

TABLE XI1 Specificity of Subtilisin for Hydrolysis of Peptide Bonds in Proteins and Polypeptides ~

Type of bond

Type of bond

Type of bond

Type of bond

Type of bond

-Ah-Ala , . . L ~ s - ~ -CySAla . . Ser-t -Val-Ala . . Try-d -Ser-Arg . . . Arg-c -Glu-Arg . . . Glyf -A.rg-Arg , . . Val-* -Met-Asn . . . Ala-d -CMC-A4sn . . . Asp-d -Val-Asn . . . Gh-f -Ser-Ssn . . . Phe-d . . . Ser-d -Ile~-~4sn -Arg-Asn . . Thr-d -Met-Asn . . Thr-COO-c -Am-Asp . . Gly-d -Asp-Asp . Alas -Val-,l-Asp . . . Asp> -Gly-Asp . . . Gly-d

-Val-CMC . . . Ma-d -Arg-CMC . . . Am-d -Leu-CMC . . . Asn-d -Leu-CyS03H . . Glyf -Val-CYSO3H . . Ser-e -Val-CyS03H . . Glyf -CySCyS . . , Alaf -Val-CyS . . . Serf -Val-Glu . . . Ala-f -Leu-Glu . . . Asn-f CyS03H-Glu . . . Gly-e -Thr-Gln . . . Ala-d -Val-Gln . . . -Ah-Gln . Asp-$ -Glu-Gln . . . CySS-f -Am-Gln . . . GluJ -Ser-Gln . . . Glu-C

-Am-Gln . , . His-’ -Arg-Gln . . Phe-h -Glu-Gly . . . Gly-e CySOaH-Gly . . . Glu-f -Ser-Gly . . . Lys-f CySOaH-Gly . . . Serf -Arg-Gly . . T h r - b -Gln-His . . Leu3 -His-Leu . . . CyS0sH-f -Gln-Le~ . Gluf -1leu-Leu . . Gln-d -Ser-Leu . . Gly-d -Ala-Leu . . Tyr-f -Ser-Leu . Tyrf -His-Leu . Val-f -Tyr-Leu . . Valf -Pro-Lys . . Ma-COO-f

-Met-Lys . . . hrg-d -Leu-Met . . Asp-C -Lys-Met . . . Glu-. -Ma-Met . . , Ly& H3+N-Phe . . . Val-’ -His-Phe . . . -irg-” -Gly-Phe . . Phe-f -Thr-Phe . . Thr-e -Phe-Phe . . . Tyr-f -Asp-Phe . . . V a P -Gly-Pro . . . Val-. -CMC-Ser . . Ah-t-d -Thr-Ser . . . Asp-“ -Gly-Ser . . His-f -Tyr-Ser . . . Lys-c -Gly-Ser . . . Thr-d -1leu-Thr . . . Ala-d

-Sla-Thr . . . 4sn-d -Asp-Ser . . . Gly-c -Arg-Thr . . . Val-b -ilrg-Try . , Gly-a -Ala-Try . . . Ileu-d -Gln-Try . . . Leu-c -Am-Try . . Val-d -Arg-Tyr . . . Asn-b -Am-Tyr . . CyS-f -Asp-Tyr . Gly-d -Leu-Tyr . . . Leu-f -Lys-Tyr . . . L e u - c -Pro-Tyr . . . Lys-* -Gly-Tyr . . . Ser-d -Phe-Tyr . . . Thr-f -Hs+N-Phe-Val , Asn-f -Leu-Val . . CySOaHl -Pro-Val . . . CySOIH-e

. .

. . . . .

. .

.

..

b-MSH (Harris and Roos, 1959b). mosaic virus (Tsugita et al., 1960). c Glucagon (Bromer et al., 1957a,b). a

6 Tobacco

Egg-white lysozyme (Canfield, 1963). Diisopropyltrypsin peptide (Dixon el aZ., 1958a,b). f Insulin (Tuppy, 1958; Haugaard and Haugaard, 1955). d

c

.

.

. . . . . . . . .. . .

.

.

.

.

.

.

. .

. .

.

. .

z

3

3 0

2

s 0 q

z

0

c3

z

3

82

ROBEltT L. IIILL

velocity data which are necessary to determine the relative rates of hydrolysis of various substrates are not available, but it would appear that only dipeptides of a few amino acids resist hydrolysis. Thus, in 24 hr under optimal conditions glycylproline resisted hydrolysis and glycylglycine was hydrolyzed only to the extent of about 3 %. More thorough studies will be required to determine which bonds are most susceptible. At this time only a few bonds formed by glycine or proline appear resistant. It is interesting that only 3 0 4 0 yo of the bonds are hydrolyzed in gelatin, a protein rich in glycine, proline, and hydroxyproline.

n

4000

k!

3000

2000

1000

0

24

48 TIME-Hours

72

96

24

48

72

96

TIME- Hours

FIG.4. Hydrolysis of casein by several proteinases. I. Hydrolysis of casein by trypsin ( A ) , chymotrypsin ( O ) ,or subtilisin ( 0 )followed by S. griseus protease (0). Curves B, C, and D indicate the extent of hydrolysis by individual enzymes without addition of S. griseus protease. Curve A indicates the extent of hydrolysis when IS.griseus protense is added t o the hydrolyzate of one of the other proteinases after approximately 47 hr. 11. Hydrolysis of casein by S. pisezis protease followed by hydrolysis with trypsin, chymotrypsin, subtilisin, or pepsin. The latter enzymes were added to the protease hydrolyzate after approximately 47 hr. From Nomoto et al. (1960a,b).

It can be concluded that the bacterial proteinases can be applied to many proplems where extensive enzymatic proteolysis is required. Because of their wide substrate specificity, they are not useful for producing one of the major sets of peptides which are required to provide overlapping sequences within large polypeptides or proteins. On the other hand, where structural studies of small peptides require extensive cleavage at a variety of bonds, the bacterial proteinases are excellent hydrolytic agents. The products of proteinase action are often quite analogous to the products

HYDROLYSIS O F PROTEINS

83

of partial acid hydrolysis, but they are obtained in higher yields than can be expected in acid hydrolyzates.

E. Papain Studies with a variety of synthetic substrates have demonstrated a very wide specificity for papain (reviewed by Kimmel and Smith, 1957; Smith and Kimmel, 1960). Peptide bonds formed by the carboxyl groups of a-amino substituted arginine and lysine are most susceptible t o papain. It also hydrolyzes similar derivatives of glutamine, histidine, glutamic acid, leucine, glycine, and tyrosine. In addition, substrates which are considered typical for chymotrypsin, pepsin, aminopeptidase, and carboxypeptidase also are hydrolyzed. Although the relative rates of hydrolysis for different synthetic substrates may differ by as much as two to three thousandfold, it is evident that even the least susceptible bonds are hydrolyzed to some extent on prolonged hydrolysis. Consideration of the types of bonds in several peptides of known structure that have been hydrolyzed by papain (Table X I I I ) would suggest this to be the case. In addition to lysyl and histidyl bonds which are expected to be split readily, less susceptible bonds formed by other residues also are split. Thus, the broad specificity of papain makes it an ideal choice for degradation of peptides from tryptic or chymotryptic digests into smaller peptides which are more amenable to sequence analysis. Valuable information concerning the specificity of papain and its use for degradation of tryptic, chymotryptic, or peptic pepetides was obtained by Konigsberg and Hill (1962, 1963) in studies on the sequence of the a- and @-chains of human hemoglobin. Figure 5 lists the products that were identified in papain digests of peptides of known sequence. Several features of these data are noteworthy. (1) It is striking that the yields of split products account for a high proportion of each of the residues in the parent peptide. Thus, in peptide C, peptide 5C accounts for 95 yo of the residues a t positions 8, 9, and 10; peptides 3C and 4C account for 95 % of residues 5, 6, and 7 ; peptides 1C and 4C account for 70 % of residues 3 and 4; and peptide 2C accounts for 55% of the first two residues. Similarly, in peptide E, an undecapeptide, all residues could be accounted for in the digestion products in yields between 70 and 100 %. (2) A large number of bonds were often split in one peptide, but seldom was it possible to isolate or detect all of the products that could result from hydrolysis a t these bonds. Thus, peptide A yielded only three of the five peptides which might be obtained. I n peptide C, five of the nine possible peptides were isolated. These results show that some bonds are hydrolyzed more extensively than others in the same peptide. The extensive cleavage of the -His-Ala- bond in peptide C and the -Ser-His- and -Gly-Ser- bonds

TABLE XI11 Specijicity of Papain for Hydrolysis of Peptide Bonds in Proteins and Polypeptidesa Type of bond

Type of bond

-Asp-Ala . . . Aan-d -Val-Ala . . . Am-* -Pro-Ala . Glu-b -Val-Ala . Gly-b -Ha+N-Val-Ala . . Hieb -Am-Ala . . Leu3 -Glu-Ala . . Leu-b -H8+N-Ser-Ala . Leu-b -Pro-Ala . . Val-b

-Pro-ilsn . . Ma-d -Pro-Asn . . Leu-d -Glu-Asn . . . Phe-0 -Val-Asp . Asp-b -ksp-A~p . . Met-’ -Leu-Glu . . . A m - d -Pro-Glu . . -Pro-Glu . . . Asn-c -Pro-Glu . . Glu-c

.. . . . . . .. .

. . . . . . .

Type of bond

.

-Glu-Glu . . LYS-COO-C -Val-Gly . . . Ala-b H8+N-Gly . . Alas -Lys-Gly . . Ileu-d -His-Gly . . . Sewb -&a-Gly . . . VaLb -Leu-His . . Ala-b -Ma-His . . LeuJ -Val-His . . . Leu-c

.

. .

.

Type of bond

-Gly-Lys . . . Ly& -Lys-Ileu . . . Ph& -Pro-Lys . . . Lys-d -Ala-Leu . . GluJ HfN-Leu . . . Glu-d -Lys-Lys . . . Tyr-d H3+N-Lys . . . Thr-d -Ala-Lsu . . . S w b -Glu-Phe . . . Thr-b,” -His-Lsu . . . Thr-c Ht+N-Ser . . . Alas -Am-Lys . . Asn-d -GIu-LYs . . . G ~ Y - ~-Leu-Ser . . . Asps H ~ + N - L ~.s. . Gly-d -Leu-Ser . . His-b -Lys-Lys . . . Ileu-d -Ileu-Thr . . . Tyr-d -Thr-Tyr . . . Phe-b

.

.

.

Of the peptides examined, none contained arginine. Bonds formed by this amino acid should be very susceptible to hydrolysis. Human hemoglobin, a-chain (Konigsberg and Hill, 1962). 6 Human hemoglobin, @-chain(Konigsberg et al., 1963). Cytochrome c (Margoliash, 1962). a

0 W

M

2 r

z

s

Peptide sequences

Peptide

Papain 1A Papain 2A Papain 3A

B

C

I

95 70 90

His-Gly

Val-Asp-Pro-Val-Asn

1

75 95

Phe-Lys

Ser-Ala-Leu-Ser-Asp- Leu-His-Ala-His-Lys 2C

3C 4C 5C

I Leu-Ser I.

I

a5

Ser-Ala Asp-Leu-His Leu-Ser-Asp-LCu-His Ala-His-Lys

55 50 45 95

Gly-Ala-Glu-Ala-Leu-Glu-Arg

D

l ? ! k C 1

GlY

Not m e a s u r e d

A rg

Ala-Glu- Ala Ala- o r Ala

20

Leu-Glu-Arg Ala-Glu-Ala-Leu

Val-Ala-His-Val-Asp-Asp-Met-Pro-Asn-Ala-Leu

E Papain 1E Papain 2E Papain 3E Papain 4E Papain 5E

I 1

1

I Ala-4Leu

Val-Ala Asp-Met-Pro-Asn-Ala His-Val-Asp His-Val-Asp- A s p

20

75 70

45 55

Val-His-Leu-Thr-Pro-Glu-Glu-Lys

F Papain IF Papain 2F Papain 3F Papain 4F Papain 5F Papain 6F Papain 7F

I C

I Glu1

Leu Leu-Thr-Pro-Glu Thr-Pro-Glu Glu-Lys LYs

Val-His

36 33

34 20 40 42 82

Val-Val-Ala-Gly-Val-Ala-Asn-Ala

G

4

I GIv I

1G

Am-Ala

2G

3G 4G

Val- Ala Val-Val-Ala

95 75 65 70

His-Val-Asp-Pro-Glu-Am-Phe-Arg

H Papain Papain Papain Papain

+

Ser-Ala

1C

Papain ID Papain 2D Papain 3D Papain 4D Papain 5D Papain 6D

Papain Papain Papain Papain

Asp-Leu-Ser

Val-Asp-Pro-Val-Asn-PheLys

Papain 1B Papain 2B

Papain Papain Papain Papain Papain

Yield (%)

Asp-Leu-Ser-His-Gly-Ser-Ala

A

1H 2H 3H

4H

I Papain 11 Papain 21

I

Asn His- Val-Asp- Pro-Glu His-Val-Asp-Pro-Glu-Asn

I

Phe-Arg

30 40 30 80

Thr-Tyr;Phe-Pro-His-Phe 'Phe-Pro-His-Phe Thr-Tvr -.I

95 80

FIG.5. The hydrolysis of several peptides from a- and /3-chains of human hemoglobin by papain. Hydrolyses were performed a t 37"-40"C for 15-18 hr at pH 5.5, with papain concentrations of 0.01-0.05%. The peptides have been numbered arbitrarily (Konigsberg and Hill, 1962, 1963). 85

86

ROBERT L. HILL

in peptide A are examples of marked susceptibility. (3) The large number of the products which were identified in a few cases suggests a low degree of specificity for certain bonds. Thus, in peptide H, the -Am-Phebond was hydrolyzed almost completely as judged by yields of the products with amino-terminal phcnylalanine, but hydrolysis of the Glu-Asn- bond was only 3 0 4 0 yo as judged by the yields of papain peptides 1H and 2H. I n general, a large number of products will be formed when a low order of specificity exists for the bonds in the substrate. Other examples of this type of action are observed with peptides C, D, E, and F. (4) The consideration of yields of the products offer a means for evaluating the susceptibilities of certain types of bonds. Table XIV lists the extent of TABLEXIV Extent of Hydrolysis of Some Peplide Bonds by Papain Amount of hydrolysis Type of bonda

-Seryl-R-Glycyl-R-Asparginyl-R-Alanyl-R-Histidyl-R-Leucyl-R-Tyros ylphenylalan yl0

Total numher of bonds 4

3 4 8 7 5

1

Extensive

Moderate

1 3 2

1 0 1 0 0 2 0

7 2 0 1

None 2 0

1 1

5 3 0

R represents all other amino acids.

hydrolysis of the susceptible bonds shown in Fig. 5. It is evident that the extent of hydrolysis varies considerably for bonds of a similar type. Thus, of the four seryl bonds two are resistant, one is extensively hydrolyzed, and one partially hydrolyzed; of the eight asparaginyl bonds all are split extensively but one; of the five leucyl bonds three are resistant and two are split only partially; and so forth. It is possible that the susceptibilities of the bonds formed by the same residue differ as a result of the other structures near the bond. Of the two resistant seryl bonds, one is adjacent to a free amino group (peptide C), which might be expected to reduce the rate of hydrolysis of the seryl bond. The second resistant seryl bond (peptide A) is adjacent to a glycyl bond which is hydrloyzed extensively. The appearance of an amino-terminal glycyl residue next to the seryl bond may render the bond less susceptible to hydrolysis. Similarly, the single resistant alanyl bond (peptide C) is adjacent to a histidyl bond, which is extensively hydrolyzed. Thus, the susceptibility of a bond is influenced partly by the ease with which other bonds in its immediate vicinity are hydrolyzed. ( 5 ) Bonds formed by the carboxyl group of glutamic acid

HYDROLYSIS OF PROTEINS

87

are not hydrolyzed as rapidly above p H 6 as they are a t pH 4. Thus, when two peptides, F and H, were hydrolyzed at p H 4.25, significant hydrolysis a t glutamyl bonds was noted. This effect of p H is consistent with earlier studies on synthetic substrates (Kimmel and Smith, 1957), a peptide from the p-chain of hemoglobin G (Hill, Swenson and Schwartz, 1960), and peptides from cytochrome c (Margoliash, 1962). Presumably, the sensitivity of glutamyl bonds varies with the degree of ionization of the 7-carboxyl group; a t low pH values where the un-ionized form is present, more rapid hydrolysis is observed. This effect of pH on the specificity of papain can often be advantagous in peptide sequence studies. It can be concluded that the wide specificity of papain observed in studies with synthetic substrates is in agreement with the results of studies on small polypeptides. On the other hand, it will be difficult to predict which types of bonds will be hydrolyzed in a peptide of known composition, inasmuch as several structural features around a bond formed by a n amino acid with a structurally favorable side chain must determine the rate of hydrolysis. Because of its wide specificity, papain will degrade most protein substrates more extensively than trypsin, pepsin, or chymotrypsin and its action is quite comparable to that of subtilisin (Hill and Schmidt, 1962; Nomoto et al., 1960a,b). Many free amino acids are liberated from proteins by papain, but it would not appear to produce as extensive hydrolysis as S. griseus protease (French et al., 1963).

F . Carboxypeptidase A and B The action of these two pancreatic exopeptidases on synthetic substrates, proteins, and peptides has been reviewed in detail by Neurath (1960). The specificity requirements which were deduced from studies with synthetic peptides have been confirmed by studies with polypeptides. The structural requirements of specific substrates for both types of carboxypeptidase are analogous except for the nature of the amino acids which contain the free, ionized a-carboxyl group a t the terminus of the substrate. Carboxypeptidase B hydrolyzes most rapidly those bonds formed by terminal lysyl and arginyl residues, whereas carboxypeptidase A hydrolyzes terminal bonds formed by a variety of aromatic, neutral, or acidic amino acids. Of the natural amino acids only carboxyl-terminal prolyl residues are resistant to the action of the enzyme. The rate of hydrolysis depends upon the nature of the side chains of the amino acids which form the susceptible bonds. Thus, differences in the rate of hydrolysis of different substrates may vary several thousandfold. The methods for application of these peptidases to hydrolysis of proteins have been discussed in detail by Canfield and Anfinsen (1963).

88

ROBERT L. HILL

Both carboxypeptidases have been applied successfully to problems in sequence analysis of several proteins and polypeptides. Thus, measurement of the order of appearance of amino acids that are liberated in the course of digestion reflect the sequence of amino acids in the substrate. Hydrolysis will continue until a specific structure prevents further degradation. Inasmuch as bonds formed by lysine or arginine are not hydrolyzed to a significant extent by carboxypeptidase A, use of the A and B forms together often is helpful for obtaining more complete hydrolysis. This method was employed successfully by Guidotti el al. (1962) for sequence analysis of tryptic peptides. Peptides with carboxyl-terminal lysine or arginine were incubated with carboxypeptidase B for 60 to 90 min and then carboxypeptidase A was added. Lysine and arginine were removed extensively after 30-60 min digestion with carboxypeptidase B, but the other amino acids were observed only after addition of carboxypeptidase A. A partial sequence for the carboxyl-terminal portions of most of the peptides was obtained in this manner. In the absence of good chemical end-group methods for determination of carboxyl-terminal residues or sequences, use of the carboxypeptidases remains the best means for analyses of this kind.

G. Leucine Aminopeptidase The specificity of this enzyme from swine kidney has been established from detailed studies with synthetic substrates (reviewed by Smith and Hill, 1960). All peptide bonds formed by L-amino acids which are adjacent to a free a-amino group are susceptible to hydrolysis, although the rates of hydrolysis vary over a several thousandfold range. The best substrates are those which contain amino-terminal leucine and the poorest are those which contain the amino nitrogen of proline in peptide linkage, e.g., glycylproline (Hill and Schmidt, 1962). The action of leucine aminopeptidase 011 protein a nd polypeptide substrates (Hill and Smith, 1958, 1959) agrees with the specificity established with synthetic substrates. 1,eucine aminopeptidase has been applied in many ways to particular problems in structural analysis of peptides and proteins. Sequences in the amino-terminal portion of a peptide can often be established by measurement of the order of appearance of amino acids that are released during hydrolysis. The procedure has been used with a variety of proteins and peptides, induding ribonuclease (Hirs et al., 1960), hemoglobin (Konigsberg and Hill, 1062, 1963; Schroeder et al., 1963), cytochrome c (Margoliash, 1062; Matsubara and Smith, 1963), and lysozyme (Canfield, 1963). Dixon et al. (1958b) 'have used aminopeptidase in a subtractive method of sequence analysis. The peptides which remain after limited hydrolysis are purified and hydrolyzed completely with acid. From the compositions

HYDROLYSIS OF PROTEINS

89

of the degraded peptides, the sequence of residues in the amino-terminal region can be deduced. I n addition t o its use for sequence analysis, leucine aminopeptidase has been applied routinely in many studies for the estimation of the tryptophan, glutamine, and asparagirie content of peptides. Prolonged hydrolysis with aminopeptidase results in complete hydrolysis of peptides as large as glucagon (Hill and Smith, 1958) without destruction of the acidlabile residues. Another useful feature is provided by the incomplete hydrolysis of bonds formed by the imino group of proline. Peptides which contain this type of bond are degraded completely to amino acids up to the residue peiiultimate to proline. Isolation of the resulting peptide offers a new starting point for sequeiitial degradation (Hill et al., 1960; Schroeder et al., 1963). Degradation of peptides which contain aspartic acid often results in limited hydrolysis of the same type observed with peptides that coiitaiii proline. Schroeder et al. (1963) noticed low yields of amino acids carboxyl terminal to aspartic acid. They suggest that the low yields are caused by the presence of P-aspartyl linkages (see Section 111,B14) which are not susceptible to hydrolysis. Usually &linkages are not formed without exposure of peptides to acid for some long periods of time. Thus, resistance t o hydrolysis of peptides containing aspartic acid would be expected only if the peptides were altered during preparation or isolation. When optically active amino acids are released from peptides by aminopeptidase, they must be of the L-configuration. For this reason, complete hydrolysis of synthetic polypeptides with aminopeptidase provides a convenient means for evaluating the sterochemical homogeneity of synthetic peptides and peptide derivatives (Hofmann et al., 1962). Because several excellent, nonenzymatic methods are available for aminoterminal end group or sequence analysis, aminopeptidase is not the first choice by many workers for sequence determinations. Aminopeptidase is difficult and expensive to prepare and it remains one of the few proteolytic enzymes used in structural studies that has not been crystallized or prepared in homogeneous form. Finally, it should be emphasized that preparations of aminopeptidase which have a low specific activity often contain other proteolytic activities and care should be exercised in their use (Hill and Smith, 1958; Smyth et al., 1962).

ENZYMATIC HYDROLYSIS VII. TOTAL Several proteolytic enzymes have a broad substrate specificity, but none are known which will hydrolyze all of the types of peptide bonds found in proteins. The S. griseus proteinase, papain, and the subtilisins extensively hydrolyze most proteins with liberation of free amino acids, but each enzyme also leaves many peptide bonds intact. For total enzymatic

DO

IiOB19RT L. HILL

hydrolysis of proteins, it is necessary to clmploy mixtures of enzymes with several different sperificities. Complete enzymatic hydrolysis has some advantages O T W acid hydrolytic. procedures. On total enzymatic hydrolysis, the acid-labile amino acids such as asparagine, glutamine, tryptophan, and the phospho- or sulfoesters of certain amino acids are not destroyed. The amino arids such as seriiit and threonine which are destroyed partially by acid, as well as those whirh are released incompletely by acid hydrolysis, should be present in theoretical yields in enzymatic hydrolyzates. In addition, enzymatic hydrolysis may be very useful for the elucidation of bonds which are involved in linkages between proteins and prosthetic groups, certain types of inhibitors or coenzymes. Finally, because of the specificity of most proteiriases for bonds formed by amino acids of the L-configuration, total enzymatic hydrolysis provides a means for determining the stereochemical homogeneity of polypeptides and proteins. Exhaustive enzymatic hydrolysis was employed by several investigators in the late nineteenth and early twentieth centuries in studies on the isolation and characterization of acid- or alkali-labile constituents of proteins. Thus, cystine (Kulz, 1890), tryptophan (Hopkins and Cole, 1902), asparagine (Damodaran, 1932), glutamine (Damodaran et al., 1932), and the polysaccharide from ovalalbumin (Neuberger, 1938), to mention only a few substances, were isolated from enzymatic digests. Crude mixtures of the pancreatic proteolytic enzymes (often called pancreatin) were used in these studies. Subsequently, methods for determination of protein-bound substances such as glutamine and asparagine (Tower et al., 1062) and thyr0xine-1~~~ (Tong and Chaikoff, 1958) have been devised and employ hydrolyzates prepared on prolonged digestion with crude mixtures of pancreatic enzymes. One of the earliest suggestions that total enzymatic hydrolysis was possible came from the studies of Frankel (1916), who showed that over 90 % of the bonds in several proteins could be broken when proteolysis with pepsin, trypsin, and chymotrypsin was followed by prolonged hydrolysis with the erepsin preparation of Cohnheim (1901). The recognition in later years of several peptidases in intestinal extrarts 1vhic.h will specifi(#allyact upon bonds that are not susceptible to the endopcptidases (Bergmann, 1942) probably accounts for these observations. The specific peptidases such as prolidase, iminodipeptidase (proliiiase), glycylglycine dipeptidase, tripeptidase, and leucine aminopeptidase, which are present in mucosa, attack many of the bonds that resist the action of endopeptidases. I n a recent study on complete enzymatic hydrolysis (Hill arid Srhmidt, 1962), methods similar to those of Frankel were employed, but other endopeptidases as well as highly purified exopeptidases were used instead

HYDROLYSIS OF PROTEINS

91

of the impure mixtiires of pancreatic, gastric, and intestinal enzymes. For the initial proteolysis, the most desirable eridopeptidase is one which will degrade a protein most extensively into small peptides. I-nder optimal conditions, papain was found to hydrolyze most proteins more extensively than pepsin, subtilisin, or mixtures of trypsin and ehymotrypsin. After initial proteolysis with papain, hydrolysis was completed with leucine aminopeptidase (Hill et al., 1058) and prolidase (Davis and Smith, 1953). On the basis of specificity studies (see Section VI,G) leucine amiriopeptidase should hydrolyze all peptide bonds that remain intact in papain hydrolyzates, except those which contain the imino nitrogen of proliiie. Since endopeptidases do not attack this bond extensively, such peptides would be present in almost all partial hydrolyzates. Hydrolysis of proline peptides of this type would be achieved by prolidase. Corticotropin, oxidized ribonuclease, papain, carboxypeptidase, and enolase (Hill and Schmidt, 1962) as well as horse heart cytovhrome c (Margoliash et nl., 1962) and human myoglobin (Perkoff et al., 1962) have been submitted t o complete enzymatic hydrolysis. The amino acid composition (including proline) of total enzymatic hydrolyzates of each protein was in close accord with compositions established earlier by analysis of acid hydrolyzates. In some cases, however, certain amino acids were not detected in theoretical amounts, whereas in others a few residues were found in somewhat more than the expected yields. It is difficult to evaluate why theoretical yields were not obtained in all cases, but high yields would be expected if amino acids were liberated from the partially pure exopeptidase preparations. Low yields suggest incomplete hydrolysis or nonspecific degradation. Although peptides could not be detected in complete enzymatic hydrolyzates, some free amino acids might be altered under the conditions that are required for complete hydrolysis. For example, glutamine is converted to pyrrolidorie carboxylic acid and ran be estimated only after conversion to glutamic acid. Thus, complete eiizymatic hydrolysis might have many applications t o specaial problems in hydrolysis of proteins, but cannot at this time replace total acid hydrolysis in protein analysis. Many amino acids are estimated more accurately by use of acid hydrolyzates without encountering the experimental variations that often exist when enzymes are used for quantitative studies. Tower et nl. (1962) have employed enzymatic hydrolysis with pancreatin preparations for liberation of glutamine and asparagine from proteins. Under the conditions employed, proteins were not hydrolyzed to a n extent of more than 50-80 %, but after correcting for incomplete hydrolysis, yields of the two amides were in excellent agreement with theoretical values. I n all probability refinement of methods such as those of Hill and

92

ROBERT L. HILL

Schmidt (1962) or Tower et al. (1962) may lead to development of better methods for complete enzymatic hydrolysis. Endopeptidases other than papain and crude mixtures of pancreatiri have not been evaluated thoroughly for the initial proteolytic step. The wide specificity of S. griseus proteinase suggests it would be useful for this purpose, although the studies of Dawid et al. (1963) show that large amounts of digestion products seem to inhibit its action. Whether this inhibition is a serious problem cannot be ascertained, although a similar type of inhibition is known with other proteolytic enzymes, including papain (Kimmel et aE., 1962). Inasmuch as papain must be maintained in its active form throughout hydrolysis with reducing agents such as cyanide, mercaptoethanol, or other thiol compounds, the bacterial enzymes, which do not require activation, might be more desirable. It would seem that leucine aminopeptidase is the best choice for the second stage of proteolysis in which the small peptides are degraded to amino acids. As mentioned earlier the only peptide bonds which might resist its action are those containing the imino group of proline. Such bonds are broken slowly (Hill and Schmidt, 1962; Hofmann et al., 1962), but more extensive hydrolysis is achieved by prolidase. Peptides with the sequence It1-R2-li3-Pro-It4-* would be hydrolyzed rapidly by aminopeptidase with liberation of Ii', It2, and the peptide R3-Pro-It4--. The It3-Pro- bond is hydrolyzed very slowly by aminopeptidase but somewhat more rapidly by prolidase. In this regard prolidase is not strictly a dipeptidase, although it acts on dipeptides of the type B-Pro a t rates several thousand times those of the type R3-Pro-R4-. This type of specificity is substantiated by the results of Nolan and Smith (1962) who demonstrated that only lysine was liberated from a glycopeptide with a sequence Lys-Pro-Arg-Glu-Glu-Gln-Phe-Asp (CHO). Peptides in endopeptidase digests of the type R1-R2-Prowould offer little resistance to the combined action of aminopeptidase and prolidase, since the R1-K2 bond would be susceptible to aminopeptidase and the resulting dipeptide R2-Pro would be hydrolyzed readily by prolidase. Evaluation of other exopeptidases has not been made. Carboxypeptidase A did not seem to alter yields of free amino acids when used in conjunction with aminopeptidase and prolidase (Hill and Schmidt, 1962). On the other hand, some proteins with intact disulfide bridges cannot be degraded completely with enzymes. Use of peptidases that have a high specificity for bonds formed by cystine would be helpful, although this problem often can be overcome by prior cleavage of disulfide bonds on oxidation (Hirs, 1956) or reduction, followed by carboxymethylation (Crestfield et al., 1963). * R refers to any other amino acid.

TABLE XV Enzymatic Hydrolysis of Several Conjugated Proteins Protein Azaserine labeled enzyme Rabbit y-globulin Bovine globulin of colostrum Human y-globulin Cytochrome c Ovalbumin Ovalbumin Ovalbumin Chondroitin sulfate complex Fetuin Chromatium heme protein Phosphorylase

Enzymes

Products isolated

Papain, pronase, aminopeptidase Papain Papain Papain Pepsin, trypsin Pepsin, trypsin, chymotrypsin, mold protease Pancreatin Trypsin, chymotrypsin Papain Papain, trypsin, chymotrypsin, pepsin, subtilisin Pepsin Chymotrypsin

CI4-labeledazaserinepeptide Glycopeptides Glycopeptides Glycopeptides Heme peptides Glycopeptides

Dawid et al. (1963) Nolan and Smith (1962) Nolan and Smith (1962) Rosevear and Smith (1961) ~ P P (1958) Y Johansen et d.(1961)

Glycopeptide Glycopeptide Glycopeptide Glycopeptides

Jevons (1958) Cunningham et al. (1957) Muir (1958); Anderson et al. (1963) Spiro (1962)

Heme peptide Pyridoxal peptide

Dus et al. (1962) Fischer et al. (1958)

Reference

r 4

E

0

r v

m

0

e

8

m

94

ROBERT L. HILL

Co\dently bound prosthetic groups of proteins have been obtained in good yields from enzymatic hydrolyzates. Tables XV list several conjugated proteins that have been exhaustively degraded with enzymes. In each case prosthetic groups of the parent protein were isolated from the enzymatic hydrolyzates and were found t o be linked to amino acid or peptides.

VIII. ENZYMATIC HYDROLYSIS O F NATIVEPROTEINS Examiiiation of the action of protcolytic enzymes on native proteins (or biologically active peptides) can yield two important types of information. First, detcrrnination of the susceptibility of particular bonds in a protein substrate offers a means for evaluation of certaiii features of the conformation of the protein (Liiiderstrom-Lang, 1052; Mihalyi and Harrington, 19511). Second, proteolysis call serve as a n important method for modification of the covalent structure of biologically active proteins (Anfinsen and Itedfield, 1956). Table XVI gives a partial list of native proteins that have been hydrolyzed with proteolytic enzymes. A discussion of the interpretation of each example listed is beyond the scope of this review, but a few comments concerning certain features of proteolysis are warranted. The mechanism of enzymatic hydrolysis of native proteins was studied in detail by Tiselius and I3riksson-Quensel (1939), who examined the action of pepsin on ovalbumin. Two mechanisms of proteolysis were considered by these workers. In the first mechanism the enzyme hydrolyzes all susceptible peptide bonds in one substrate molccwle before hydrolysis of a second molecule begiris. This type of mechanism has been described by Linderstr@m-Lang (1952) as the “all or 11011e” type. I n the second mechanism, the enzyme hydrolyzes the single, most susceptible bond in all substrate molecules before hydrolysis of other bonds occurs. This mechanism is called the “zipper” type. Hydrolysis of a protein can proceed by either of the two mechanisms or by a mec*haiiismwhich has features of both types. General aspects of the problem have been reviewed and theoretical equations which describe the kinetics of earh mevhanism have been derived (Liaderstr~m-Larig, 1952, 1953). A number of studies have been designed to determine the nature of the mechanism of proteolysis with a specific enzyme and substrate. Giiisberg aiid Schachman (1960a,b) concluded that chymotryptic hydrolysis of insulin probaldy proceeds by the “all or none” mechanism, whereas peptic hydrolysis of riborruclease follows a “zipper” mechanism. In other studies it appears that the kinetics of digestion of human serum albumin by pepsin arid chymotrypsin follow the “all or none” mechanism, whereas tryptic action is of the “zipper” type (Kaminski aiid Tanner,

HYDROLYSIS OF PEOTEINS

95

1959). Ottesen and Schroeder (1961) found that subtilisin acts on native human hemoglobin by an “all or none” mechanism. It is interesting that the peptides from undenatured hemoglobin differ from those of heatdenatured hemoglobin. Therefore, if an enzyme must denature a native protein prior to proteolysis, as suggested by LinderstrGm-Lang (1952), the denaturation resulting from subtilisin action must differ from that produced by heat. On the basis of the studies available a t this time, it is impossible to predict which mechanism of proteolysis will occur in any given system. On the other hand, consideration of these mechanisms suggests that several structural features of a native protein determine the nature of the hydrolytic products. The covalent structures which provide the specific groups required by a particular enzyme must be partly responsible for the nature of the hydrolytic products, but features of the conformation must also be important. The folding of the polypeptide chains can allow a potentially susceptible bond to resist cleavage by restricting the availability of the groups that must react with the enzyme. Conversely, other bonds might be maintained in a configuration which make them readily available at the surface of the protein and thereby easily hydrolyzed by enzymes of the appropriate specificity. The effects of conformation on the extent of proteolysis are demolistrated most vividly by observations that proteolytic enzymes generally hydrolyze denatured proteins more rapidly than native proteins. This has been reported for trypsin and chymotrypsin (Anson and Mirsky, 1934), papain (Lineweaver and Hoover, 1941), carboxypeptidase (Fraenkel-Conrat et al., 1955), leucine aminopeptidase (Hill and Smith, 1957), and subtilisin (Ottesen and Schroeder, 1961). Thus, it is evident that examination of the action of proteolytic enzymes 011 native proteins may serve as one means for evaluation of the conformation of proteins in solution (Harrington et al., 1959; Mihalyi and Godfrcy, 1963). Several studies have demonstrated that the conformation of a native protein renders particular bonds in the peptide backbone of the molecule more susceptible to proteolysis than others. One of the best documented examples of this efiect is the action of proteolytic enzymes 011 y-globulins or specific antibodies. A number of workers have examined the proteolysis of yglobulin after the original observations of peptic digestion of horse diphtheria antitoxin by Pope (1939). However, the studies of Porter (1‘350, 1959) and Fleischmaii ef al. (1963) on the fragments of y-globulin produced after digestion with papain are the most thorough studies to date and have scrved as a guide to many of the later studies on the immune globulins. It appears that y-globulin from a variety of mammals is composed of four polypeptide chains, two with a moleciilar weight of 50,000 (A-chains) and two with a weight of 25,000 (R-chains). The four chains

T.4BLE XVI Enzymatic Hydrolysis of Proteins and Polypeptides

Enzyme Trypsin

Kative protein Human 7-globulin Horse diphtheria antitoxin Human serum albumin Insulin Trypsin

Enolase Catalase Adolase Ribonuclease Ribonuclease S Fibrinogen Myosin Tropomyosin Bovine plasma albumin Papain

Rabbit ?-globulin Human 7-globulin

Reference Schrohenloher (1963) Northrop (1941-1942), Rothen (1941-1942) Lapresle et al. (1959), Kaminski and Tanner (1959) Xicol (1960), Carpenter and Baum (1962) Bressler et aE. (1954), Chernikov (1956), Hess and Wainfon (1958) Malmstrom (1958) Anan (1958) Bresler et al. (1954) Ooi et al. (1963) dllende and Richards (1962) Mihalyi and Godfrey (1963) Mihalyi and Harrington (1959) de Milstein and Bailey (1961) Richard et al. (1960) Porter (1950, 1959), Putnam et al. (1962), Fleishman et at. (1963) Hsiao and Putnam (1961)

Enzyme Pepsin

Chymotrypsin

Sative protein

W

Q,

Reference

Horse diphtheria Pope (1939), Peterman Papenheimer (1941) Pepsin Perlmann (1954), Tokuyasu and Funatsu (1962) Botulinum toxin Wagman (1963) Catalase Anan (1958) ACTH Li et al. (1955) Ribonuclease Anfinsen (1956), Ginsberg and Schachman (1960a,b) Human serum Kaminski and Tanner (1959) albumin Human Hanson and Johansson r-globulin (1960) Growth hormone Li et al. (1959) Oxytocin Golubow and du Vigneaud (1963) Insulin Ginsberg and Schachman (1960a,b), Butler et al. (1950) Ribonuclease Rupley and Scheraga (1963) Human Serum Kaminski and Tanner (1959) albumin Chymotrypsin Gladner and Neurath (1954) Growth hormone Harris et al. (1954) Ribonuclease Anfinsen and Richards [quoted in Anfinsen (1956)l Ribonuclease TI Takahashi (1962)

m

3

r

8F

Human serum globulins Lipovitellin Thyroglobulin Carboxypeptidase A

Leucine Insulin aminopeptidase Oxytocin

Deutsch et al. (1961) Glick (1963) O’Donnell et al. (1958)

Papain ACTH Enolase

Insulin

Lens (1949), Harris (1952), Harris and Li (1952), Slobin and Carpenter (1963) Enolase Malmstrom (1958) Hemoglobin. h t o n i n i et al. (1961) ACTH Harris and Li (1955) Soybean trypsin Davie and Neurath (1955) inhibitor Tobacco mosaic Harris and Knight (1955) virus Aldolase Drechsler et al. (1959) Crotoxin Fraenkel-Conrat and Singer (1956)

Subtilisin

Ribonuclease

Ovalbumin Human hemoglobin Cytochrome c

I

Hill and Smith (1957), Smith et al. (1958) Golubow and du Vigneaud (1963) Hill and Smith (1960) White (1955) Malmstrom (1958) Richards and Vithayathil, (1959), Gordillo et al. (1962) Ottesen (1958) Ottesen and Schroeder (1961) Nozaki et al. (1957)

*zu 2

E m

S. yriseus protease Taka-amylase

Toda and Akabori (1963)

0

Collagenase

von Hippel and Harrington (1959)

8

Collagen

I3

98

1iOBERT I.. MILL

are combiiied through disulfide bonds (k'ig. 6). Hydrolysis by papain results in a rapid cleavage of the A-chains, so that in the presence of reducing agents which split one disulfide bond between two A-chains, two fragments are produced. One fragment is composed of one R-rhain and half of one A-chain, and another fragment is composed of the other two halves of A-chains. Although prolonged proteolysis degrades these fragments, it appears that the coiiformation of 7-globulin allows a particular segment in the A-chains to he highly susceptible t o the action of enzymes. Other examples of this type of fragment production have been described for myosin (Mihalyi and Harriiigton, 1959), thyroglobuliii (O'Donnell et al., 1958), fibrinogen (Rlihalyi and Godfrey, 1963), and collagen (von Hippel and Harrington, 1959). B

A

A

B

B

A

A

A

Is-l

+ Small

2

Peptldes

Rapid Profeolysis by Papain, Pepsin Trypsln or Chymofrypsin. Fragment I

Fragment lU

Rabbif 7 - g l o b u l l n

Fie. 6. Schematic rrpresrnlation of the hytlrolysis of natlvr rahbit y-globulln or specific rabbit antibodies by proteolytic enzymes y-Globulin contains four polypeptide chains. Two thnins h a t e a molrcular weight of 50,000 ( A ) and two others (B) a molecular weight of 25,000. Protcolysis and cleavage of one disulfide bond rrsults in prefercntid rlraiagc of the two A-chains with the production of tmo types of subunits. After Bleischman et al. (1963).

Several studies xith riboiiuclease siiggost tlhat cwtaiii peptide bonds of a given kind are more available to proteolysis than others (reviewed by Scheraga and lbpley, 1962). Thus, prpsiii hydrolyzes oiic boiid betwwi residues 120 and 121 which leads to inat tivation of the molecule (Aiifinsen, 1956). Sitbtilisiii (Richards mid Vithayathil, 1959) rapidly splits a single bond between alariiiie and seririe a t residues 20 a i d 21. C'arboxypcptidase

HYDROLYSIS O F PROTEINS

99

slowly removes two to three carboxyl-terminal residues, whereas no hydrolysis is observed with aminopeptidase (Hill and Smith, 1957). Thermally unfolded ribonuclease becomes susceptible to trypsin and chymotrypsin in contrast to the resistance to proteolysis exhibited a t low temperatures (Itupley and Scheraga, 1963; Ooi et aZ., 1063). Thus, with the aid of enzymes, some insight has been gained into the folding of certain portions of the ribonuclease molecule. In addition, these studies have given valuable information relating the structure of ribonuclease to its enzymatic function. Other examples are given in Table XVI of enzymatic modification of the covalent structure of biologically active proteins. It is difficult to make a general conclusion about such studies other than to state that no single enzyme appears to be more generally useful than others in experiments of this kind. Each protein with its specific conformation must have different portions of its polypeptide chain available for proteolysis. For example, some globular proteins appear to have the amino- or carboxyl-terminal residues near the surface as judged by the ease of removal of the terminal amino acids with proteolytic enzymes. Others appear to have end groups in a configuration which renders them inaccessible to the exopeptidases. It is also evident that the absence of detectable splitting does not definitely prove that all peptide bonds are unavailable to proteolysis. Before hydrolysis of exposed bonds can be obtained, it is necessary to employ an enzyme with the proper specificity.

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THE UNUSUAL LINKS AND CROSS-LINKS OF CQLLAGEN By JOHN J . HARDING The Gelatine and Glue Research Association. Halloway. London. England

I. Introduction . . . . . . . . . . . . . . I1. The Cross-Links of Collagen . . . . . . . . . . A . Physical Evidence for Crom-Links in Collagen . . . . . B. The Relationship between Collagen and Gelatin . . . . . C . Soluble Collagens and Their Subunits . . . . . . . . D . Lathyrism . . . . . . . . . . . . . . I11. y-Glutamyl and P-Aspartyl Peptide Linkages . . . . . . A . Naturally Occurring y-Glutamyl Peptides . . . . . . . B . Naturally Occurring P-Aspartyl Peptides . . . . . . . C . y-Glutamyl and p-Aspartyl Peptide Linkages in Polypeptides and Proteins, Especially Collagen and Gelatin . . . . . . . D . Discussion . . . . . . . . . . . . . . E . Conclusions . . . . . . . . . . . . . . IV . €-Amino Peptide Linkages . . . . . . . . . . . V . Ester-Like Linkages in Collagen . . . . . . . . . . A . Determination of Esters in Collagen Using Lithium Borohydride . B. Determination of Ester-Like Links in Collagen and Gelatin Using Hydroxylamine . . . . . . . . . . . C . Determination of the Ester-Like Linkages of Collagen Using Hydrazine . . . . . . . . . . . . . . D . Conclusions . . . . . . . . . . . . . . VI . Carbohydrate Linkages . . . . . . . . . . . . A . Attempted Removal of Carbohydrate from Collagen . . . . B . Residues from Enzymatic Digests of Denatured Collagen . . . C . Studies of Carbohydrates in Collagen Using Periodic Acid . . . D . Conclusions . . . . . . . . . . . . . . VII . The Interrelationship between Carbohydrate and Ester Links and the Cross-Links of Collagen . . . . . . . . . . . . A . Collagen . . . . . . . . . . . . . . . B. Discussion . . . . . . . . . . . . . . VIII . Other Unusual Links and Cross-Links in Collagen . . . . . . References . . . . . . . . . . . . . . .

109 110 111 114 116 119 120 121 122 123 131 136 136 144 146 149 158 161 162 162 164 165 169 169 170 176 178 181

I. INTRODUCTION This review concerns the work on the unusual links and cross-links of collagen . The unusual linkages may exist as branching points or cross109

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links or may be built into the peptide backbone itself. Pertinent studies on other proteins have been included. Section I1 contaiiis a general discussion of the cross-links of collagen. In subsequent sections the evidence for different unusual links in collagen is reviewed. Certain of these linkages appear to be involved in the cross-links. 11. THECHOSS-LINKS OF COLLAGEN Proteins are normally visualized as consisting of long chains of amino acids joined by way of their a-amino and a-carboxyl groups, forming linkages. These chains can either be stretched peptide (--CO-M-) out or folded. Further bonds between adjacent chains, or adjacent parts of the samc chain after folding, are incorporated into the structure to stabilize the protein configuration which is essential for its biological function. These honds could be hydrogen bonds, hydrophobic bonds, electrostatic bonds, or covalent bonds and, indeed, bonds of the first three types probably ocmr in all proteins. In addition to these, cystine disulfide bridges arc knomn to occur in certain cases. Such disulfide links probably have little importance in collagen as the cystine content found is less than 0.05 yo (Eastoe, 195.5) and may not be an intrinsic part of the collagen molecule, but a constituent of associated impurities (Leach, 1960). Fischer (1906) recognized the possibility of linkages between active side chains. I'auling and Nicmann (1939) suggested that covalent cross-links such as disulfide bridgcs, side-chain ester links, and side-chain peptide links might bc partly responsible for the configurational stability of proteins. Only covaltnt cbross-links excluding disulfides will be considered in the present revie\$. The preserwe of cross-links not involving disulfide (S-S) bridges has bccii suggested in elastin (Partridgr, 1962; Partridge et nl., 1963; Walford rt al., 1961) and resilin (Andersen, 1963). In two recent papers publishcd a t the same time (Thomas et al., 1963; Bedford and Katritzky, 1963) further data have been presented that establish the precisc natnre of the moieties forming the cross-links. By chromatography of an acid hydrolyzate of elastin on ion-exchange resins of differing crosslink density, two (*ompotleiitswere isolated, each containing a heterocyclic nucleus attached to several amino acids. These so-called H-peptides were studied by chemical methods as well as by ultraviolet and proton resoi1anc.e spectra. The results lead to the conclusioii that the bridging componcwt is a pyridinium salt with peptide chains linked a t four positions arouiid the heterocyclic nucleiis. In the case of collagen one must consider that the collagen monomer, often called tropocollagen, appears to consist of three helical polypeptide chairis wound around each other to form a coiled coil (Rich arid Crick,

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1961). It behaves as a rigid rod with a molecular weight of approximately 350,000 (Roedtker and Doty, 1956; Hannig and Engel, 196l), a diameter of about 14 A, and a length of 3000 A (Roedtker and Doty, 1956), irrespective of its source. It is therefore necessary to distinguish between inter- and intramolecular cross-links. The latter are links between the individual chains of a triple helical collagen monomer; whereas the former are links from one collagen monomer t o another

Intramolecular cross-links

Intermolecular cross-links

The bulk of the physical evidence for cross-links depends on the existence of a complete lattice formed by intermolecular cross-links. The separation of components of denatured collagen sheds light on both types of linkage. The physical evidence for cross-links in collagen will first be presented, followed by a discussion of the importance of cross-linking in the collagen $ gelatin transformations. Finally, the blocking of the cross-linking mechanism by lathyrogenic reagents will be considered.

A. Physical Evidence for Cross-Links in Collagen The insolubility of all but a small proportion of mature collagen in any aqueous or organic solvent that does not attack it chemically is generally indicative of a cross-linked system. Collagen remains largely insoluble even in slightly acidic aqueous media, where carboxyl groups would be un-ionized and no electrostatic linking could occur, and even in the presence of agents for breaking hydrogen bonds in aqueous solution. With increasing temperature weak bonds such as hydrogen bonds become less effective, and yet the bulk of the collagenous material remains insoluble. Ames (1952a) discussed imide cross-linking (-CO-NH-CO-) to

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explain the release of ammonia during the conversion of collagen to gelatin. In the same year Wiederhorn and Reardon (1952) postulated the presence of cross-links to explain the stress-strain properties of heat-shrunk collagen. They calculated the chain molecular weight of the residues between crosslinks to be 55,000. This value was not greatly influenced by the use of media having different dielectric constants and a t different temperatures; this argues in favor of the covalent nature of the cross-links. The swelling of collagen in buffers of p H below 4 or above 11 has been taken as evidence (Harkness, 1961) that no covalent cross-links exist in collagen, because keratin, containing many disulfide cross-links, does not swell in this way. It must be remembered, however, that the amount of swelling is limited when fairly mild conditions are used. Swelling increases during liming, and Fysh (1958) suggested that this, in conjunction with the decrease of shrinkage temperature, indicated the breaking of cross-links. The increase of the shrinkage temperature of collagen is well established as a n indication of the extent of cross-linking by tanning agents. It seems reasonable that collagenous materials with a shrinkage temperature less than that of mature insoluble collagen, e.g., soluble collagen and alkalitreated collagen, will have a correspondingly lesser degree of cross-linking provided that their formation from collagen does not involve a great deal of nonspecific degradation. Gustavson (1946) demonstrated that the shrinkage temperature of collagen was lowered only 11°C by carrying out the determination in 0.1 M p-naphthalenesulfonic acid a t pH 1.6. Under these conditions all the electrostatic links would be broken, and Gustavson suggests that the relatively small fall of shrinkage temperature proves that electrostatic links are not the main cohesive forces of the collagen structure. Flory (1956a,b) considered the shrinkage of collagen in terms of statistical mechanics and concluded that thermal shrinkage is a phase transition similar to melting. The elevation of the melting temperature with inrreasing cross-link density was treated theoretically. Brown et aE. (1958) demonstrated that the shrinkage temperatures of immature collagens, including collagen from young children as well as that from uteri, were lower than that of adult collagen. They found that the action of salicylate did not reduce the shrinkage temperature of adult collagen to that of fetal collagen, and concluded that either the stabilizing links are not hydrogen bonds or they are hydrogen bonds that are tucked away within the molecule. The increase of shrinkage temperature with age has been reported (Joseph and Bose, 1962; Verxhr, 1963) as well as its decrease in certain pathological conditions (Stringer and Highton, 1960). Considering that both insoluble and soluble collagen have very similar

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amino acid compositions (Bowes et al., 1953), it is unlikely that electrostatic bonding can explain the difference between them in this instance. Alkali treatment reduces shrinkage temperature (Fysh, 1958), and Courts (1960) suggests a minimum figure of approximately 44°C for limed ossein (demineralized bone). At this stage few intermolecular cross-links can be present because the material can be dissolved in water a t 60°C. Gustavson (1962) has shown that treatment with periodate also lowers the shrinkage temperature (T,) by as much as 20°C. This could suggest that alkali and periodate attack the same cross-link, but other side reactions under the conditions used will also have some effect. Zahn and Nischwitz (1960) cross-linked collagen with p,p'-difluoro-m,m'dinitrodiphenyl sulfone and by analysis found the number of cross-links introduced. From this and shrinkage-temperature determinations they estimated that approximately 6.5 cross-links per lo6 gm are required to increase T , by 10°C. If this relation could be applied to decreases of shrinkage temperature, assuming long-limed collagen has no cross-links and bearing in mind the reservations expressed above, one can estimate that mature ox collagen has about fifteen cross-links per lo5 gm. Cater (1963) has studied the cross-links of collagen by the stress-strain method adopted by Wiederhorn and Reardon (1952). The treated collagen gave a value similar to that of the previous authors, but fresh kangaroo or wallaby tendon gave a lower value of only one cross-link per 4 X lo5gm of collagen. Cater emphasizes that this method only estimates the number of cross-links in excess of those required to give a complete network. He also studied the collagen samples after cross-links have been introduced by a number of tanning agents. The shrinkage temperature of these samples is clearly related to the number of cross-links introduced as determined by the stress-strain method (see Fig. l), although shrinkage-temperature determinations are very dependent on pH, solvent, and rate of heating. These results suggest that approximately nine cross-links are required t o raise the shrinkage temperature by 30°C. This is about half the value suggested by Zahn and Nischwitz (1960) and, making the same assumptions as before, leads to an estimate of six cross-links per lo5 gm in collagen. These estimates are of the same order as the number of ester links said to exist in mature collagen (see Section V). Stress-strain measurements also indicate that the cross-link density in ratskin collagen increases with age (Kulonen et al., 1963). The apparent relation between shrinkage temperature and total imino acid content (Gustavson, 1956b; Piez and Gross, 1960; Harrington and von Hippel, 1961b) is not relevant to the above discussion which is concerned only with a comparison of collagens and modified collagens having the same imino acid content.

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t

Shrinkage temperaturePC)

FIG.1. Plot of the number of cross-links introduced by glyoxal or glutaraldehyde against the shrinkage temperature of the product. Data taken from Cater (1963).

Thus, the physical data discussed establish with considerable certainty that cross-links occur in collagen. The nature of these cross-links, their abundance, and their distribution in the collagen molecule remain to be established. The relationship between collagen and gelatin will now be considered before proceeding to a discussion of the subunits of the collagen molecule.

B. The Relationship between Collagen and Gelatin 1 . The Collagen 3 Gelatin Transformation

Ames (1952a) discussed the relative merits of cross-linked and single(+hainmodels for collagen to explain the conversion to gelatin. The multichain model gave gelatin by a cleavage of the cross-links, whereas conversioii of the single-chain model depended on peptide cleavage. Later Ames (1952b) concluded from the titration curves of gelatins that neither peptide cleavage nor the release of ammonia played a major role in the conversion of collagen t o gelatin. Following on this many other authors have found it necessary to assume cross-links in collagen in order t o explain the results of studies on the collagen -+ gelatin transformation (Gustavson, 1955b,c; Veis and Cohen, 1956; Veis et al., 1958; Courts, 1960). Veis et al. (1958) have discussed the differences between acid-processed and alkali-processed gelatins in

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terms of cross-linking. On the basis of light-scattering, titration, and electrophoretic mobility data they suggested that alkali-processed gelatins exist as single-chain random coils, whereas acid-processed gelatins are multichain molecules still held together by the covalent cross-links of the original molecule. They proposed that the collagen + gelatin transformation involves three processes: (a) melting of the polypeptide helical coils, (b) severance of the interchain bonds, (c) hydrolysis of peptide linkages. Although (a) always occurs first, they suggested that ( b ) precedes (c) during alkali treatment, whereas during acid extraction (b) and (c) occur concomitantly. The idea of single-chain molecules for alkali-processed gelatins is not in accord with the work of Courts and Stainsby (1958), who have postulated a multichain structure for both types of gelatin on the basis of the relationship between molecular weight, determined by light scattering, and chain molecular weight. The existence of a multichain structure for gelatin is very powerful evidence for the existence of cross-links in collagen. It was not clear from the results of Courts and Stainsby (1958) if the structure were branched or cross-linked, but Heyns and Legler (1958) found the same number of carboxyl- and amino-terminal amino acids which suggests that gelatin consists of chains held together by cross-links. As Ward (1960) points out the extraction of gelatin becomes less difficult with increased liming of collagen and this is indicative of cross-link rupture. This was apparent from Courts’ work on eucollagen (alkali-treated collagen) (Courts, 1960) showing that the long-limed collagen is held together only by electrovalent links and hydrogen bonding (see also Kuhn et al., 1963b). Crosby and Stainsby (1962) have discussed the differences between eucollagen and soluble collagen a t some length. I n recent years a number of authors have reported the specific enzymatic cleavage of the cross-links in collagen (Barkin and Oneson, 1961; Highberger, 1961b; Kuhn et at., 1961; Nishihara, 1962; Schmitt, 1963; Kiihn et al., 1963a; Rubin et al., 1963; Hafter and Hormann, 1963). The enzymes used include pepsin, ficin, trypsin, and unpurified pancreas extracts; of these pepsin is the most effective. As pepsin has no esterase or other nonproteolytic activity it appears that the cross-links include, even if they are not entirely, sections of peptide chains. 2. The Gelatin -+Collagen Transformation

The reversion of gelatins to collagen has also been discussed with refer ence t o cross-links. The denaturation of collagen results in marked changes of viscosity, optical rotation, molecular weight, volume, kinetics of proteolysis, and other properties. Under certain conditions a partial reversal of these changes can be achieved (Flory and Garrett, 1958; von Hippel

116

JOHN J. HARDING

and Harrington, 1959; Flory and Weaver, 1960; Harrington and von Hippel, 1961a; Courts, 1962; Engel, 1962a; Courts and Little, 1963; Higgs and Reed, 1963; Reed et al., 1963). A proportion of the denatured material has been shown to have undergone true reconstitution by the fact that its denaturation temperature (Flory and Garrett, 1958), electron microscope forms (Rice, 1960; Veis and Cohen, 1960), and resistance to enzymes (Kuhn, 1963) are the same as those of the original collagen. ' I he steps involved in the reversion process have been discussed by von Hippel and Harrington (1959) and Flory and Weaver (1960). A very interesting aspect of the reversion to collagen has arisen from the studies of Grasbmann et al. (1!)60). They digested denatured acid-soluble collagen with trypsin and Sound that a number of the peptides had a threechain structure. The denatured material must consist of single chains and chain pairs (see Section II,C) with very few triple-chain molecules. This state of affairs presumably persisted during proteolysis, and therefore the resultant peptides must then take on a helival form and associate to form triple-chain molecules. This demonstrates the favorableness of the threechain structure for imino acid-rich peptides. Veis and Cohen (1960) found that reconstitution to collagen, as shown by electron micrographs, only occurred for multichain gelatins. They suggested that the chains must be held in the collagen form for a small region near the cross-links. These regions form the nuclei for subsequent reversion. It has been established that a proportion of denatured collagen [about 10 % ' according to Engel (1962a)l can revert fully to the collagen structure. The most ready reversion seems to occur with multichain gelatins. The reversion of the components of denatured collagen is also closely related to the presence of cross-links and is discussed in the next subsection.

C. Soluble Collagens and Thew Subunzts Certain solutions, such as dilute alkali buffers, salt solutions, and acid buffers, are each able to extract a small proportion of collagen from collagenous material. These solutions contain collagen as the individual triple helices. Such soluble collagens can have no covalent intermoIecular caross-links. This has been brought out in Table I taken from a review on collagen by IIormann (196Ob). Similar cbonclusionswere made by Gustavson (1962) after a detailed comparison of bovine collagen u ith cod collagen, which is largely soluble iii weak acid solutions. After denaturation these three-chain macromolecules can be separated into several subunits. The molecular weights and compositions of these

T H E UNUSUAL LINKS AND CROSS-LINKS O F COLLAGEN

117

TABLEI Comparison of Collagen and Procollagenl* Property or effect

Soluble collagcn

Collagen

Amino acid composition Identical Electron microscope spacing Identical Shrinks at G2"-64"C Warming in water Forms gelatin a t 54°C Acid buffers Dissolves in native form Swells Hydrogen-bond breakers Dissolves with denaturaSwells tion Stabilization of the fibers Hydrogen bonds and elec- Hydrogen bonds, electrotrostatic forces static and covalent links From Hormann (IY60b).

subunits and the interrelationships of these to each other and to the molecular weight and composition of the collagen macromolecule itself provide extremely powerful evidence for the existence of intramolecular cross-links in collagen. A complete review of the extensive literature concerned with these subunits is not available, although several brief accounts have appeared (see Crosby and Stainsby, 1962; also Hannig and Engel, 1961; Harrington and von Hippel, 1961b). It has been shown that denatured acid-soluble collagen is a mixture of three components with molecular weights Ma, 2M,, and 3Ma, where M , is the molecular weight of the smallest subunit. The proportion of the largest component ( 7 ) is relatively small (Altgelt et al., 1961; Grassmann et al., 1961). The relative proportions of the two smaller components (aand p) found in different collagens are summarized in Table 11. Alkali- and salt-soluble collagens contain little or none of the 0-subunit (Masurov and Orekhovitch, 1960; Nikkari and Kulonen, 1962; Piez et al., 1961, 1963; Jackson, 1962; Wood, 1962). Later studies (Piez et al., 1961; Schleyer, 1962; Piez et al., 1963) have shown that the a- and @-componentscan each be separated into two components with somewhat different amino acid compositions. The interrelationships of these are such that the so-called pl-component consists of one d-and one a2-component, whereas the 02-component consists of two al-components. The y-component clearly consists of all three chains linked together. The collagen monomer without intramolecular cross-links consists of two a1- and one a2-subunit. This is the form in which the collagen is originally laid down and in this form it is extractable by dilute alkali buffers and dilute sodium chloride; a t a later stage intramolecular cross-links are introduced and the resulting collagen can only be extracted by acid buffers.

118

J O H N J. HARDING

TABLEI1 Relative Proportions of the a- and &Components of Denatured AcidSoluble Collagen Tissue

Molar ratio

Weight ratio

Ratskin

2: 1

1:l

Rat tendon Rat tendon Calfskin

4:s 1:l 1:l

2 :3 1:2 1:2

Calfskin Human fetal skin Codskin Dogfish sharkskin Ichthyocol Spiny dogfish skin

2: 1 2:l 2: 1 2: 1 5: 1 1:l

1:l 1:l 1:l 1:1 5:2 1:2

References Orekhovitch and Shpikiter, (1958bc), Orekhovitch et al. (1959), Masurov and Orekhovitch (106O), Piez el al. (l961), Martinet al. (1%3), Piez el al. (1963) Nikkari and I
Iizvestigatioiis of the reversion of the a-, p-, and y-components to collagen indicate that the y-component, with all three chains linked together, undergoes the most ready and complete return to the collagen structure (Vcis et al., 1961; Altgelt et al., 1961; see also Engel, 1962a,b), presumably because the chain adjacent to the cross-links is held in the correct configuratioii arid acts as a nudeus for reversion. It is important to emphasize that a- and /3- and probably also y-components can be demonstrated in acid-soluble collagens obtained from a variety of tissues of a number of different animals and fish. Those studied so far inrlude rabbitskin (Mathews et al., 1954), ratskin (Orekhovitch and Shpikiter, 1955b, 195%; Orekhovitch et al., 1959, 1960; Masurov and Orekhovitch, 1960; Piez et al., 1961, 1963; Wood, 1962; Martin et al., 1963; Orekhovitch et al., 1962), calfskiri (Doty and Nishihara, 1958; Piez et al., 1960; Haririig and Engel, 1961; Grassniann et al., 1961; Altgelt et al., 1961; Highberger, 1961a; Wood, 1962; Schleyer, 1962; Engel et al., 1962; Speakniari, 1963), huinari skin (Rakerinan, 196la,b; Bakerman and Hersh, 1963), rat tail tendon (Kessler et al., 1959, 1960; Rosen et al., 1960a,b; Piez et al., 1963), carp ichthyocol (Chun and Doty, 1958; Doty and Nishihara, 1958; Orekhovitch arid Shpikiter, 1958c; Piez et al., 1963), codskin (Doty arid Nishihara, 1958; Orekhovitch and Shpikiter, 1 9 5 8 ~ ;Young and Loririicr, 1960), dogfish sharkskin (Lewis and Piez, 1961), and spiny dogfish skin (Piez et al., 1963). It is clear that the formation of a t least two dis-

T H E UNUSUAL LINKS AND CROSS-LINKS O F COLLAGEN

119

crete components or1 denaturation of acid-soluble collagen is a property of collagen from all species. It has been shown that the intramolecular links in P-components are split by heat or mild alkali treatments (Chun and Doty, 1958; Doty and Nishihara, 1958; Kuhn et at., 1963b), although this action is rapidly followed by a more general breakdown (Altgelt et d . , 1961; Schleyer, 1962). A similar cleavage by pepsin and trypsin has been reported by Kuhn (Kuhn et al., 1963a) and by Schmitt (Rubin et al., 1963), whereas it was not found by Hafter and Hormann (1963). The existence of these subunits establishes with certainty that intramolecular cross-links exist between the individual chains of the collagen macromolecule. Isolat,ion of even larger components from insoluble collagens (Veis et d.,1960, 1961) establishes that in mature collagen intermolecular cross-links are also present.

D. Lalhyrism Certain simple organic compounds, notably p-aminopropionitrile and aminoacetonitrile, when admiriistcred t o growing animals, bring about a considerable weakening of the connective tissue (Levene and Gross, 1959) together with a vastly increased extractability of the collagen into neutral salt solutions (Levene and Gross, 1959; Wirtschafter and Bentley, 1962; Martin and Goldhaber, 1963). No differences have been found between this lathyritic collagen and normal soluble collagen by amino acid analysis, intrinsic viscosity, electron microscopy, sedimentation, carbohydrate content, denaturation temperature, optical rotatory dispersion, flow birefringence, X-ray, infrared, a m i n o group availability, and ester-link studies (Levene and Gross, 1959; Nikkari and Kulonen, 1962; Gross, 1963). It is now apparent that the lathyrogen does not attack mature collagen but rather prevents the formation of the cross-links (Wirtschafter and Rentley, 1962; Martin and Goldhaber, 1963). Levene (1962) has shown that certain carbony1 compounds are able to reverse the avtion of lathyrogcns. He thereforc concludes that the latter block rarbonyl groups of the collagen necessary for normal maturation, and discusses this in relation to the aldehyde groups said to exist in collagen (Landucci, Pouradier and Durante, 1958). The necessary carbonyl groups could, however, be part of sugar molecules and might be of interest when considering carbohydrate cross-linkages. It is clear that these relatively simple organic molecules can prevent the intermolecular cross-linking of collagen. The increased solubility in neutral salt solution as well as iri acid buffers indicates that intramolecular cross-linkiiig may also be prevented. This has been confirmed by studies of the a- and P-components of lathyritic collagen.

120

JOHN J. HARDING

Martin and assoviates (1961) took a single acid extract from the skins of lathyritic rats. After denaturation they subjected the collagen to ultracentrifugation and found that only a small proportion of the @-component was present, i.e., the collagen resembled salt-soluble rather than acidsoluble collagen. Carboxymethyl cellulose chromatography also revealed a much greater proportion of the a-component. It appears therefore that the lathyrogen prevents the formation of acid-soluble collagen from saltsoluble collagen, i e., it prevents the 201 + @ step. Nikkari arid Kulonen (1962) noted that acid-soluble lathyritic collagen exhibited an elevated a l p ratio oiily when a prior NaCl extraction was omitted. Lathyrism, therefore, only causes an increased amount of saltsoluble c,ollagen and does not affect any acid-soluble collagen that may already be present. Very recently, Martin et al. (1963) foulid a rnucah slower incorporation of C14-glycine into the @-romponents prepared from lathyritic collagen compared with normal collagen, as well as the lesser amounts of the @-cornponent found in the former case. They could detect no differences in the amino acid compositions of corresponding subunits isolated from lathyritic and normal collagens. The lathyrogenic compounds can therefore be seen to inhibit both intraand intermolecular cross-linking. This would indicate that either the intramolecular linking is an cssential prerequisite for intermolecular crosslinking, or, more likely, that both processes occur by a very similar reaction or sequence of reactions. Further studies with different lathyrogens and compounds able to reverse thcir artion may well give a n insight into the nature of the cross-links themselves. Interference with the natural cross-linking of collagen must also be considered in relation to pathological collagens as prevention of stabilization m a y occur by meails other than the administration of lathyrogens.

111. 7-GLIJTAMY L ANI) P-ASPARTYL PEI’TIDE LINKAGES One possible variation on the accepted a-peptide structure of proteins would be the presence of prptide linkages involving the amino acid sidechain carboxyl and amino groups. A discussion of links involving E-amiiio groups will be considered in Section IV. Here @-asparty1and y-glutamyl peptide linkages will be considered. These could occur in either of two ways: (a) as an alternative to a-peptide linking in the peptide backbone

n = l or 2

THE U N U S U A L LINKS AND CROSS-LINKS OF COLLAGEN

121

or (b) a s a n intrachain link between a 0-or y-carboxyl group of one chain and a n amino group of an adjacent chain R

R

R

I

co I

n = l or 2

The amino group in this structure could be an eamino group of lysine or hydroxylysine or the terminal a-amino group of the second chain. In the latter case the 0-or y-carboxyl peptide link would constitute a branching point rather than a cross-link. y-Glutamyl and P-aspartyl links have been reported in many small peptides and these will be considered briefly before proceeding t o the evidence for such links in proteins and, in particular, in collagen.

A. Naturally Occurring y-Glutamyl Peptides The first peptide shown to have a r-glutamyl peptide link was glutathione or y-glutamylcysteinylglycine. Between 1920 and 1935 a great deal of evidence was accumulated in favor of this structure and was finally substantiated by the synthesis of glutathione accomplished by Harington and Mead (1935). Reviews of the evidence for the structure are given briefly by Harington and Mead (1935) and more recently by Fox (1945) and by Bricas and Fromageot (1953). The latter authors, also reviewed the work demonstrating the presence of y-glutamyl linkages in a number of other naturally occurring peptides and polypeptides including the fermentation Lactobacillus casei factor [(pteroyl-y-glutamy1)-y-glutamylglutamicacid] and poly-D-glutamic acid. A single volume devoted entirely to glutathione has been published [ "Glutathione," Biochem. SOC.Symp. (Cambridge, Engl.) 17 (1959)l. Since that time a very considerable number of small peptides containing y-glutamyl linkages have been reported. Some of those found very recently are included in Table 111, together with those mentioned above. The recent papers refer to some naturally occurring y-glutamyl peptides which had been found previously, and these are also reported by WaldSchmidt-Leita and Reicheneder (1961), and Franzblau (1962).

122

JOHN J. HARDING

TABLE 111 y-Glutamyl Peptides Pcptide Glutathione or 7-glutamyl cysteinylglycine (Pteroyl-y-glutamy1)-y-glutamylglutamic acid or fermentation L. casei factor y-L-GIu-L-rrYr and y-L-Glu-L-Phe

y-~-Gln-S-(prop-l-cnyl)-L-cysteine y-Glu-Leu 7-Glu-Ileu 7-Glu-Val S-(a,@-Dicarboxyethy1)glutathione

Source

Reference

Very widespread; yeast and most animal tissues Corynebactel zum

Harington arid Mead (1935)

Soya beans

Morris and Thompson (1962) Mutikkala and Virtanen (1962)

Seeds of chives

Bricas and Fromageot (1953)

Human urine

Buchanan et al. (1962)

Calf lens

Calam and Walcy (1963) Dunnill and Fowden (1 963) Morris et al. (1963)

y-L-Glutamyl-@-pyrazol-1-yl-1,-alanine

Cucumber seeds

y-Glu-Leu and y-Glu-Met

Kidney bean seeds

In all the y-glutamyl peptides reported glutamic acid residues linked via the y-carboxyl groups have the a-carboxyl free and therefore do not ronstitute branching points or cross-links. Several approaches have been used to identify the presence of the y-peptide linkage. Demonstration of a free a-amino and a-carboxyl group on the same carbon atom has been performed using oxidation by hydrogen peroxide or chloramine-T in the rase of glutathione (see Harington and Mead, 1935). The terminal y-glutamyl residue was thus oxidized to a succinyl residue and succinic acid could be found after hydrolysis. In other cases the ninhydrin reaction has shown the same structure (Buchanan et al., 1962; Matikkala and Virtanen, 1962; Morris et al., 1963). Dunnill and Fowden (1963) demonstrated the presence of a y-glutamyl peptide linkage by the reactions with nitrous acid and with N-bromosuccinimide. The most authoritative method, that of complete synthesis, has been carried out in the case of glutathione (Harington and Mead, 1935); y-L-glutamyl-Ltyrosine and y-L-glutamyl-L-phenylalanine (Morris and Thompson, 1962) ; y-glutamylleucine (Buchanan et al., 1962; Morris et al., 1963), S-(a,Pdicarboxyethy1)glutathiorie (Calam and Waley, 1063) and y-glutamylmethionine (Morris et al., 1963).

B . A'nturally Occurring P-Aspartyl Peptides P-Aspartyl peptides are of far less frequent occurrence in nature than those containing y-glutamyl linkages. The first peptide shown to contain

THE UNUSUAL LINKS AND CROSS-LINKS OF COLLAGEN

123

a 6-aspartyl peptide link was the antibiotic bacitracin A (Lockhart and Abraham, 1956; Swallow and Abraham, 1959). Bacitracin A is of further interest in that it also contains a peptide link involving the eamino group of lysine (Haussmann et al., 1955; Lockhart and Abraham, 1956). More recently, P-aspartyl peptides have been isolated from human urine. The first of these was P-aspartylhistidine (Kakimoto and Armstrong, 1961) in which the P-aspartyl linkage was identified by the ninhydrin reaction in solution and on paper, by hydrazinolysis which yielded the @-acidhydrazide of aspartic acid, and by comparison with synthetic a- and p-aspartylhistidine with respect to melting point, solubility, crystalline form, paper chromatographic behavior, and ninhydrin reaction. Buchanan et al. (1962) have produced evidence to suggest that many other P-aspartyl peptides occur in human urine. They used the evolution of ammonia on reaction with ninhydrin a t 37°C to distinguish between p- and a-aspartyl peptides. 0-Aspartylglycine and P-aspartylleucine isolated from urine were compared with the synthetic compounds in their behavior on paper chromatography. It was shown that no a - t p conversion of aspartylglycine could have occurred during isolation by addition of labeled a-aspartylglycine to urine samples prior to fractionation. The authors concluded that a t least sixteen P-aspartyl di- and tripeptides occur in human urine. I n all these the aspartic acid residue is NHz-terminal. I n all the 0-aspartyl and y-glutamyl peptides mentioned, with the exception of bacitracin A, the residues bound by the w-carboxyl group have their a-carboxyl groups free. In most of the peptides the residue is NHz-terminal and these facts greatly facilitated the identification of the 0- or y-peptide linkage.

C. y-Glutamyl and @-Asparty1 Peptide Linkages in Polypeptides and Proteins, Especially Collagen and Gelatin Polypeptides can be obtained from Bacillus subtilis or Bacillus anthracis that are composed entirely of D-glutamic acid residues. To determine whether the polypeptide from B. subtilis was PO~Y-Y-D- or poly-a-D-glutamic acid, Bovarnick (1942) studied the optical rotation in alkali. Dakin (1912) had suggested that only amino acid residues with the a-carboxyl group in peptide linkage would be able to racemize in alkaline solution. Consequently, poly-a-D-glutamic acid would be expected to undergo mutarotation under these conditions, whereas poly-7-D-glutamic acid should not do so. Bovarnick found that no change of optical rotation could be observed in an alkaline solution of the natural polypeptide and he concluded on this evidence and on the basis of a negative biuret reaction that the polypeptide from B. subtilis was poly-y-glutamic acid. Haurowitz and Bursa (1949) carried out a study on a number of proteins and the capsular polyglutamic acid of B. anthracis to determine what

124

JOHN J. HARDING

y-glutamyl links were present. Their procedure involved a n initial digestion with trypsin to produce some glutamyl residues in NH2-terminal positions. The digest was then treated with NaOBr to oxidize any terminal y-glutamyl residues to succinyl residues. Any succinic acid formed a t this stage from free glutamic acid and glutamine was extracted with ether and rejected. The aqueous layer was then hydrolyzed, and the succinic acid released was determined. This succinic acid gave a minimum figure for the number of y-glutamyl links present. When it is considered that the specificity of trypsin is such that it opens links involving the carboxyl groups of lysine and arginine and that this action is decreased if glutamic acid is the other amino acid involved iii the linkage (see Dixon and Webb, 1958), it is surprising that any formation of terminal y-glutamyl residues occurs in proteins or that trypsin attacks polyglutamic acid a t all. It is possible that the large amount of trypsin contained sufficient quantities of other enzymes to cause more general breakdown. Joseph and Bose (1960, 1962) used the same method for gelatin, collagen, and elastin. They concluded that the number of y-glutamyl linkages increases with age (Joseph and Bose, l962), but the very uncertain nature of this method renders it unsuitable for such studies. Kovitcs and Bruckner (1952) and KovAcs et al. (1953a) converted synthetic poly-a-glutamic acid and the poly-wglutamic acid from B. subtilis to the polyhydrazide and then degraded them by the Curtius rearrangement. The reaction is very similar to the Hofmann rearrangement (see Fig. 2 ) . Poly-a-glutamic acid gave the expected a-diaminobutyric acid after subsequent hydrolysis, whereas the natural compound gave p-formylpropionic acid. This again suggests that the polyglutamic acid of 13. subtilis is y-linked. Hofmann rearrangement on the ester of the natural polypeptide again gave P-formylpropionic acid in yields suggesting that no a-peptide linking was present (Bruckner et al., 1953a); synthetic poly-aglutamic acid yielded a,y-diaminobutyric acid (Bruckner et al., 195313). Williams et al. (1955) have shown that a n enzyme from R. subtilis can catalyze the transfer of a y-glutamyl residue from the polyglutamic acid to u-glutamic acid to form y-glutamylglutamic acid. Waley (1955) first synthesized poly-y-glutamic acid. He compared the infrared spectra, titration, and solubility properties of the synthetic polyy-glutamic avid with polyglutamic arid from B. anthracis and R. licheniformis and with synthetic poly-a-glutamic acid. For the properties studied the natural polypeptide resembled synthetic poly-y-glutamic arid and not poly-a-glutamic acid. In addition he isolated y-glutamylglutamic acid from a partial hydrolyzate of the natural polypeptide. Fromageot and Jutisz (1953) claimed to have demonstrated the presencse of y-glutamyl links in hemoglobin and edestin by reduction with lithium

125

THE UNUSUAL LINKS AND CROSS-LINKS O F COLLAGEN

r-Glutamyl residue

-NH-

COOH I CH- CH,-

a-Glutamyl residue CH,-

CHz-CO

-

i

I

1

COOR I -NH-CH-CH,-CHz-CO-

I

CH,-CH,-COOR I

-NH-CH-CO-

'

CH,-

-NH-CH-CH,-

CH,-CO

-

I

"

Fa

-NH-CH-CHz-

NH,- CH-CHz-

CHO-CH2-

CONH,

NaOBr

]

CH2- CO

-

CH2-CO

-

hydrolysis

""'

CH,-

-NH-CH-CO-

NaOBr

-NH-CHNCO CH,-

-

esterification

esterification

CONH,

CHz- COOH

-NH-CH-CO

CH,-

1

CHz- NCO

[-NH-&H-C!O-

CHZI -NH-CH-CO-

CHZ-

NHZ

hydrolysis CH,-CCHZ-NHZ

C H r COOH

I NH,- CH -COOH

a, y-Diamino butyric acid CH2- COOH

P-Formylpropionic acid (succinic semialdehyde)

FIQ.2. Identification of y-glutamyl peptide links by the Hofmann rearrangement.

aluminum hydride and subsequent identification of r-amino-bhydroxyvaleric acid in the acid hydrolyzate (see Fig. 3). This product, however, could also be formed by reductive cleavage of at-glutamyl peptide links. Chibnall and Rees (1958), having found that asparagine and glutamine residues are especially labiie to reductive cleavage, strongly criticize this approach to the study of p- and y-peptide links in proteins. Chibnail et al. (1958b) have reviewed the previous work on natural polyglutamic->'acid and have also described results of the reduction of

12G

JOHN J. HARDING

y-Glutamyl residue

a-Glutamyl residue

COOH

CH,-CH,-

I

COOH

I

-NH-CH-CO-

- NH-CH-CH,-CH,--0-

esterification FOOR

co-

- NH-.CH-CH,-CH,-

esterification YH,-NH-CH-CO-

I

I

Li BH, CH,OH I -NH-CHCH,-

CH,CH,-

CO-

1

I

CH,OH

I

CH,OH

1

CH,OH

hydrolysis

CH,-

CH,-COOH

iLiBH4

CH,-

-NH-CH-CO-

hydrolysis

NH,--CH-CH,-

CH,-COOR

CH,-

I NH,- CH-COOH

FIG. 3. Identification of y-glutamyl peptide links by lithium borohydride or lithium aluminum hydride reduction.

esterified polyglutamic acid from B. subtilis with lithium borohydride (see Fig. 3). Their results show that the u-glutamic acid residues are exclusively y-linked. Thus it is now clear that the polyglutamic acids produced by B. subtilis and B. anthracis are entirely poly-y-u-glutamic acid. Kovhcs et al. (1955) have studied the effect of NaOCl on the amidated dicarboxylic acid residues of gliadin and chymotrypsin. The amides undergo Hofmann rearrangement to yield the corresponding amino compounds (Fig. 2 ) . Thus asparagiriyl and glutaminyl residues would yield alp-diaminopropionic acid and a,y-diaminobutyric acid, respectively, after hydrolysis, whereas isoasparaginyl and isoglutaminyl residues would yield acetaldehyde and /3-formylpropionic acid. Neither aldehydic compound could be found after Hofmann rearrangement and hydrolysis of chymotrypsin. Gliadin, however, yielded both acetaldehyde and p-formylpropionic acid. The authors point out that the acetaldehyde could have been produced by oxidation of NH2-terminal aspartic acid that they had shown to be present in gliadin after treatment under the same alkaline conditions as used for the Hofmann rearrangement. They conclude, however, that isoglutaminyl residues exist in gliadin. Haurowitz and associates (1957) using the thiohydantoin method of Schlack and Kumpf (1926) but with labeled ammonium thiocyanate found

THE UNUSUAL LINKS AND CROSS-LINKS O F COLLAGEN

127

more thiocyanate was bound to many proteins than could be explained by the number of terminal carboxyl groups present. They demonstrated that the excess thiocyanate bound was not due to any of the active sidechain groups known t o be present in proteins and concluded it was caused by binding with the a-carboxyls of aspartir and/or glutamic acid residues that were present in 0-and y-linkage, respectively. This was supported by ( a ) the small incorporation of labeled thiocyanate after esterification or lithium aluminum hydride reduction of the proteins, (b) the small amount of thiocyanate bound by fibroin which has a low content of dicarboxylic acids, (c) the much greater incorporation exhibited by poly-y-glutamic acid compared with poly-a-glutamic acid (Haurowitz and Horowitz, 1956). The latter fact also shows that the results were not due to a! -+y conversion under the anhydrous conditions of the reaction with ammonium thiocyanate. Collagen and gelatin behaved in a very similar way to most of the other proteins studied. This method is clearly riot quantitative as poly-y-glutamic acid only binds an excess of 4.2 moles per molecular weight of 12,000 gm, whereas if a reaction occurred with each free a-carboxyl group along the chain the excess should be of the order of 100 moles. The authors suggest that the low yield in this case may be due to steric hindrance or y --$ a rearrangement. The results for the proteins could be due to either 0-aspartyl or y-glutamyl linking in the peptide backbone. As y-glutamyl peptides appeared to be of far more frequent occurrence in nature, Haurowitz et al. concluded that the excess binding was the result of y-glutamyl linking. Waldschmidt-Leitz and Reicheneder (1961) identified a n NHz-terminal glutamic acid residue in a wheat protein bound by way of its y-carboxyl group. They inferred this from the evolution of carbon dioxide during reaction with ninhydrin. In view of the fact that ninhydrin is able to split many peptide bonds (Dowmont and Fruton, 1952; Yanari, 1956) its use in this way is open to some question; however, since glutamic acid could be identified as the NHz-terminal residue by the Sanger method and yet was not released by aminopeptidase, it seems probable that a y-glutamyl link is present. Very recently, Czerkawski et al. (1963), studying the composition and structure of the cell wall mucopeptide of Micrococcus lysodeikticus paid special attention to the number of free basic and acidic groups present, using titration and dye-absorption methods. They found that 40 % of the y-carboxyl groups and a similar proportion of the t-amino groups were not free. As the mucopeptide was known to be extensively cross-linked, the authors concluded that this cross-linking involves side-chain peptide bonds between lysine and glutamic acid.

128

J O H N J. HARDING

The use of the Lossen rearrangement of hydroxamic acids was first brought t o bear on this problem by Gallop et al. (1960). The basis of the method is shown in Fig. 4. The Lossen rearrangement has been discussed in a general review on hydroxamic acids by Yale (1943). Gallop et al. (1960) studied the reaction with a number of model compounds, poly-a-glutamic acid and an alkali-processed!pigskin gelatin. The dinitrophenylation step allows the 70-NH-

(7Hz)n CH- COOH

MeOH/Ac,O esterification

-

-

-NH-CH-

COOCH,

30 min

FDNB 1

-NH-&H-

CONH-0-DNP Lossen rearrangement 0.05 N NaOH 100°C

coI

(CH,), -NH-LH--NH,

-NH-

acid hydrolysis6 A' HC1 24 hr

/

n'/ //

COOH I

J'

( y M 2

CHO F r o m y-glutamyl links

COOH

/'

100°C

CH-CO-NH-OH

/'

= 2

I (7Hz)n

+

2 NH,

CHO I

In=l !decarboxylation

t

CH,CHO From P-aspartyl links

FIG.4. Identification of y-glutamyl and P-aspartyl residues using the Lossen warrangcment.

rearrangement to be carried out under milder conditions than could be used otherwise. The rearrangement step itself was shown to be quantitative, although the rate of rearrangement differed for the different model compounds. From the reaction sequence shown glutamic acid residues in ylinkage will, after esterification, Lossen rearrangement, and hydrolysis, yield succinic semialdehyde (P-formylpropionic acid), whereas acetaldehyde would be formed from P-aspartyl linkages. Glutamic aeid and aspartic acid inca-peptidellinkage would, however, yield a,y-diaminobutyric acid

THE UNUSUAL LINKS AND CROSS-LINKS O F COLLAGEN

129

(DAB) and a,p-diamiiiopropioriic acid (DAP), respectively. The methyl ester of poly-a-glutamic acid resulted in the formation of a,p-diaminobutyric acid as expected. The over-all process was not quantitative, apparently owing to hydrolysis of hydroxamic acid groups during the dialysis carried out prior to diiiitrophenylation, but the totals of glutamic acid plus a,y-diaminobutyric acid before and after heating to promote Lossen rearrangement were the same showing that no side reaction had occurred and no y-glutamyl linking was present. The gelatin was subjected to amino acid analysis before and after reaction. I n the hydrolyzate of the reaction product both a,y-diaminobutyric acid and alp-diaminopropionic acid were found, and also a decrease in content of aspartic and glutamic acids was noted. Other amino acids were unaffected. The decrease in the content of aspartic acid was only slightly greater than the amount of a#-diaminopropionic acid formed, but a decrease of 39.6 residues of glutamic acid was found compared with the formation of only 8.4 residues of diaminobutyric acid. The authors suggest that the remaining 31 residues of glutamic acid must have been present in the gelatin ester in y-peptide linkage. This was confirmed by the identification of succinic semialdehyde irt the hydrolyzate of the reaction product. The additional ammonia that should be released by formation of succinic semialdehyde was also noted. Gallop et aE. (1960) are careful to point out that they have only demonstrated the presence of y-glutamyl links in the gelatin ester. Such links may have been introduced during esterification, which was carried out using methanol/acetic anhydride. These anhydrous conditions could promote ring closure by the y-carboxyl groups with subsequent reopening to form either a- or y-peptide links. The results for aspartic acid in gelatin and those for poly-a-glutamic acid, however, tend to suggest that y-glutamyl links do ocrur in gelatin and therefore probably in collageii also. I'ememberiiig that the whole reacltion is not quantitative it would appear from this work that perhaps half the glutamic acid present in gelatin is in y-peptide linkage. To avoid any possible a -+y conversion promoted by the anhydrous conditions during esterificatiori Pranzblau (1962) and Franzblau et al. (1963) developed a method by which the hydroxamic acids could be formed directly from the unmodified proteins in an aqueous system. This was achieved using a water-soluble carbodiimide, l-c~yclohexyl-3[2-morpholinyl(4)-ethyl]-carbodiimide metho-p-toluene sulfoiiate (Fig. 5). The reaction conditions (p1-T 4, 25"C, in aqueous medium) eliminated any possibility of a p conversion. These conditions also precluded the possibility of hydroxyamiriolysis of the natural ester linkages of collagen which will be discussed iii Section V. This method, in common with all the others used to study y-glutamyl links, was not quantitative. After subsequent dini---f

130

J O H N J. HARDING

-COOH

R I N II

f C

II N I R’

R

-

I

N

-coo-c

It

I NH

I R’

NH,OH f -CO-NH-OH R-NH-CO-NH-R’ FIG.5 . Tlir iisc of a carhodiimide for the direct hydroxyamidation of carboxyl groups.

trophenylation, Lossen rearrangement, and hydrolysis, a,y-diaminobutyric wid, a,p-diaminopropionic acid, and succiiiic semialdehyde were estimated quantitatively. Poly-a-glutamic acid after the reaction sequence showed a 30 yo decrease of glutamic acid content which was exactly accounted for by the a ,y-diaminobutyric acid produced. Siiccinic semialdehyde was not detectable. Poly-y-glutamic acid showed a 54 yoloss of glutamic acid; a,@-diaminopropionicacid was not determined, but no a,y-diaminobutyric acid could be found. The hydrolyzate gave a strong positive test for succinic semialdehyde, aiid the ammonia in the hydrolyzate indicated that the lost glutamic acid had undergone reaction as expected. The same series of reactions was carried out on gelatins derived from the soluble collagens of ichthyocol and calfskin. I n both cases the loss of aspartic acid was balanced fairly well by the amount of a,@-diaminopropionic acid formed suggesting that the aspartic acid that reacted was originally in a-peptide linkage. The figures for glutamic acid and a?ybutyric acid did not balance, however, and succinic semialdehyde could be detected in the hydrolyzed products. In the case of ichthyocol the succinic semialdehyde formed was estimated quantitatively and its value (22 moles/ 1000 residues) plus that of the a,y-diaminobutyric acid (4.3 moles) accounts very well for the loss of glutamic acid (25.4 moles). The formation of sucrinic semialdehyde is supported by the extra formation of ammonia found in both oases. Sedimentation studies suggest that the natural ester linkages are still intact. The conditions of reaction do not promote a -+ y conversion and this was borne out adequately by the experiments with poly-a-glutamic acid arid poly-y-glutamic acid. The reactions were not quantitative and give minimal values indicating that 30 % of the glutamic acid in ichthyocol collagen and 13 % of the glut,amic acid in soluble calfskin collagen is in y-peptide linkage. The mild thermal conditions for the formation of the gelatins from the respective soluble collagens preclude any suggestion that the y-glutamyl links could have been introduced at-this stage.

THE UNUSUAL LINKS AND CROSS-LINKS OF COLLAGEN

131

Franzblau (1962) points out that y-glutamyl linking introduces two methylene groups into the peptide chain that must be of great importance in relation to the configuration of the chains. He goes further to suggest that such links, which presumably occur in the polar (disordered regions) of the collagen molecule, may in fact be largely responsible for that disorder. Franzblau (1962) and Franzblau et al. (1963) have drawn attention to evidence suggesting the existence of y-glutamyl links in gelatin, that was obtained by Dakin (1912) 50 years ago. Dakin postulated that amino acids bound by their a-carboxyl groups would racemize in dilute alkali, whereas those with the a-carboxyl group free, being unable to undergo keto-enol tautomerism, could not do so. He followed this up by a study of the action of dilute alkali on gelatin. After treatment the amino acids were separated and studied for racemization. Most of the amino acids, including aspartic acid, were found t o be completely racemized. Glutamic acid and lysine, however, retained their activity. This supports the findings of Franzblau (1962) and Franzblau et al. (1963) that P-aspartyl links are not present in collagen, whereas y-glutamyl links are present. Dakin's results for lysine are difficult to explain. His results for glutamic acid suggest that not only are y-glutamyl links present in gelatin but further that most of the glutamic acid present is linked in this way. In later papers Dakin carried out analogous experiments on casein, cow and sheep caseinogen, hen albumin, and duck albumin, and found complete racemization of aspartic and glutamic acid in every case except sheep caseinogen where only partial racemization of' glutamic acid was observed. These results have been summarized in a paper by Levene and Bass (1929). The essential facts are supported by findings of Bovarnick (1942) that natural poly-y-glutamic acid does not racemize under these conditions. Very recently, Rojkind has isolated y-glutamyl peptides from a collagenase digest of ichthyocol (see Gallop, 1964).

D. Discussion The main methods used for the detection of y-glutamyl linkages in proteins, including collagen and gelatin, depend on the a-carboxyl group being free. It is, therefore, a t first surprising that gelatin and collagen do not exhibit a great preponderance of glutamic acid a s the apparent COOHterminal residue. To resolve this problem one must consider what COOHterminal methods have been applied to these proteins. Grassmann and Hormann (1953) determined the COOH-terminal amino acids of collagen and gelatin using a modification of the thiohydantoin method of Schlack and Kumpf (1926). They first benzoylated the amino groups and then reacted the proteins with ammonium thiocyanate in acetic anhydride and acetic acid to form thiohydantoin derivatives. These could

132

J O H N J . HARDING

be split off and hydrolyzed back to the amino acids which in turn were separated and identified by paper chromatography. The main COOH-terminal amino acids detwted in gelatin were glyrine, threonine, and alanine. No COOH-termiual amino acids could be detected in the case of collagen. Deasy (1956) usiiig the Baptist and Bull (1953) modification of the Schlack and Kumpf (1926) method found glycine, alanine, arid leuciiie as COOHterminals of six collagens and a gelatin. Joseph and Bose (1959) using the same method, found valine and isoleucine in addition to these three. In relation t o the probability of free a-carboxyl groups of glutamic acid all along the peptide chains of collagen, it is important to note that the thiohydantoin method does not determine COOH-terminal aspartic acid, glutamic acid, or glutamine (Baptist and Bull, 1953) and so presumably will not determine y-glutamyl residues either. This is borne out by the results obtained by Baptist and Bull (1953) for glutathione. The second method applied to collagen and gelatin has been the lithium borohydride reduction method. This was first carried out on procollagen by Grassmarin and co-workers (1954) who found glycine and valine as COOH-terminals. In collagen glycine and alariine were found as COOHterminal amino acids by Grassmann and Endres (published in a review by Grassmann, 1955). The method in this case involves esterification and reduction to form an amino alcohol a t the COOH-terminal. Complete hydrolysis then results in the release of the amino alcohols in the presence of a great excess of amino acids. The mixture is then dinitrophenylated and the DNP-amino alcohols (DNP, dinitrophenyl) are extracted from the alkaline solution by ether or ethyl acetate (cf. Fig. 9). y-Glutamyl residues after esterification, reduction, hydrolysis, and dinitrophenylation will be present as DNP-NH-CH-CH2-CH2-COOH

In alkaline solution this compound still has a charged carboxyl group and therefore remains behind with the DNP-amino acids when the other DNP-amino alcohols are extracted, thus escaping detection. One might proceed along the lines set out by Chibnall et al. (1958a) in their study of asparaginyl and glutaminyl residues in the protein chain. Clearly, the lithium borohydride method by the procedure applied to collagen would not be affected by y-glutamyl linking, but it is apparent that modifications of this method could have very useful applications to this problem. The third COOH-terminal method applied to gelatin has been the hydraziriolysis method, carried out by Heyns and Legler (1957, 1958). This method (Akabori et al., 1956) involves treatment of the protein with

T H E UNUSUAL LINKS AND CROSS-LINKS O F COLLAGEN

133

anhydrous hydrazine at elevated temperatures, whereby all the amino acid residues except the COOH-terminal are released as hydrazide (Fig. 6). The COOH-terminal amino acid is released as the free amino acid. The mixture can then be either dinitrophenylated or treated with a n aldehyde. In both cases the hydrazides of most amino acids form compounds that are uncharged in alkaline solution and thus can be separated from the amino acids or DNP-amino acids by ether extraction. This method, however, caused problems because the products from aspartic and glutamic acids COOH-terminal glutamic acid

y-glutamyl residues CH,I

CO -

CHz-

I

I

7%

I

CH,-

COOH

I

CH-COOH

COOH

7%

7%

NH,-

I

NH,NHz

NH,NH,

CH,I

COOH

-NH-CH-CO-

-NH-CH-COOH

NH,NH,

CH,- CO-NH-NH, I ?HZ NHz- CH- COOH

CH,I

COOH

7%

7% -NH-CH-COOH

a-glutamyl residues

NHz-

CH-CO-NH-NHZ

FIG.6. The reactions of a- and y-glutamyl residues subjected to the hydrazinolysis method for the dctermination of COOH-terminal amino acids.

remained behind owing to their free carboxyl groups. Heyns and Legler, therefore, used a strongly polar aldehyde to form polar hydrazides which remained behind when the DNP-amino acids were extracted with ether from the acidified solution. The situation is thus analogous to that found in the case of the reduction method. The products from a- and y-glutamyl residues in the chain, being closely similar, will remain with the mixed hydrazides and will not be determined by the usual methods. Nevertheless, a hydrazinolysis procedure as carried out by Ohno (1954) on lysozyme with separation of all the possible products of aspartic and glutamic acids in different types of linkage could again shed further light on this problem. A closer consideration of the methods as yet applied for COOH-terminal amino acid determination in gelatin and collagen shows that they would not differentiate between a-, @-, a i d y-peptide linkages. It is, however, possible that modifications of the lithium borohydride and the hydrazinolysis methods could be used for a quantitative study of @-asparty1 and y-glutamyl residues in proteins and especially in collagen and gelatiu. A further problem that warrants some discussion is the possibility of 01 + y interconversion under the conditions used for isolation or reaction

134

JOHN J. HARDING

of the proteins by the different methods. Clayton and Kenner (1953) and Clayton et al. (1956) have shown that a-glutamyl peptides, on treatment with thionyl chloride and a tertiary base in an anhydrous medium, undergo ring closure. Subsequent alkaline hydrolysis opens the ring to give mainly the y-glutamyl peptide

-OC-HN-H~,

C

,NH-CH-CO-

II

0

I R

SOCI, /pyridine in dimethyl formamide I

(alkaline hydrolysis

-0C-HN-HC,

I

COOH

R

Similar results using different reagents in other anhydrous media have been found by Battersby and Robinson (1955, 1956). Consequently, any studies of y-glutamyl and P-aspartyl linkages in proteiiis or peptides where prior treatment in anhydrous media has occurred is open to criticism on the grounds of possible a ---f y conversion. The criticism will be stronger where a dehydrating agent has also been included in the system. I n the experiments of Haurowitz et al. (1957) proteins and peptides were in an acetic anhydridelacetic acid medium during thiohydantoin formation. Their results with a-peptides and poly-a-glutamic acid indicated that very little a + y interconversion took place, but they accepted that it could occur if glutamic acid residues were adjacent to certain other amino acid residues. These authors also suggested that possible y -+ a conversion

THE UNUSUAL L I N K S AND CXOSS-LINKS

O F COLLAGEN

135

may occur, thus explaining the low figures obtained with poly-y-glutamic acid. Gallop et al. (1960) esterified the carboxyl groups prior to reaction with hydroxylamine and Lossen rearrangement. They point out themselves that in the anhydrous and dehydrating medium used for esterification, methanol/acetic anhydride, a -+ y conversion is a distinct possibility. Their results for poly-a-glutamic acid and for the aspartic acid in collagen make this less likely, but in later work they developed a method to avoid the esterification step (E’ranzblau, 1962; Franzblau et al., 1963). It appears that a + y or a --f p conversion has not interfered in studies of these links as much as might have been expected, but it is obviously better to avoid anhydrous media at any stage in these experiments. This applies equally well to anhydrous media used during the preparation of the proteins, e.g., organic solvents used for fat extraction or for drying materials should be in contact with the proteins for as short a period as possible. After due coiisideration of the control experiments carried out in the studies on collagen and gelatin and of the fact that no p-aspartyl linkages have been reported, it seems unlikely that any a + y conversion has occurred. In the case of other proteins it also seems unlikely; but, in view of the fairly low values obtained and the small but significant value given by poly-a-glutamic acid by the method of Hauromitz et al. (1957), confirmatory experiments using methods similar to those of Franzblau (1962) and Franzblau et aZ. (1963) are necessary before any definite conclusions ran be drawn. A further point worth considering is the hydrolysis of y-glutamyl links. Hopkins (1929) found that prolonged boiling of glutathione in water resulted in the formation of glutamic acid and glycylcysteine anhydride. Kendall et al. (1930) hydrolyzed glutathione t o glutamic acid and cysteinylglycine by heating to 62°C in water for 5 days. It appears, therefore, that y-glutamyl peptide linkages are far more readily hydrolyzable than are a-peptide links (see also Shiba and Kaneko, 1960). This indicates that adequate precautions must be taken to avoid hydrolyzing conditions during the processes prior to determination. This point was considered by Haurowitz and Bursa (1949) who used large amounts of trypsin during digestion in order to reduce the time spent a t 38°C a t pH 8.5. Ovalbumin and serum albumin were, however, denatured a t 100°C for 30 min prior to digestion and also were digested for 2 days instead of one. It is purely speculative that this could be partly why these two proteins gave the lowest yields of succinic acid. Joseph and Bose (1960) using the same method found that collagen gave ten times as much succinic acid as an alkali-processed gelatin. It is significant in this respect that Gallop et al. (1960) found strong indications of y-glutamyl linking in

136

JOHN J. IIAHDING

a commercial “alkali-provessed” pigskin gelatin, i.e., a gelatin that has oiily been extracted after a prolonged treatment of the collagenous material with alkali. It is possible that y-glutamyl links in collagen are a great deal more stable than the one in glutathione. The recent work of Franzblau (1962) aiid Yranzblau et al. (1963) employed gelatins prepared from soluble collagens by heat denaturation a t 60°C for o~ily10 min, conditions uiilikely to have ruptured many y-glutamyl linkages.

It. Conclusions It is clear that a great deal of the criticism applied t o the earlier work has been fully (Ionsidered in more recent studies, and it must now be accepted that a considerable number of y-glutamyl linkages exist in collagen arid gelatin. Their existence in other proteins would seem probable, but requires some further research. I t must be stressed that the glutamic arid residues in rollagen that are involved in the y-peptide linkages are iiot responsible for the cross-linking of collagen. They are present solely as an alternative mode of linkage of the amino acids. Such linkages constitute the greatest deviation from the a-peptide theory of protein structure yet established. Their importance iii bringing about the unusual structure of c,ollagen that is shown by physical studies cannot be readily assessed, but the interjertion of pairs of carbon atoms a t intervals along the peptide backbone is bound to produce some interesting features. The present rvidence indicates that no significant amount of P-aspartyl peptide bonding exists in collagen or gelatin. IV. €-AMINOPEPTIDE LINKAGES Another simple deviation from the accepted a-peptide structure of proteins would be the presence of peptide linkages involving the 6-amino groups of lysirie and hydroxylysine. e-Amino peptide links, like p- or y-rarboxyl peptide links, could be incorporated into a single chain or could form cross-links or branching points with both amino groups bound. Very few known c-amino peptide compounds are known to occur naturally. There is evidence that such linkages may occur in collagen and gelatin, but it is mostly inferential, being based on the incompleteness of reaction of various reagents with the c-amino groups. The only direct evidence is the isolation, from a partial acid hydrolyzate of collagen, of an t-lysine tripeptide. The evidence indicating the presence of e-amino peptide links in collagen has been disrussed by Joseph and Bose (1962). Relatively few natural e-lysine peptides have been reported. Biocytin has been shown to be c-N-biotinyl-L-lysine by degradative and synthetic studies (Peck et al., 1952). Another antibiotic, bacitracin A, is a large peptide which appears to

THE UNUSUAL LINKS AND CROSS-LINKS OF COLLAGEN

137

contain a number of unusual links including a n E-lysine peptide link, a 0-aspartyl peptide link, a thiazoline ring, and a strange bond between isoleucine and phenylalanine (Haussmann et al., 1955; Fraenkel-Conrat, 1956; Lockhart and Abraham, 1956). Very recently, it has been suggested that some of the e-amino groups of lysine are linked to the y-carboxyl groups of glutamic acid in mucopeptides isolated from the cell walls of Micrococcus Zysodeilcticus (Czerkawski et al., 1963). 1. c-Lysine Tripeptide

The elysine tripeptide was isolated from bovine achilles tendon after its partial hydrolysis in 7 M HCl. It was identified very thoroughly by both degradative and synthetic studies as L,L-W-(glycyl-a-glutamy1)-lysine hW,-CH,-CO-NH

-CH-CO-NH-(CH,)~-CH I (p’z COOH

--COOH I NHZ

In view of the strong acid hydrolysis used for its preparation it is unlikely that the tripeptide is an artifact. The amount of the tripeptide isolated accounted for only a fraction of a per cent of the total lysine. Mechanic and Levy (1959), however, recalling the incompleteness of reaction of the c-amino groups of collagen and gelatin with l-fluoro-2,4-dinitrobenzene (FDNB) reported by many authors (e.g., Bowes and Moss, 1953) suggested that the 40 yo of lysine reported to be unassailable has its t-amino group in peptide linkage. Bowes and Kenten (1948) had first suggested a similar possibility on the basis of the discrepancy between the values obtained analytically for hydroxylysine and lysine and those suggested by titration curve and Van Slyke determinations. In an endeavor to clarify the situation let us consider the availability of the c-amino groups to FDNB and other reagents. 2. Reaction of FDNR with Collagen and Gelatin

In Table IV the availability of the eamino groups to FDNB found by different authors is tabulated in chronological order. This reagent is considered first because ideas of covalent masking of €-amino groups have largely sprung from the FDNB reaction. The original availabilities given by Bowes and Moss (1951) and Sykes (1952) have been increased in view of a vorrcction of the original analytical figures for the lysine content of gelatin and collagen (Bowes et al., 1955). Bowes and Moss (1953) carried out a number of subsidiary experiments to find the reason for the low availability. They found that very little

TABLEIV The Availability of the €-Amino Groups of Collagen and Gelatin to F D N B ~~~

~~

Availability" Material Collagen Gelatin Hide powder Collagen IJrea-treated collagen Heat-shrunk collagen Alkali-treated collagen Formic acid-treated collagen Hyaluronidase-treated collagen Gelatin Procollagen Gelatin

(%) 50 50 75-80 55 59 48 55 55 55 55 68 70

Acid-treated gelatin

u p to 94

Alkali-processed ox bone gelatin Alkali-processed calfskin gelatin Alkali-processed oxhide gelatin Acid-processed pigskin gelatin Acid-processed ox bone gelatin Gelatin Dentine

97-100 97 96 88-97 90 u p to 100 9

Ox bone

10

Decalcified dentine

96

1)ccalcified ox bone

93

Hide or tendon

64-70

Collagen

70

Lathyritic collagen

70

Procollagen

56

Collagen

82

Procollagen determined in 2 M NaClOa Collagen determined in 2 M NaC104

96

Gelatin from newborn ratskin Gelatin from mature ratskin

92 70

93

(60) (60) (95-100) (67) (75) (60) (67) (67) (67) (67)

Reference Bowes and Moss (1951) Bowes and Moss (1951) Sykes (1952) Bowes and Moss (1953) Bowes and Moss (1953) Bowes and Moss (1953) Bowes and Moss (1953) Bowes and Moss (1053) Bowes and Moss (1953) Bowes and Moss (1953) Bowes and Moss (1953) Heyns and Konigsdorf (1953) Heyns and Konigsdorf (1953) Courts (1954) Courts (1954) Courts (1954) Courts (1954) Courts (1954) Heyns and Wolff (1956) Solomons and Irving (1958) Solomons and Irving (1958) Solomons and Irving (1958) Solomons and Irving (1958) Solomons and Irving (1958) Nikkari and Kulonen (1962) Nikkari and Kulonen (1962) Hormann and Wilm (see Hormann, 1962) Hormann and Wilm (see Hormann, 1962) Hormann and Wilm (see Hormann, 1962) Hormann and Wilm (see Hormann, 1962) Joseph and Bose (19G2) Joseph and Bose (1062) ~~

~-

Figures in parentheses are the revised figures in accordance with later analytical figures (Bowes et al., 1955). 138 (I

T H E U N U S U A L LINKS A N D CROSS-LINKS OF COLLAGEN

139

nitrogen was evolved when the DNP-proteins were treated with nitrous acid. No evidence could be obtained of a-DNP-lysine, a,€-di-DNP-lysine, or acid-resistant DNP-lysine peptides in the hydrolyzates of the DNPproteins. Very little free lysine could be found in the hydrolyzates. If some of the lysine were in epeptide linkage one would have expected this to have been released on hydrolysis of the DNP-protein. It would also have been released if these lysine residues were unable to react owing to steric hindrance. Bowes and Moss suggest that all the e-amino groups may have reacted with FDNB, but on hydrolysis part of the DNP-lysine was destroyed. Such behavior has not been found with other proteins. The €-amino groups of other proteins appear to react quantitatively with FDNB, although in many cases the proteins must be denatured first (Porter, 1948). Certain proteins exhibited a greater reactivity to ketene than to FDNB, presumably because FDNB is a more bulky molecule. Heyns and Konigsdorf (1953) showed that by treating gelatin with 0.1 N HC1 for 38 hr the t-amino availability rose from 70 to 94 %, but a more prolonged treatment caused a decrease in availability again. Later Heyns and Wolff (1956) went thoroughly into the question of the pH of the medium for dinitrophenylation. Their results indicated that in the dilute bicarbonate solutions usually used the H F released during reaction lowers the p H sufficiently to slow down substitution. In higher concentrations of bicarbonate they found that further eamino groups reacted. By using a p H 8.6 sodium bicarbonate-sodium carbonate buffer they achieved complete reaction. Under these conditions no indication of a-peptide hydrolysis was obtained. Using borate buffers a p H of 8.9 was required to allow complete reaction, whereas very slight a-peptide hydrolysis only occurred at pH values in excess of 9.2. These results may partly resolve the anomalies found for the reaction of FDNB with collagen and gelatin, although they do not explain the results of Bowes and Moss (1953). The work of Joseph and Bose (1962) indicates that the availability of the €-amino groups t o FDNB decreases with age. In summary, although the reaction of FDNB with collagen and gelatin shows certain peculiarities, it clearly does not lend support to the hypothesis that 40 % of the €-amino groups are in peptide linkage. This is clearly demonstrated by the almost complete reaction achieved by Heyns and Konigsdorf (1953), Courts (1954) , Heyns and Wolff (1956), Solomons and Irving (1958), and Hormann and Wilm (see Hormann, 1962) for a number of different preparations. 3. Reaetion of Other Reagents with Collagen and GeEatin

I n Table V are listed the availabilities of the e-amino groups to other reagents. I n some cases the availabilities were not compared with the total

140

JOHN J. HARDING

TABLEV The Availability of the e-Amino Groups of Collagen and Gelatin to a Number oj Reagents Availability. Reaction or reagent

Material

(76)

Reference

-~

~~

-

Acetylation

Hide powder Calfskin Gelatin Buffalohide collagen Collagen Tropocollagen

100 100 100 100 100 100

Gustavson (1955a) Gustavson (1955a) Kenchington (1958) Bose and Joseph (1958) Gustavson (1962) Bensusan et al. (1962a)

Benzoylation

Buffalohide collagen

100

Bose and Joseph (1958)

Benzene sulfonylation

Gelatin Gelatin

98

Gurin and Clarke (1934) Leach et al. (1962)

Surcinylation

Hide powder Limed calfskin

Guanidination

Gelatin Several gelatins Ichthyocol Gelatin

Sodium bromoacetate

Hide powder

p,p’-Difluoro-m,m’dinitrodiphenyl sulfone

Tendon collagen

1,5-l)ifluoro-2,4-dinitrobenzene

Collagen

Nitrous acid

Oxhide collagen Limed sheepskin Several collagens Hide powder Collagen

Nitrosyl chloride

Tendon collagen Ichthyocol

75 65

Ninhydrin

Several collagens Several gelatins Collagen

100 100 91

Betheil and Gallop (1960) Betheil and Gallop (1960) Bensusan et al. (196213)

Trypsin

Denatured collagen Gelatin

72 100

Joseph and Bose (1960) Betheil and Gallop (1960)

>94 90-95 90-95

97 100 87 94

Gustavson (1961) Gustavson (1961) Kenchington (1958) Janus (1958) Betheil and Gallop (1960) Betheil and Gallop (1960)

75 (95) Sykes (1952)

>60 55-75

Zahn and Nischwitz (1960) Zahn et al. (1962)

82 (100) Bowes and Kenten (1948) 100 Bowes and Kenten (1949) 100 Salo (1950) 75 (95) Sykes (1952) 100 Gustavson (1962)

Levy et al. (1960) Levy et al. (1960)

Figures in parentheses are revised figures in accordance with later analytical figures (Bowes et al., 1955). Q

THE UNUSUAL LINKS AND CROSS-LINKS OF COLLAGEN

141

lysine and hydroxylysine content but with the free amino groups determined by the Van Slyke method. This is permissible as nitrous acid has been shown to attack the e-amino groups quantitatively (Table V). The results tabulated in Table V make the possibility that 30 or 40 % of the c-amino groups are in peptide linkage extremely improbable. Most of the reactions, especially acylation, seem to go to completion quite readily. It is, of course, arguable that these reagents may split the e-amino peptide links; however, many of the reactions take place under very mild conditions. Also, in arid conditions at least, c-amino peptide linkages are more stable than a-peptide links (Lockhart and Abraham, 1956). Gustavson (1962), recalling that the shrinkage temperature of collagen is unaltered by complete acetylation or deamination, points out that the cross-links in collagen cannot involve the e-amino groups of lysine and hydroxylysine. A method that has been proposed for the detection of bound groups in proteins is the use of titration curves (Kenchington, 1960). The titration curve of collagen by Bowes and Kenten (1948) gives a value for the E-amino groups in agreement with the Van Slyke method and with a later determination of the total lysine and hydroxylysine (Bowes et al., 1955). Later titration curves by Ames (1952b) and Sykes (1952) also show that no masked eamino groups are present. Kenchington and Ward (1954) determined the titration curve of an alkali-processed gelatin. The figures for free eamino groups found by titration agreed closely with the known content of lysine and hydroxylysine. After titration in one direction Kenchington added an equal amount of the complementary reagent which resulted in restoration of the original pH. This establishes with certainty that no release of amino groups had occurred during titration. This work clearly demonstrates that the €-amino groups are not masked in gelatin. At this poirit it must be emphasized that none of the methods mentioned for the estimation of free amino groups is sufficiently sensitive to establish that no e-amino peptide links exist in collagen or gelatin. It is apparent, however, that if they are present they are but few. A further aspect to consider is the acid stability of e-peptide linkages. Lockhart and Abraham (1956) found that after 43 hr of hydrolysis with 11 N HCI a t 80°C no significant release of free lysine and very little free L-aspartic acid could be detected demonstrating that the link between the c-amino group of lysine and the a-carboxyl group of aspartic acid is extraordinarily stable. One would expect from this that e-amino peptide links in proteins would be at least as stable. In collagen and gelatin, however, complete release of lysine is accomplished by hydrolysis with 5.5 N HC1 a t 100°C for 24 hr (Eastoe, 1955). It therefore seems unlikely that a large proportion of the lysine could be in epeptide linkage.

142

JOHN J. HARDING

The maximum number of e-amino peptide linkages that could be present in gelatin or collagen may be deduced b y consideration of the way such linkages could be incorporated into the protein molecule. The main possible structures are depicted in Fig. 7. A closer examination of these structures severely limits the number of a. Branching point R I

R

R I

,,,---NH-CH-COOH

I (YH2)4

NH

I CO-

R I H NH -----CO-CH I - ---R NHZ

Y

b. Cross-link to a side-chain carboxyl g r o u p

R NH,-

R

I

CH-CO

----_--NH-CH-CO

-------NH-CH-

I

COOH

I (YH2)4 NH

[

I

E-R] CO

R I

R (YH2)z I N H r CH-CO-------NH-CH-CO-------NH-CH-COOH = 0,1, 2, ... x=lor2

n c . Straight chain i.

R NH,-

d H-

co--- - NH-

(CHJ~-

R I

cH-NH --- - CO- cHI

NH,

COOH

ii. NH,-

R I

CH-CO----

R

NH-(CHJ4-

I CH- CO----NH-CH-COOH

I

NH2

FIG.7. Possible ways for the incorporation of €-amino peptide ,links in proteins.

THE UNUSUAL LINKS AND CROSS-LINKS O F COLLAGEN

143

r-amino peptide links that can be envisaged to occur in collagen or gelatin. The branched structure (Fig. 7a) requires an extra NH2-terminal amino acid for every branching point present. Gelatin has only one or two NH2-terminals per lo6gm (Courts, 1954) which is probably the maximum chain weight for collagen (see Section 11). It is therefore very unlikely that many branch points of this nature can be present in gelatin or collagen. The same argument applies to the first straight-chain structure shown (Fig. 7c,i). The other straight-chain structure (Fig. 7c,ii) would have an apparent NH2-terminal lysirie or hydroxylysine residue for each lysine or hydroxylysine incorporated in this way. Heyns and Legler (1958), however, found that only about 0.2 % of the lysine residues in gelatin occur as NH2-terminals. The final possibility is that the r-bound lysine forms cross-links as in Fig. 7b. Extensive cross-linking of this type, however, would involve an equally extensive masking of carboxyl groups. Absence of such masking has been shown by titration studies, e.g., those of Kenchington and Ward (1954). The y-glutamyl peptide links reviewed in Section I11 concern glutamyl residues with the a-carboxyl groups free, and r-peptide links to such residues would behave as the branched structure of Fig. 7a. €-Peptide linkage of the lysine and hydroxylysine of collagen and gelatin can therefore only occur to a very limited extent. This limit is the experimental error of the methods cited to establish the absence of such links. Evidence that encourages one to think that r-amino lysine linkages may occur in collagen, probably as cross-links, is that found by Franzblau (1962) studying a collagenase digest of ichthyocol. I n the undialyzed digest 12 yoof the €-amino groups of lysine were not available to FDNB. After dialysis the nondialyzable fraction was enriched with respect to lysine and of this portion 25 yo did not react with FDNB. It is unlikely that nonavailability in such small peptides could be due to anything except covalent masking. I n view of the difficulties found with the application of the FDNB method to the €-amino groups in gelatin and collagen (Table IV), however, further evidence will be necessary to establish whether or not r-peptide links occur in this fraction. The evidence for e-amino peptide links in collagen rests almost entirely on the isolation of the t-lysine tripeptide by Mechanic and Levy (1959). That this tripeptide was ~,~-N~-(glycyl-a-glutamyl)-lysine seems to be adequately established. The only possible criticism is that the tripeptide may be a n artifact. Peptides are known to be able to rearrange especially during dilute acid hydrolysis; for example, Sanger and Thompson (1952) found that during hydrolysis of glycylvaline in 0.1 N HCl a t 100°C some valylglycine was formed. The inversion did not occur using 1 2 N HC1 a t 37°C. It is therefore unlikely that a similar transfer reaction could

144

J O H N J. HARDING

have occurred under the reaction conditions (7 N HC1 a t 24°C) used by Mechanir and Levy, but the possibility remains, especially in view of the small quantity recovered. Peptide formation by proteolytic enzymes is a well-known phenomenon (Desnuelle, 1953) and could be considered in relation to the apparent blocking of €-amino groups in a collagenase digest of collagen. I t is, however, extremely unlikely that collagenase could ratalyze the formation or cleavage of c-peptide linkages.

6.Conclusions The suggestion that €-amino lysine peptide linkages owur in collagen is based almost entirely on the evidence of the tripeptide isolated by Mechanic and Levy (1959). The quantity of the tripeptide obtained accounted for only a fraction of a per cent of the lysine present. Their suggestion that 30 or 40 % of the lysine may be bound via its €-amino groups seems most improbable. From the evidence presented above no more than a few per cent of the lysine can be involved in this way.

V, ESTER-LIKE LINKAGES IN COLLAGEN The possibility that ester links might exist in proteins is not a new concept. In 1906, Emil Fischer (1906) pointed out that peptide linkages were probably not the only form of c.oupling in proteins arid suggested that sidechain ester links and other links were probably present, a s well as piperazirie rings. Paulirig and Niemann (1939) suggested that side-chain peptide, ester, or disulfide bonds might be partly responsible for stabilizing protein configurations. The same three possibilities were discussed by Wiederhorn and Reardon (1952) in relation to the cross-linking of collagen. Chibnall (1942), having decided that p-lactoglobulin consisted of three chains, proposed side-chain esters, imides, and thiol esters as possible cross-links. Ester linkages in proteins were also suggested by Haurowitz and Bursa (1949). More recently, Smith (1958) has proposed that links between side-chain carboxyl groups and the active groups of tyrosine, cysteine, and histidine may stabilize the globular structure of proteins. He considers this to be the difference between globular and fibrous proteins because the latter contain little of any of these amino acids. Fruton (1961) has discussed the possible nonpeptide bonds that might occur in proteins, including esters, thiol esters, oxazaline, and thiazaline rings. Ester links between threonine and the terminal carboxyl of a peptide chain forming a lactone have been found in artinomycin (Bullock and Johnson, 1957), while Dekker et al. (1949) have found a n imide link in the form of a pyrrolidonyl ring involving the N-terminal glutamic acid residue in a peptide isolated from algae. In teichoic acids (polymers present in

the cell walls of some bacteria) i d a i i i n c oc'c~ursbouiid iii wtcr linkage to hydroxyl groups of glycerol or ribitol (Raddiley, 1964). Phosphodiestcr cross-links have hren idriitified iri P-caascin and pcpsiii (Pcrlmann, 1!k54). Extcr cwxs-links in elastin via hexoscs have hrcii suggested, on little evideiiw, by Walford et al. (1961). The structures of some of these linkages are given in Fig. 8. 12elatively few proteins, or even peptides, have been shown to have ester-like linkages incorporated into their structure. The evidence for such links in collagen will now he reviewed. Pkter linkages iii caollagen were first proposed by Schneider (194'3) as the means by which sugar bridges were incorporated into the peptide chain. Thr first real rvidence for ester linkages in collagen was obtained by Grass-

1 CH-(CH2),j-COI

Simple ester link from the w-carboxyl of aspartic or glutamic acids to the hydroxyl group of any hydroxyamino acid

I

O-----CH

I

I I

I

Simple thioester link from aspartic or glutamic acid to the thiol group of cysteine

C H- (CH2),i- CO -S -CH2- C H

I

CH-(CHJn-

I

I

CO-NH-CO-(C%),j-CH

I

Simple imide link from asparagine or glutamine to aspartic o r glutamic acids

I

Phosphcdiester between two hydroxyamino acid residues

Lactone ring from terminal carboxyl to a hydroxyamino acid

Terminal pyrrolidonyl ring

I

C€I-(C&),j-CO-

I

0

04s(cH2)n-

OH

I

7

Ester cross-link via a hexose molecule

OH Examplrs of cstpr links in proteins. FIG.8.

146

JOHN J. HARDING

man et al. (1854) using lithium borohydride reduction of procollagen. In the following year Alexander (1955) suggested that ester cross-linking was the cause of the insolubility of collagen in concentrated lithium bromide. In the same year Gustavson (l955a), discussing the relationship between shrinkage temperature and hydroxyproline content, proposed that the hydroxyl groups of hydroxyproliiie might participate in ester cross-links. He also proposed ester linkages to account for discrepancies in the basebinding capacities of collagen and alkali-processed gelatin (Gustavson, 1955b,c), Grassmann (1955) has drawn a clear distinction between the ester links he found and those Gustavson suggested in that the former are present in the chain itself, whereas the latter form cross-links between the chains. Gustavson (1962) has discussed ester links in collagen in relation to the incomplete methylation of its carboxyl groups, the incompleteness of acetylation of its hydroxyl groups, and the thermal stability of such substituted collagens. The estimation of ester-like linkages using hydroxylamine or hydrazine, which have since been the methods most widely used for ester determination in collagen, was first carried out by Gallop et al. (1959). Each of the methods used has been strongly criticized on various grounds, so each will be discussed separately along with appropriate critical studies.

A . Determination of Esters in Collagen Using Lithium Borohydride Lithium borohydride is very widely used for the specific cleavage of ester linkages. It is generally thought not to reduce free carboxyl groups or amide groups. On this basis it was introduced into protein chemistry as a reagent to be used in the determination of COOH-terminal amino acids (Chibnall and Rees, 1951). The chemical basis of its use is summarized in Fig. 9. The amino alcohols produced can be separated and determined as such (Blumenfeld and Gallop, 1962b), or other products may be determined after the additional reactions shown. Grassmarin et al. (1954) first used lithium borohydride to determine the COOH-terminal amino acids of procollagen. They carried out the determination using method (ii) and found about 40 end groups per 1000 residues. In another experiment without esterification they obtained the same yields of DNP-amino alcohols. Consequently, on the basis of the specificity of lithium borohydride known at the time and the fact that no COOH-terminal amino acids could be detected by the thiohydantoin method, they suggested that the terminal carboxyls of procollagen were in cster linkage. Later, similar results were obtained using this method on collagen and on limed collagen (Grassmann and Endres, unpublished data; see Grassmann and Kuhn, 1’355). Grassmann and Kuhn (1955) also found

T H E UNUSUAL LINKS AND CROSS-LINKS O F COLLAGEN

147

that collagen oxidized with phenyliodoso acetate gives the same quantities of amino alcohols on subsequent reduction whether esterified or not and is also unaltered when the oxidized collagen is treated with alkali a t pH 10.5 for 30 hr. Grassmann (1960) has also claimed that some of the carboxyl groups of aspartic and glutamic acids are also reduced by lithium borohydride. Konno and Altman (1958) isolated a glycine-carbohydrate fragment from a carboxypeptidase digest of muscle collagen; treatment with lithium borohydride followed by the FDNB method showed that glycine was joined to the carbohydrate by an ester linkage. R

I

-CO-NH-CH-COOH esterification

e. g., MeOH/Ac,O RI -CO-NH-CH--COOR’

t

Protein suspended

I R - co \NH-cH-cH,oH I

in tetrahydrofuran andreduced by LiBI&

acid hydrolysis R

R-CHO

+ CH,O + NH,

RI DNP-NH-CH-CH,OH

NH,-

I

CH-CH,OH

/

FIG.9. Use of lithium borohydride to study COOH-terminal amino acids

Blumenfeld and Gallop (1962b) have used lithium borohydride reduction, with subsequent chromatographic separation of the amino alcohols produced, to identify the carboxyl donor of the ester links previously found by Gallop et al. (1959) using hydroxylamine and hydrazine. The peaks obtained on the chromatogram for the two products in question, namely homoserine and p-amino-yhydroxybutyric acid, are very small, but nonetheless seem to establish that a- and p-carboxyl groups of aspartic acid participate in the hydroxylamine-sensitive links. 1.Control Studies o n L i t h i u m Borohydride Identification of Esters in Collagen

Grassmann et al. (1955) studied the validity of their method by carrying it out on a number of amino acids, di- and tripeptides, and insulin with and without previous esterification. Nonesterified amino acids suffered 1 yo reduction. The yields of DNP-amino alcohols from the peptides were up

138

JOIIK J . HAHDING

to 11.5 yo. Yields from the cstcrified peptides and amiiio acids were close to !I0 Yo. The yields with esterificd and nonesterified iiisuliii were 120 aiid 25 yo of the czaldated figure, respcctively. Crawhall aiid I1:lliott (1955b) carried out a similar study under lcss vigorous caoiiditions. Using peptides prepared from silk fibroin thcy obtained theoretical rrduction after 12 hr, but further reductioii oclciirred thereafter. When a similar rrdiwtion was carried out on the peptide mixture withotit previous esterifimtion the samc amiiio alcohols (ethanolamiiic, alaiiiiiol, and serinol) were obtained in about one-third of the quantity obtained from the estcrified pcptides. That this was iiot ciitirely cmiscd by the reductioti of free carbosyl groups was shomii by the fact that hippiiric acid only gave 12 yo ethaiiolamiiie iindcr the sa'ne cbonditioiis. Hrduction of lysozyme gave i i i decwasiiig order of abundance alaiiiiiol, cthaiiolaminc, leuc+iol, scrinol, valinol, mid phenylala~iinol. A11 of thesr, n.ith the possible cxccptioii of leiwitiol, have thercforc ariscn from deavagc of the peptide vhaiii. Crawhall aiid Elliott (l(365b) coiicluded that peptide bonds iiivolviiig glycirie atid alaiiitie were more siweptible to reductive caleavage that1 other peptide bonds. This point is especially inttrcstiiig h a u s e these ivcrc also the amiiio alcohols found hy Grassmaiiii atid Kuhii (l!L%). <:rawhall and 1i:lliott (1955b) suggest that part of the D N P alaiiiiiol that Grassmann et al. (1955) fotiiid by reduction of uiiesterified iiisiilin also rcsultcd from pcptide fission. Chihnall, Fees, and their co-workers (Chibnall and Hces, 19ri8; Chihnall r.1 d ,1958a; Chihiall el al., 1!158h) have c.ritically assessed the usefuliirss of the method they thcmsclvcs had introduced. With insulin, besides tho cxpcctcd alaninol, they also found cthanolamine, leuciiiol, arid tyrosiiiol as prodwts of pcptide cleavage (Chihiiall arid Rees, 1958). F:xperimeiits with lysozymc and P-lactoglobulin provided similar evidence. They thcrrfore support ('rawhall and Elliott (1'355a,b) aiid strongly (~ritkizethe rontrary assertions of Grassmatiti et al. (195.5). The error in C-terminal d(>tcrmiiiatioticlearly increases with thc size of thc protein coiiccriied, arid C'hibiiall suggests a uscfid uppcr limit of 10,000 molecular weight. This applies eqiially t o thc determination of a small number of estcr-like linkages. With reference to the identification of products from reduction of ester linkages involving aspartic acid the only control experiments were those of Rhimenfeld and Gallop themselves (1962b) using poly-a,P-aspartic* avid which reacted as expccted. Heductive peptide cleavage will clearly only affect the valucs for a-boiuid esters. I t is, however, noteworthy that peptide links involving aspartic acid are particwlarly resistant t o lithium t )orohydridc. Thcl results of Grassmann and his co-workers (Grassmann et al., 1!)54; Grassmanti aiid Kuhii, 1955) show several features that suggest that

T H E UNUSIJAL LINKS AND CROSS-LINKS

O F COIAIAAGEX

149

peptide cleavage may have beeti a n important facator in their studies: ( a ) The amino alcohols found were the very ones shown to he formed most readily by peptide cleavage. ( b ) Esterificatioii of the protein made 110 difference at all t o the vaIucs obtained. (c) The quantities of amino alcohols vorresporid t o approximately 10 yo cleavagc of links iiivolvirig alaniiie and glycinc as the carboxyl donors. The fact that these were the only two amino acids found as the amino alcohols is probably due to their preferential cleavage and their considerable abundance in collagen. It would be interesting to repeat the work of Grassmann after attempting t o de-esterify the collagen or procollageii hy the action of hydroxylamine.

B. Determanataon of Ester-Lake Lanlcs an Collagen and Gelatin TJszng Hydroxylamzne The basis of the method for the determination of ester linkages by hydroxylamine is set out i i i the following schrme. NH,OH R--0-OR’

R-CO-NH-H

Purplish-red complex

1

NH,OH

+

sulfanilic acid a-naphthylamine

t

Diazo dye

indole

Colored product

The protein is treated with hydroxylamine which splits any ester linkages forming a hydroxamic acid. The amount of hydroxamic acid present may be determined colorimetrically by: ( a ) formation of a ferric complex, (b) oxidation to nitrous acid which is then reacted with sulfariilic acid and a-naphthylamine to give a diazo dye, or (c) hydrolyzing off the hydroxylamiiie which then reacts with indole. In methods ( b ) arid (c) excess hydroxylamiiie must be removed before the determination. This is normally achieved by dialysis. The use of the term “ester-like” is deliberate, as it is known that hydroxylamiiie will react with many other groupings t o form hydroxamic acids. Among these are imides, anhydrides, arid chlorides, amides, and certain peptide linkages (Yale, 1943; Gallop et al., 1959; Williams et al.,

150

JOHN J . HARDING

1!155). For this reason the conditions used for hydroxyamidation are of the utmost importance. This method was first applied to gelatin by Gallop et al. (1959). They used acid- and alkali-processed commercial gelatins as well as two prepared by mild denaturation of acid-soluble collagens from calfskin and carp swim bladder. They determined hydroxamate by all three methods and also studied the reaction kinetically and by sedimentation studies. They found that the binding of hydroxamate occurred in a fast and then a slow reaction. During the fast reaction no loss of amide nitrogen was observed and no release of amino groups was apparent by the ninhydrin method, although both occurred during the slow reaction. The results show that about 5 moles of hydroxamate were bound per 100,000 gm during the fast reaction for all except the alkali-processed gelatin, which bound about half this amount. This gelatin also bound less during the slow reaction. This is due to a fission of ester and amide linkages, respectively, during alkali treatment. Their sedimentation studies showed a fall of molecular weight to 20,000 suggesting polypeptide units in collagen held together by esterlike linkages. They could observe no change in the hydroxylamine reaction or the sedimentation results after treatment of the acid-processed gelatin with LiBH, and suggest that the latter was unable to penetrate the insoluble gelatin particles to attack the ester linkages. This observation clearly distinguished between the ester links postulated by Grassmann et al. (1954) and the hydroxylamine-sensitive links (Gallop et al., 1959). Hormarin and Klenk (see Hormann, 1960a) confirmed the work of Gallop and co-workers by finding 6.4 moles of hydroxamate per thousand residues and a fall of molecular weight to 20,000 in studies on procollagen. The work of these and later workers has been summarized in Table VI along with the conditions used. The reason for the different valucs obtained is not readily discernible except for the extremely high values (45 moles/105 gm) of Lieflander and Wacker (1962) which must be due to the high pH used. It may be that the lower pH used by Bello in his determinations (Bello, 1960; Rello and Bello, 1963) is the reason that his values are particularly low (0.5-1.5 moles/l05 gm). It is of interest that Graham et al. (1963) could observe no splitting of the ester links in ovine submaxillary gland mucoprotein during treatment with 1 M hydroxylamine at pH 8 and 37°C. Hydroxyamidation of simple esters occurs most readily only a t pH values greater than 9 (Botvinik et al., 1962). Hormann and Klenk (Hormann, 1960a) and also Bello (1960) and Gustavson (1962) have shown that mature collagen can be brought completely into solution very rapidly by hydroxylamine in the presence of a

THE IJNIJSUAL L I N K S AND CROSS-LINKS O F COLLAGEN

151

hydrogen-bond breaker. For example, 2 M potassium thiocyanate plus 0.75 M hydroxylamine dissolved hide collagen completely within 2 hr a t 20°C (Hormann, 1960a). This suggests that esters are involved in the cross-links of collagen. Hormann and Klenk (published by Hormann, 1960b) also claim that the pH dependence and the kinetics of the formation of hydroxamic acids in collagen show that the linkages involved are indeed esters. The difference of 4.3 links per 1000 residues between the value for mature collagen and that for soluble collagen they ascribe to intermolecular cross-linking (Hormann, 1960b). The residue after trypsin digestion of heat-denatured collagen had a tenfold increase of ester linkages suggesting that the esters are involved in the more cross-linked and unassailable part of the collagen molecule. Hormann et nl. (1961) have reported on the decrease in the number of ester-like linkages during liming thus relating the alkali-labile links, that must be broken to produce gelatin, t o the hydroxylamine-sensitive linkages. Ester links not split by liming can be found in the gelatin produced. I n the presence of hydrogen-bond breakers during liming the collagen dissolves completely within a few days, aiid the collagen produced contains no esterlike linkages. Grassmann et al. (1962) were able t o solubilize hide collagen in 9 hr using hydroxylamine with hydrogen-bond breakers. Their figures (see Table VI) show that ten ester-like links per lo6 gm are split during liming. During the same period only 0.6 mole of peptide bonds per lo5 gm were broken. Gallop and Seifter (1962) have reported that calf or fish gelatin treated with hydroxylamine can be separated into two components by electrophoresis on starch. Both components have molecular weights of 20,000 and similar amino acid compositions. Joseph and Bose (1962) have found the number of ester-like linkages increases with the age of the tissue. Hormann (1962) has compared the ester content of procollagen, calf collagen, and adult ox collagen (see Table VI). The latter had about 4 moles/105 gm of ester links more than either calf collagen or procollagen indicating that cross-linking by ester linkages does not produce the initial insolubility in acid buffers. Nikkari arid Kulonen (1962) tried to relate the impaired stability of lathyritic collagen to the hydroxylamine-sensitive linkages. They found values of 0.5-1.5 moles/105 gm for both lathyritic arid normal collagen, but in view of the threefold range quoted and the lack of analytical data it is difficult to assess the value of this work. These values are close to those determined by Bello (1960) and later Bello and Bello (1963). The latter authors also noted a fall of molecular weight to 32,000 during the reaction. Rlumenfeld and Gallop (1962b) confirmed their previous determination

L

bl &

TABLEVI Determination o j Ester-Like Linkages in Collagen and Gelatin V s i n g Hydroxylainine Temperat iirc

Referrrice

("C) 25 40 25 40 25 40

Time (hr)

Conrentration of NH,OH

(*I[)

3 1.5 3 1.5 3 1.5

PH 10 10 10 10 10 10

Hiirmann (196Oa,h)

.lG AG G G

sc SC

8C IC 25 4 25 4

4 1-5b

Hormann et al. (1961)

Bello (1960)

Materiala

Ester links (moles/l06 gm) 2.5 2.5 5 5 5 5 6.4

10.7

1-5b

1.5 1.5 1.5 1.5

8.6 8 .6 8.6 8.6

IC IC G G

0.5-1.5 0.5-1.5

37 37

1330 13-40

0.75 0.75

0.55 9.55

IC LC

12.4 3.5

Sikkari and Kulonen (1962)

40

1.5

0.5

9

sc

0.5-1.5

Bello and Bello (1963)

25 25

8.6 8.6

IC G

0.34.5 0.4

Blumenfeld and Gallop (196%)

40

9-10

sc

4

16 16

1.5

1

2 2

6

4

C

z

z

?

z

P z Ei zc

Grassmann et al. (1962)

Lieflander and Wacker (1962)

Horinann (1962)

25 25 25

16 16 16

0.14 0.14 0.14

9 9 9

40 40 40 40

15-25 1-2 15-25 1-2

0.12 0.9 0.12 0.9

> 13 > 14 > 13 > 14

37 37 37

10 10 10

1.5 1.5 1.5

25 25 25

3 3 3

9.5 9.5 9.5

IC LC I C after lime/LiCl treatment

16 6

9G

11

AG Esterified AG Esterified -1G

45 i5 82

ox IC Calf I C

sc

0 ii

6.2 6 .0

51

i;:

2

s d

5E

~.

Joseph and Bose (1962)

2 2

10 10

C

C

1.1 2.5

2

10

c

3.5

AG, Alkali-processed gelatin; G, acid-extracted gelatin; SC, soluble collagen; IC, insoluble collagen; LC, limed collagen.

* Time in days.

These three samples represent collagen from ratskins of increasing ages.

+Z

U n

!5

$ ? !2

z

E C

I54

JOHN J. HAHDING

of the bound hydroxamate. They also observed a loss of almost 50 % ' of bound hydroxamate ori dialysis. Gallop et al. (1960) had already shown that the cu-hydroxnmate groups of poly-a,P-aspartyl hydroxamic acid were unstable t o dialysis against distilled water, whereas the b-hydroxamate groups were stable. These findings suggest that values for bound hydroxamate determilied after dialysis should be lower than those determiiied without dialysis, but this has not been found in pracatice (Gallop et a/., 195'3). These authors have carried out experiments to determine the nature of the csrboxyl donor of the ester linkages. As already mentioned lithium borohydride studies suggest that the a- and p-carboxyl groups of aspartic acid are involved. This was also demonstrated by the Losseri rearrangement. In general terms the Lossen rearrangement results from the thermal decomposition of hydroxamic acids, whereby the group initially attached to the carbon atom is found in the product attached t o the iiitrogeii atom (Yale, 1943) (see Pig. 10). R-CO-

I

OR

NH,OH

R-CO-

FDNB

NH -OH

R-CO-

NH- 0-DNP

I

-

OH

oI1 - t p

R-C-N-0-DNP O II R-C-N

t

R-N=C=O

i

+ O--DNP Hf

Dinitrophenol

1

heat H,O R-NH, E s t e r originally on (Y

-COOH

Expected products Acetaldehyde from malonic semialdehyde*

Aspartic acid

fl -COOH

a,fl-Diaminopropionicacid (DAP)

u!- COOH

Succinic semialdehyde

y -COOH

a ,y -Diaminobutyric acid (DAB)

Glutamic acid *Acetaldehyde is formed by decarboxylation of malonic semialdehyde during subsequent acid hydrolysis.

FIG.10. The Loswn rearrangement.

155

T H E UNUSUAL LINKS AND CROSS-LINKS O F COLLAGEN

The reaction is closely similar to the Hofmann, Curtius, and Beckmann rearrangements. The over-all reaction consists of the conversion of RCOOR‘ to RNH2, therefore where the carboxyl donor is the cY-carboxyl group of an amino acid the resulting product would have two amino groups on the a-carbon atom. Consequently, ammonia is evolved and the corresponding aldehyde remains. On this basis the expected products from various esters are given in Fig. 10. Hydrolyzates of the modified protein were examined for aldehydes and for the other possible products by column chromatography. The results are given in Table VII. TABLEVII Analysis of Material Following Lossen Rearrangement of the Dinitrophenyl Hydroxamate Ilerivative of Gelatin f r o m Ichthyocol as Compared with the Analysis of a Suitable Control Gelatin% ~~

Residues/1000 residues of amino acids

Component Hydroxamic acid Glutamic acid DAB Aspartic acid DAP A 1deh y de

Control gelatin (A)

Treated gelatin

0 71.0 0 46.0 0 4.9

4.9 71.4 0 41.5 2.6 6.7

(R)

Difference (B - A) f4.9

f0.4 0 -4.5 +1.8

From Blumenfeld and Gallop (1962b).

First, there is no loss of glutamic acid and no formation of DAB, therefore neither of the carboxyl groups of glutamic acid can participate in the ester linkages. In the case of aspartie acid, however, there is a significant decrease after Lossen Rearrangement and also DAP is found. The sum of the formation of DAP and aldehyde equals approximately the loss of aspartic acid. These results indicate that both the a- and P-carboxyl groups of aspartic acid are involved in the hydroxylamine-sensitive linkages. 1 . Control Studies of the H y ~ r o x ~ l a Method ~~ne

Several procedures based on the three methods given for the determination of hydroxamates have been developed by different authors (Czaky, 1948; Hill, 1947; Rergmarin and Segal, 1956; Yashpe et al., 1960; Gutirikov and Schenk, 1962; Lipmann and Tuttle, 1945; Seifter et al., 1960). The iodine-oxidation method and the indole method are more sensitive than method ( a ) based on the ferric complex (Bergmann and Segal, 1956; Yashpe et al., 1960; Seifter et al., 1960). The claim of Bergmann and Segal (1956)

156

JOHN J. HARDING

that their iodinc method (mi be used without prior hydrolysis has been questioned by Yashpe et al. (1960). All three methods have been compared by Seifter el (11. (1960). The most important point to be established, however, is that hydroxamate formation could not occur to the extent found without assuming ester-like linkages. I t is therefore vital t o ascertain what effect, if any, ran be attributed to such reactions as peptide cleavage and loss of amide nitrogen with coilcornitant formation of hydroxamic acids. The results of some relrvant studies are given in Table VIII. It has bccn shown that y-glutamyl peptide links are split by hydroxylamine in slightly alkaline conditions with the formation of hydroxamic acids (Williams and Thorne, 1954; Williams et al., 1955). Braunitzer (1!)56) using very harsh and prolonged conditions found no a-peptide cleavage for several proteins, whereas Ramachandrari and Narita (1958) using very similar conditions found approximately 1 mole of 1ieu7rnd groups per 18,000 gm of tobacac-omosaic virus ( T M V ) protein. Thc latter authors also fourid complete hydroxyamidation of amide groups. Gallop and ro-workers (1959) had themselves studied peptidc cleavagr aiid deamidation under their own, much milder, conditions. Their use of the ninhydrin method to study the release of a-amino groups was unsuitable for this type of study. The fast and slow binding of hydroxamate which thry reported could vorrcspoiid to the ester reaction and the side reactions, rcspcctively. Their results suggestcd that only an insignificant amount of peptidr atid amide cleavagc occurred during the fast rcaction. Only after 24 hr wider the same vonditions was any dialyzable material found. They also applied their method to other proteins arid found that some gavc a slight reaction, wherras others gave no detectable reaction. Bovine serum albumin, among those giving a slight reaction, showed no fall of molecwlar weight afterward. Hormann et al. (1961) found no slow reartion u hell the hydroxamate was determined after dialysis. They suggested that the c+oiitiiiuedreaction found in undialyzed material might be due to impurities in the collagen preparation. Their finding, that collagen dissolved by limc and lithium chloride contains no hydroxylamine-sensitive linkages, is iiiterestiiig as the material produced was still of high molecwlar weight aiid so had almost all the original peptide links intact, even though it may have lost most of its amide groups. Halls arid Wood (1956) studied the hydroxyamidatiori of a single ester group mtroduc.ed into chymotrypsin by acetylatioii. Estimation of the extent of reaction was carried out using ferric chloride. In the pH range 5.5-11.7 thc reacstioii was quantitative, i.e., no side reactions had ocrurred. Recently, Liefliinder arid Wacker (1962) reported on the reaction of hydroxylamine with esters, peptides, glycopeptides, amides, sugar deriva-

2m

r: z

TABLE VIII Amide and Peptide Cleavage by Hydroxylamine Reference

Molarity of NH&H

Braunitzer (1956)

3

7

Rarnachandran and Narita (1958)

3

6-9

Gallop et al. (1959)

1

10

3

25

1

10

1.5

40

pH

Reaction time (hr)

Temperature

1-120

60-100

("C)

d

2 P r

Material

Peptide cleavage Arnidc cleavage

Several proteins

No

TMV protein

About 0.5%

-

E 2

xm

P

Lieflander and Wacker (1962)

24

60

Up to 100c/c

Z

U

0

0.12 0.5

> 13

-

>13

-

40 10

Gelatins and soluble collagen Gelatins and soluble collagen Model compoiinds Model compounds

s

Very little

Very little

Very little

Very little

$

: CI

Z

z m NO Yes

Yes Yes

0

r

n

158

JOHN .J. WARDING

tixres, and proteins. They concluded that use of concentrations of hydroxylamirie greater than 0.12 M (final concentration) causes the reaction to lose its specificity. Even with 0.12 M hydroxylamine hydroxyamidation of amides occurred, whereas some esters studied did not react. The most notable fact about their determinations is the high pH used which appears to excaeed pH 13 in all cases. In view of the high pH it is difficult to relate this work to the ester determinations of other authors. This is borne out by the exceedingly high value obtained by these authors for gelatin (see Table VI). Also, although a greatly increased effect of side reactions was shown on model compounds on passing from the lower to the higher concentrations of hydroxylamine, with the proteins studied very little change was observed. It would appear that the fast and slow reactions of Gallop et al. (1959) correspond to (1) ester cleavage arid (2) peptide and amide cleavage, respectively, SO that it should be possible to correct for these factors. The knowledge that other proteins gave, at most, a slight reaction and no fall in molecular weight (Gallop et al., 1959) encourages the view that, although quantitatively other reactions are causing a loss of accuracy of the method, the links being estimated are certainly not labile amides or peptides. Strong evidence in support of this assertion comes from the findings of Hormariii and Kleiik (see Hormann, 1960b) and Franzblau (1962) showing that the hydroxylamine-sensitive linkages are concentrated in larger moieties remaiiiiiig after enzymatic digestion of gelatin and collagen. The high proportion of sensitive links in this case could not possibly be accounted for in terms of amide and peptide cleavage. The work of Blumenfeld and Gallop (1962b) on the determination of products after Lossen rearrangement is less liable to error than straightforward determinations because the only rionester reactions to interfere will be those which lead to the formation of the same products, i.e., the rupture of peptide and amide groups involving the carboxyl groups of aspartic arid. As the corresponding products from glutamic acid were not found, it indicates that such reactions are of little relevance under these conditions. Even if all the amide groups were present as asparagine the a-compounds have to be accounted for by ester-like linkages.

C. Determination of the Ester-Lzke Lznkages of Collagen Using Hydrazzne The protein sample is treated with hydrazine a t an alkaline pH whereby any esters present are converted to acid hydrazides. These are most readily determined after hydrolysis by reaction with p-dimethylaminobenzaldchyde, giving a yellow color that is determined colorimetrically a t 450 mp. The last two steps are normally combined:

159

THE UNUSUAL LINKS AND CROSS-LINKS O F COLLAGEN

I

DNP-NH-NH-

CO-R

Red in alkali

Me Yellow

An alternative method of studying the reaction is by dinitrophenylation of the acid hydrazides (see above). The DNP derivatives are red in alkali and can be determined colorimetrirally. Gallop et al. (1959) first used hydrazine to demonstrate ester links, but gave few details except that the fall of molecular weight was similar to that produced by hydroxylamine. The bound hydrazides were demonstrated by the reaction with p-dimethylaminobenzaldehyde, by dinitrophenylation, and by reduction of copper salts. More recently, Rlumenfeld and Gallop (1962b) treated gelatins from calf procollagen and ichthyocol with molar hydrazine a t pH 10 for 1%hr. Excess hydrazine was removed by dialysis and the bound hydrazide determined by reaction with p-dimethylaminobenzaldehyde (see Seifter et al., 1960). The value for bound hydrazide rose rapidly in the first hour of reaction and then leveled off. At the end of the rapid stage the ichthyocol gelatin and calf gelatin had bound 6.1 and 5.5 acid hydraaide groups per 1000 residues, respectively. These values are the same as found by the hydroxylamine reaction. In the same paper they describe experiments involving digestion of hydrazine-treated gelatin with collagenase followed by separation and study of the peptides with acid hydrazide functions. After digestion the peptides were separated on phosphocellulose columns and 2-ml fractions collected which were examined for free amino groups and for acid hydrazide functions. Fractions containing acid hydrazides were pooled and treated with o-benzaldehyde sodium sulfonate. The hydrazones formed were readily separatcd from the peptides by rechromatography on phosphocellulose. The amino acid analysis of the pooled peptides with hydrazone functions was determined. The number of molcs of aspartic acid and hydrazone present were approximately equal, whereas the number of moles of glutamic acid was much lower. This indicates that the bound hydrazone

160

J O H N J . HAIZDINC:

of the pcptidcs is liiikcd to aspartic acid. Vcry similar results were obtained with calf procollagen. The oiily r\ &terminal amiiio acid of the hydrazone-coiitaining peptides was glycine. Aiialysis of the dinitrophenylated peptides showed a loss of' glyciiic, now presciit as DNP-glyrine, approximately equal to the prolitic content of the peptides. This is consistent with the known specificaity of coliagenase. The average chain length of the peptidcs is ten amino achids, and one would have expected further proline residues iii the other cight amiiio acids. Blumerifcld aiid Gallop take this as indivating that these hydt.azoiie-coiitaiiiiiig peptidcs originate from the carboxyl eiid of the polypeptide chain in thc protciris. Also, from the caomposition of these prptides it is appareiit that each peptide has two residues of aspartic acid aiid two hydrazoiie funrtions. The authors suggest that the acyl donors of the hydrazine-sensitive links in collagen are aspartic acid residues. This coincides with the coiiclusioiis obtained by the same authors 011 the basis of studies oii the reactions of hydroxylamiw and lithium borohydride with collagen. By these lattcr rcactioiis they also have shown that both the a- and the P-carboxyl groups of aspartic acid are involved. Remembering that no 0-aspartyl liiikages (mi be identified in collagen (I~ranzblau,1962) the a-carboxyl groups ('oncerned must be O H - t e r m i n a l groups, again suggesting that the hydrazone peptides originate from the carboxyl end of the polypeptide chain. Very recently, de la Burde et al. (1963) have suggested an ester value of about 2 moles/105 gm in oxhide collagen, on the basis of their studies of the hydrazine rcactioii. As this paper raises several other points, this reaction will be coiisidered more fully below. 1 . Control Sttidtes of the fieactton of Hydrazine with Proteins

Hydraziriolysis under severe coiiditions (c.g., anhydrous hydrazine :tt 100°C for 10 hr) has been widely used to determiiic COOH-termilia1 amiiio acids (Akabori et al., 1!)56) and the distribution of the amide groups t)ctwccii aspartic and glutarnica acids (Ohiio, 1054). Under these c-oiiditionsamidc and most peptide bonds react with formation of' acid hydrazides, but free carboxyl groups are unaltered (Akabori et al., 1956). lices and Singer (1956) h a w obtaiiicd evidcnce of a gradual fall iii molecular weight of 7-globulin in anhydrous hydrazine at room temperature Bradbury (1'358a,b) using an arid catalyst obtained almost vomplete peptide cleavagc iii anhydrous hydrazine caontainiiig molar hydraziiie sulfate in 16 hr at CiO'C, although Hamac~handranand Narita (1958) cmsidcrcd thew caoiiditioiis far too mild t o split all the peptide links of tobacco mosaic virus protriii. 1ie:wtioiis ruidcr such cwiditions, holvrvrr, give littlc iiidication of thc

THE U N U S U A L LINKS A N D CHOSS-LINKS O F COLLAGEN

161

effect of peptide aiid amide reactions under the much milder conditions of Elumetifeld arid Gallop (1962h) except to stress that these reactions may take place. These authors observed very little peptide cleavage using the niizhydrin method during the fast reaction with hydrazine, although this bccame significant later. An extensive study of the reaction of hydrazine with collagen under different conditions has been carried out recently by de la Burde et al. (1963). They used steer coriiim collagen before arid after a short liming and carried out hydraziiiolysis a t room temperature for 30 hr in up to 70 % aqueous hydrazine. The treated fibers examined under the electron microscope still showed the characteristic cross-striations and periodicity of native collagen provided the hydrazine concentration did not exceed 55 %. Negligible hydrazinolysis of peptide bonds occurred with hydrazine concentrations less than 30 %. They did, however, observe a loss of amide groups proportional to the concentration of hydraeirie used, although only half of this could he found as bound hydrazide. This suggests that a t least some of the amide is hydrolyzed by the alkaline conditions rather thaii hydrazinolyzed. In relating this work to that of Blumenfeld and Gallop (1962b) the conditioris brought about by the different ronccntratioix of hydrazirie must be considered. De la Rurde et al. (1963) have used concentrations of hydrazine up to 22 M for 30 hr a t 25’C. For their lowest concentrations the pH was close to pH 11, but a t higher concentrations it was off the p H scale. Iirst, from these results of de la Burde et at. (1963) we can effectively rille out peptide hydrolysis under Gallop’s coriditioiis as this only occurs a t concGeiitrationsten times greater and a t a higher pH. The loss of amide content is more interesting. At the concentrations (1 M ) used by Blumenfeld and Gallop (1962b) amide loss would account for 1mole per lo5gm except that their reaction time (90 min) was one-twentieth that of de la Burde who also used a higher p1-T. This, in conjunction with the fact, that not all the amide lost appears as hydrazide, suggests that the figure of about 6 molcs per lo5gm determined by Blumenfeld arid Gallop owcs very little to hydraziriolysis of amides. Fleisc’hman et ul. (1963) have isolated the A and B chairis of 7-globuliii by gel filtratioii after reduction of the 7-globulin with mercaptoethanol. No decrease in the size of these individual chains was observed after treatiiig them with hydrazine under the conditions of Blumenfeld and Gallop (19GBh). L).

Corictuszons

To conclude on ester links, there is clearly much to be considered in the work quoted using lithium horohydride, hydroxylamine, aiid hydrazine,

162

J O H N J. HARDING

but it would appear fairly certain that ester linkages do occur in collagen and gelatin, that they are partly cleaved during treatment with alkali, and that they take part in the intermolecular cross-linking of the polypeptide chains. The number of such links present in different collagens is, as yet, not clear because estimated values may be too high owing to amide cleavage or too low owing to alkaline hydrolysis. The work of Blumerifeld and Gallop (1962b) indicates that they occur in pairs near the COOH-terminals and involve both the CY- and ,8-carboxyls of aspartic acid. The problem of mucopolysaccharide impurities will be discussed a t the end of Section VI. This is of great importance to considerations of esterlike linkages as ester links may occur in certain mucopolysaccharides. They have been suggested as the choridroitin sulfate-protein links in a complex isolated from cartilage (Muir, 1958).

VI. CARBOHYDRATE LINKAGES The first suggestion of carbohydrate linking in collagen came from Grassmann and Schleich (1935) who, having shown the presence of glucose and galactose in equal proportions in hide, were unable to remove any of this hexose by shrinking or with alkali. Schneider (1940, 1949) also identified galactosamine after achieving a 200-fold enrichment of the sugars by breaking down the collagen with baryta. He suggested that the sugars form covalent links between successive peptide chains in collagen and mentioned esters as a possible linkage. 1,ater Grassmann et al. (1954) suggested the sugar might be bound by ester linkages. In the same year Kuntzel (1954) indicated that purified procollagen contained carbohydrates as an integral part of the protein because the other components of skin had entirely different distributions of the sugar entities. Moss (1955), however, finding variable amounts of hexoses in collagen and procollagen, suggested that the carbohydrate was present as an impurity. Whether or not it is an integral part of the collagen structure must be established before any consideration can be given t o the way in which it might be linked. The main methods used to study the binding have been ( a ) attempts t o remove all the carbohydrate from collagen, (b) study of the larger products obtained by enzymatic digestion of partially denatured collagen, and (c) study of the reaction of collagen and procollageri with periodic acid. These approaches will now be considered.

A . Aitempted Remoual oj Carbohydrate jroin Collagen The early attempts of Grassmarin and Schleich (1935) to remove the ('rrbohydrate by shrinkage and liming have already been mentioned. Both

T H E UNUSUAL LINKS AND CROSS-LINKS O F COLLAGEN

1A3

Kuntzel (1954) and Moss (1955) found very little glucosamirie in purified materials. Grassmann et al. (1957a) have demonstrated the decrease of hexoses and hexosamines by repeatedly dissolving procollagen and precipitating it again by dialysis (Table IX). By going through this process twice they removed more than 90 yo of the hexosamine but only 20 % of the hexose. TABLEIX Change in Carbohydrate Content of Calf Procollagen during Recrystallizationa Hexose Sample Procollagen, impure Recrystallized once Recrystallized twice 5

(%I 0.91 0.84 0.73

Hexosamine

(76) 0.130 0.085 0.011

From Grassmann et al. (1957a).

Hormann (1957) has shown that hyaluronidase treatment considerably reduces the hexosamine content of collagen and procollagen. Gross et al. (1958) prepared a number of extensively purified collagens and determined their hexose and hexosamine contents. They found very low values for hexosamine but a minimum of approximately 0.5 % ’ hexose. The only sugars identifiable by paper chromatography were glucose and galactose. Kuhn et aE. (1959) attempted to remove the carbohydrate from acidsoluble collagen by two nondestructive methods: (1) by repeatedly dissolving and reprecipitating the fibers by dialysis, and ( 2 ) by repeated extraction of the soluble collagen solution with chloroform whereby the protein separates out a t the water/chloroform interface while free carbohydrate remains in the aqueous layer. They confirmed that all the glucosamine and half the hexose could be removed by either method, but the remaining 0.5 % of hexose could not be reduced further by repeated use of either procedure. Oneson and Zacharias (1960) were unable to remove the carbohydrate by treatment with ficin. They found a clear relationship between the hexose content and viscosity, indicating a special role for the hexose of collagen. A similar relationship was not found for the hexosamines. Even by treatment with lime Grassmann and Zeschita (see Grassmann, 1960; Hormann, 1960a) were able to remove only the hexosamine leaving more than half of the hexose behind in the collagen. All this evidence clearly indicates that the hexosamines found in collagen are readily removable by different methods, whereas about 50 yo of the

164

JOHN J. HAIZDING

original hexose content is hrmly boniid to the peptide chains and caiinot be removed without being destroyed.

B. Resadues from flnzymatzc Dagests of Denatured Collagen The first relevant studies of digests of this typc were carried out by Pirie (1'351) and by Grassmarin aiid his co-workers (Grassmarin, 1955; Grassmann el al., 1957a; Hormann, 1957, 19604. After trypsin digestion of denatured collagen or procollagen, residues remained that consisted of up to 30 % carbohydrate, representing up t o 90 % of the hexose origiiially present The residue from collagen still contained proline and hydroxyprolirie suggesting that it is a true constituent of c.ollagen. Very similar rrsults have been ohtaiiied using dastase (Hormann, 1957) aiid panvrcatin (Pirie, 1!)51). Two rather different approaches were those of Konno arid Altiiian (1058), who isolated a glycine-carbohydrate complex from a carboxypeptidase digest of deiiatured muscle collagen, and of Oriesoii and Zacharias (1060), who have isolated an oligosaccharide containing galactose, glucose, niannose, and fucose froin a ficiri digest of tendon collagen. Hormann (19604 has continued the work 011 rcsidues from enzymatic. digests using trypsin, elastase, and chollagenasc. All three left a residue enriched with respect to hexoses but not with respect to hexosamiiie. The residue from caollagenase digestion still c.ontaint.d all the amiiio acids of tsollagcn, including hydroxyproliiie in the proportion in which it oc(wrs i n collagen This very strongly suggests that the residue is part of collagen atid riot part of any of the mucopolysaccharldes assoviated with collagen. This work also points to the fact that the hrxoses are boinid to collagen, and the fact that they occur iii the more unassailable regions of the collagcn molecule suggests that they may take part in the cross-links. Hormaiin (1!)6Oa) has pictured trypsin attack as in Fig. 11. A very important finding by Hormann and Klenk (see Hormaiiii, l96Ob) is that not only the hexoses but also the hydroxylamiric-sensitive linkages are convcntrated in the trypsin-digest residue and, indeed, remain in a very similar proportion to each other; this will be discussed later (see Section VII). Seifter et ul. (1961) have also reported a fourfold increase in the proportion of neutral sugar in the nondialyzable peptides fouiid after digestion of ichthyocol gelatin with collagenase. They do, however, still contain an appreriable amount of hydroxyproline and proline arid the usual proportioii of glycine, indicating that this amino acid may be uniformly distributed throughout the molccule. Joseph arid Bose (1962) have isolated a glycopeptide from ratskin gclatiii after digestion with trypsiii, elastin, and carboxypeptidase. The glyco-

THE r N U S I r A L LINKS AND CROSS-LINKS O F COLLAGES

165

pcptide contained oihy glycirie aiid glutamic arid with a small amount of aspartic acid as amino acid constituents. Glycirie was the C-terminal of the peptide portion and no N-terminal amino acid could be detected. The authors therefore concluded that the carbohydrate portion was joined to the a-amino group of the glutamic acid residue. They suggested that this linkage involved an N-glycosidic bond, although they accepted that the carbohydrate of the glycopeptide isolated might represent only a minority of total carbohydrate present. Native collagen

A

A

I

I I

Partially denatured collagen trypsin

t

-tFIG. 11. Trypsin att;ick on collagen. .4, cross-linking hy carbohydrate. From Hiirmmn (1960n).

Kloker (1961) has obtained mixturcs of glycopeptides from gelatin aftcr partial hydrolysis with either triethylamine or N-dimethylaminoethanol. Unfortunately, the gelatin used was a commercial preparation and was not subjected to any purification process prior to use. The percentage recovery of the original carbohydrate was extremely small (1-2 %), and the glycopeptides contained large quantities of hexosamines. In view of these facts it seems probable that the glycopeptides isolated are not characteristic of the bound carbohydrate of gelatin (or collagen) and arc more likely to be degradation products from mucoproteiri impurities.

C. Studies of Carbohydrates in Collagen Using Periodic Acid Periodic acid has been used widely in carbohydrate chemistry owing to the quantitativc and specific nature of its action in splitting 1,2-glycol or p-amino alcohol groupings. The products shown in each figure are normally produced quantitatively and together with the periodate consumpt'ion can give a very good indica-

166

JOHN J . HARDING

\cH-oH I CH-OH

,

+ HIO,

C ' H-OH

+ HIO,

I

,CH---NH,

c

-CHO -CHO

CH-OH I ,CH-OH

CH,-NH, &-OH

I

+ HIO,

(1)

3- HI03

(2)

-CHO -CHO

+

-CHO

\CH-OH

I

+ H,O

+2HI04

- HCooH

-CHO

+HIO,

HCHO CHO

I

NH3

+ 2HI03 +H,O

+NH3

+ HIO,

(3 )

(4)

tion of the linkage being attacked. Phenyliodoso acetate is thought to react, in exactly the same way. Reaction (4) shows the way in which hydroxylysine would be attacked to give ammonia and formaldehyde. After Jackson (1954) had shown that treatment of collagen with periodic acid caused a substantial decrease of shrinkage temperature, Grassmann and Kuhn (1955) showed that both periodate and phenyliodoso acetate would degrade either collagen or procollagen to peptides containing about 23 amino acids. The product from collagen was electrophoretically homogeneous, high molecular weight, riondialyzable and gelled even in dilute solution, whereas the product from procollagen had none of these properties. The amino acid compositions determined by paper electrophoresis showed no significant changes from those of the original materials. To explain these results Grassmann and Kuhn (1955) discussed the possibility of carbohydrate residues occurring as part of the peptide backbone joined by an ester linkage to the C-terminal of one chain of 23 amino acids and N-glycsosidically to the N-terminal of the next (see also Schneider, 1949). There were distiiwt disadvantages to this hypothesis esperially as the proteins had insufficient hexose to have one hexose per 23 amino acid residues. Grassmann et uZ. (1957a) discussed this difficulty a t some length. Their work, however, has been criticized because their conditions were very severe, i e . , 0.2 M periodate at pH 7-8 and 40°C,or phenyliodoso acetate at pH 3.4 for 2-3 days. In subsequent work they used far milder conditions and also conducted more effective control studies. Hormann and

THE UNUSUAL LINKS AND CROSS-LINKS OF COLLAGEN

16’7

Fries (1958) used 0.02 M periodate in 8 % acetic acid a t 20°C and followed the reaction in terms of periodate consumption, free amino group content, hexose content, ammonia evolved, formaldehyde evolved, and hydroxylysine content. 111 the initial rapid reaction 2.8 moles of periodate/100 moles of amino acids were used, but consumption continued to rise linearly thereafter. The oxidation products were of high molecular weight and completely nondialyzable even after 72 hr at 40°C or 250 hr a t 20°C. The oxidation of hydroxylysirie was shown by the fall in free amino groups and the production of ammonia and formaldehyde, all of which agreed quantitatively with each other, and with the loss of hydroxylysine determined analytically. Hexose destructioii proceeded much more rapidly, but 20 yo of the hexoses were not destroyed, presumably having no cis-glycol groups. The fact that destruction of hexoses released no amino groups (Hormann and Fries, 1958) proves the hypothesis of short chains joined by hexose bridges (Schneider, 1949; Grassmarin et al., 1957a) to be incorrect. The conditions of Grassmann and Kiihn (1955) simply caused peptide cleavage that did not occur in the work of Hormaiiri and Fries (1958). The latter authors also showed that phenyliodoso acetate was not sufficiently specific for this study. Later Hormann et al. (1959) studied the breakdown of the amino acids of procollagen by periodate under the conditions of Hormann and Fries (1958). Only a few amino acids were destroyed. By considering the breakdown of these amino acids and hexoses all the periodate used after 5 hr can be accounted for (see Table X). To account for the periodate conTABLEX Periodate Balance after 5-hr Oxidations 0xidized component

Moles HI04;consumption per mole

Amount oxidizedb (moles/100 moles)

Hexose Hydroxylysine Tyrosine Methionine

1 1 4 2

0.59 0.43 0.29 0.21

Calculated HIOI consumption (moles/100 moles) 0.5U

0.43 1.16 0.42

2.60

From Hormann et al. (1959). According to amino acid analysis. Experimental periodate consumption was 2.8 moles.

b

sumption it was assumed that each hexose destroyed took up only one molecule of periodate, i.e., it has only one pair of adjacent free hydroxyl groups. It is clear that up to ail oxidation time of 5 hr, a t least, the action of

168

J O I l N J. IIAHDINC

periodic a c d (ham be accounted for in the above terms arid is therefore 1 cry specifics. l<xptrimciitsunder the same (miditions for 140 hr (Hormaiiii ct nl., 1W)) showcd a fairly extensive destruction of frrr amino acids that was grratly r d u c ~ dwhen thry werr incorporatrd iiito prptides, rxcrpt i i i thc cast of tyrosiiir which took up about four molrcdrs of pcriodatr prr molccule whctker bound or free. It is perhaps worth rioting that Kuhn ef al. (1959) found that collagen fibers after 1 hr of oxidation, when most of the hexose had been removed, still rcsembled native collagen in electron micrographs. FIbrmaiiii aiid Kleiik (scc Hiirmaiiii, 1960a) ha\ P shown that mature vollagen caii be dissolved caompletely by periodate and a hydrogeii-bond hrcaker in acetic. acid to give mostly a high molecular weight product, provided all three reagents are present simultaneously. Therr is a c.losc similarity between these results and those obtained using hydroxylaminr and hydrogen-bond breakers (Hormaiin, 1960a), and obviously rupture of (TOSS-linksis involved in both mses. The oxidixable cross-links must involve hexoses as the amino acid side chaiiis would riot be dcstroyed by periodatr if they participated in any (*ovalentcross-linking. Blumenfeld et al. (1963b) have identified the breakdown products of the hrxosrs after treatment of ichthyocol with periodate. The carbony1 groups released reacted with 2,4-dinitrophenylhydraziliein arid solution to form 2,4-dinitropheiiylosazoiies which were separated by thin-layer chromatography. Only the 2,4-dinitrophcnylosazones of glyoxal aiid glycseraldehyde could be dettcted, demonstrating that the hexoses arc hound via the C-1 positions arid h a w the 2-, 3-, arid 4-hydroxj roups free, c.g., for glucose CH,OH

FH,OH 0

\

OHC

+ CH,O

CHO

I

OH

I

IH+

CH,oH I CHOH

I

t

+

CHO I CHO

f

R-OH

CHO

For every molc of hexose originally presciit 0.63 molc of glyoxal wrrc fouiid. A similar yield was fouiid for model compounds and it therefore appcars

T H E UNTJBUAL LINKS AND CROSS-LINKS OF COLLAGEN

1ti9

that all the hexose in ichthyocol has the 1-position substituted aiid the 2-, 3-, and 4-positions free. It is riot possible t o tell from the periodate reaction whether the C-6 hydroxyl is substituted or not. Very recently, Gallop has suggested that certain rather ill-defined sugars found in small amounts in ichthyocol may be involved in the cross-links (Gallop, 1964).

D. Concluszons Any studies of thc carbohydratc compoiierits that coiistitiite an iritriiisica part of the collagen molccule are considerably hampered by the problem of frering the material from mucopolysaccharide impurities. These impurities have heen studied by a iiiimber of authors (e.g., Eastoe aiid Eastoe, 1954; Leach, 1960). The expcrimerits described above indicate that the hexosamiiie assoriated with collagen is part of an impurity and not part of the collagcii. The preseiicc of hcxosamiiics in the mucopolysaccharides associated with collagen (e.g., Iliscahe et al., 1958) iiidicates that collagen preparations coiitaiiiirig no hexosaminc must be frcc of mucopolysaccharide impurities. Such preparations arc suitable matcrial for use in iiivestigatioiis of the carbohydrate compoiicrit of collagen itsclf. It is apparent from the work described that soluble eollagen is miwh more readily purified than insoluble collagen. The problem of the purification of collagen has been discussed a t some length by Eastoe (1955). It appears to he reasonably wrtaiii, however, that part of the hexose associated with collagcn is firmly bound to the protcin and probably takes part in the cross-linking of the polypeptide chains. The role of the hexoses in collagcii will he discuscd furthcr in

1'11.

T H E IR'TEltIlEL4TIOYSHIP BETWEEN cL\1tBOI-IYI)R4TE 22 I ) IikTEIt I,I\ KS A N D THE CI~OSS-LIYI~S o w COLLAGE.C

l'hc rcsults diwusscd iii Scctioiis V aiid VI have dcmoiistrated that hcxoses atid ester linkages play an importaiit rolc in the structure of collagen. 130th appear to be involved in thc intermolecular c*ross-linking of thc cdlageii mac~romolwules. The precise manner iii which thcy may bc iiitcrrtlsted \%illbc voiisidcred in thc presciit swtion. As this subject is vloscly relatcd to thc linkages hctwecii the carbohydrate a i d protcin compoiiciits of glyroproteiiis, these liiihages will also bc discussed. The iiivolvemciit of aspartic. acid in maiiy of these links as well as in thc cstcr liiiks of rollageii is a point of major iiiterest. Consideration of the geiieral approach t o this typc of problem is also very iiiformativr.

170

J O H N J. HARDING

A. Collagen The earliest ideas of the mode of linkage of the hexoses in collageii were those of Schiieidcr (1940, 1949), whereby the hexose was said to be bound N-glycosidically to an a-amino group arid by an ester linkage to an a-carboxyl group. Grassmarin (1955), having found evidence of ester linkages (Grassmann et al., 1954), supported this hypothesis. Grassmann, et al. (l957b) studied the osazone reaction of O-glycosides, N-glycosides, and isoglycosides in order t o gain an insight into the linkage of the hexose component. By experiments with model compounds they found that orily the two latter classes formed osazoiies. To obtain osazones with O-glycosides one must first hydrolyze the glycosidic link. Unhydrolyzed collagen and procollagen gave only a very small amouiit of the osazone (determined colorimetrically). After acid hydrolysis the amount of osazone determined corresponded to the amouiit of hexose present in the proteins as determined by other methods. Thus, the N-glycosidic links proposed by Srhneider (1949) cannot be present, nor (:an the isoglycosidic links discussed by Grassmanil el al. (1957b). They concluded that the hexoses of collagen are bound O-glycosidically either as individual hexoses or as a large molecular carbohydrate compoiient. Hormann (1957), on the basis of the accumulatioii of hexose in the residues from trypsin or elastase digestion of denatured collagen, has suggested that the hexose ocrurs in certain favored regions of the oollagen molecule. This is also indicated by electron microscope studies of collagen stained by t,he periodic acid-Schiff method. Standing apart from the rest of the work is the glycirie-carbohydrate fraction isolated by Konno and Altman (19x9, which appears to originate from the C-terminal of the protein chains rather than represent a cross-link between the chains. Oneson arid Zacharias (1960), having isolated an oligosaccharide from a partial hydrolyzate of collagen, suggested that the carbohydrate is present in the' form of a polysaccharide and not as individual hexoses distributed over the molecule. They suggest further that this oligosaccharide could still perform the function of a cross-link in the molecule and this could be the point at which ficin attacks collagen. Hormann (1960b) has collected together much of the data on the hexoses and ester links and attempted to construct a reasonable picture of their relationship t o each other and to collagen arid procollagen. First, a simple comparison is made of collagen arid procollagen in these respects (see Table XI). It is apparent that collagen and procollageii differ little in hexose content but that the riumber of ester-like links in collagen in excess of those in procollagen is approximately equal to the hexose content of

THE UNUSUAL LINKS AND CROSS-LINKS O F COLLAGEN

171

TABLE XI NHsOH-Sensitive Links and Hexoses in Collagen and Procollagena*b

u

Sample

NHpOH-Sensitive links

Hexoses

Procollagen Collagen

0.64

0.38 0.36

Difference

0.43

1.07

Data in moles/100 moles of amino acids. From Hormann (1960b).

either. If it is assumed that collagen and procollagen have the same amount of intramolecular cross-linking, the extra ester-link per hexose must be due to intermolecular cross-linking. This idea is expressed in Fig. 12. OH

Procollagen Intramolecular carbohydrate bridge

Collagen Inter- and intramolecular carbohydrate bridge

FIG.12. Structures of cross-links in procollagen and collagen. From Hiirmann (1960b).

For this hypothesis t o be strictly true, however, the ester/hexose ratio for procollagen should be unity and the ratio for collagen should be two. The ratios found are higher in both cases (Table XI); this may be due to

1i2

JOHN J . HAHDING

the ester value 1)cing high on account of partivipatioli of amides iii thc reaction. That a definite ratio exists betwccri hexosc arid ester links was showii by Hormaiiri aiid Klcnk (see Hormann, 196Ob) by comparison of the ratio in collagen arid in t h e trypxin-digest residue (sce Table XII). ‘r.\BLE

XI1

(”on,centrationof Hexose and NHnOH-Sensitioe Links an a Residue Remuining a f k r ‘I’rypsin Ihgrstion o j Heat-Denatured CollagenrLVh

Itcsiduv

Collagen

Collagen residiie

Hexose NH20H-Hensitivc links

36 102

475 lO2O

Although the ratio varies very little, it does in fact come doscr to the 2 : 1 ratio rccluired by Hormann’ssuggestcd structure. Oiic advantage of the proposed structure is that 110 additional molecules have to be introduced in order to attain the intermolecular cross-linking of collagen. Also, the strucature as it staiids can accbouiit for the a- aiid P-vompoileiits of collagen arid the possibility that the larger /3-compoiicnt gives two a-components after treatment with alkali (Doty aiid Nishihara, 1958). It is to be noted that the hexosc pivtured in procollagen by Hormariri and Nlwk has thrce a d j a w i t hydroxyl groups aiid thcreforr would take up two molcc.ulcsof periodate per hexose. This is in contrast with the fiudiirgs of Hormatiii et nl. (1959) where only oiie moleciile of periodate per hcxose was consumed in the (Lase of procollagen. This could be accounted for if the hexose were linked 1-2 or 1--4 instead of 1-6 as shown. Thc problem of the oligosaccharide found by Oricsoii arid Zacharias (1‘360) miist, however, t x ac*countcd for. Hdrmann (1(360b, discvssioii) has pointed out that the oligosawharide may represeiit the 20 of the hexosc roiitciit of collagen that is iiot attacked by periodate. I’raiizblaii (1962), E’ranzblau et al. ( l Y ( i l ) , aiid Seiftcr el al. (1!)6l) studied a caollageaase digest of ichthyocol gelatiii and found the iiondialyzable material enriched with respect to esters, hexoses, aiid tyrosine, as me11 a h polar amino acids. They found a constant ratio of these threc ciititics throughout. The ester/hexose ratio was about 2.5, whereas the tyrosiiie/ hexose ratio approximated to unity. The ester/hexose ratio is close to that fourid by Hormariii arid Klerik (see Hormarin 196Ob) for iiisoliible cdlageii aiid does not support the idea of only one ester link per hexose for soluble c~ollage~i.Tyrosine was not linked via its hydroxyl group arid therefore is presumably just preseiit in the same peptides as the hexoses and ester

T H E CNTJSUAL L I N K S AiVD CROSS-LINKS OF COLLAGEN

173

links. Wheii the gelatin was treated with hydraziiie before digestion the hydrazide-containing peptides dialyzed more rapidly suggesting that hydrazinr split the peptides under coilsideration leaving the carboxyl donor as part of a small peptide, while the hexose and tyrosine formed part of a larger moiety. From the observatioii that hydrazirie and hydroxylamine break the cbollageii molec*iileto give siibiiiiits with molecular weights close to 30,000 (Gallop el nl., 195Y), Franzblaii (1962) proposes that the a-component of collagen with a molecular weight of 120,000 coiisists of four subunits held together by pairs of ester links. This explains much of the previous work of Gallop aiid his colleagues (Gallop P t nl., 1959; Blumenfeld and Gallop, 1962b), hut does riot account for the fact that ichthyocol contains a small proportion of P-components which probably consist of a-components held together by alkali-labile linkages. This (+anbe explained by postulating traiisesterification during intramolecular cross-linking (Gallop, 1964). Hormaiiii aiid Nordwig (see Hormann, 1962) have reported on very similar experiments tising collngenase and mature collagen. They again found a 2 : l ester/hexose ratio which is compatible this time with Hormann’s hypothetical structure (1960b). Ilormaiin (1962) has also suggested that the cross-links o c ~ i i rin pairs. In this way the pairs will occur every 640-700 A in the caollageii molecule which is in agreement with the frequency of caross-links suggested by Wiederhorn and Reardoii (~ 5 2 from ) stress-strain measurements on thermally denatured collagen. This indicates that the linkages that coiitribute to the cross-linked lattice of mature collagen arise from the same region as the intramolecular links, i.e., from the polar region demonstrated by elec.tron microscope studies. Iiecently, Hormann (1962) has reported some rather unexpected results conceriiing the stage a t which estrr-hexose intermolerular links are introduced. It was expected that calf c*ollageninsoluble in acid buffers would contain almost as many ester linkages as mature csollagcn, but this was not foulid to he so (Table XIII). h lack of cross-linking was also indicated by the observatioii that a large proportion of calf caollagen could he dissolved in 4 M sodium perchlorate or in hot water. Hormaiiri (1962) suggests that procollagen is strengthened to give mature collagen via intermediates where the fiber is stabilized in a noncovalent way, possibly by mucopolysacc~haride. The recent work of Blumenfeld et al. (1963h), using periodate, iiidicated that in ichthyocol the hexoses are linked through the C-1 position while the 2-, 3-, and 4-hydroxyl groups are unsubstituted (see Section VI). To study the C-6 positioii of the galactose in ichthyocol they used galactose oxidasc which will oiily oxidize galactose residues with the C-6 position uiisubstituted. After treatment of icahthyocol with this eiizyme 82 % of

174

JOHN J. HAILDING

the original galactose was oxidized. Thus, only the C-6 positions of glucose present remain to be accounted for. A s there are six ester links and only 1.4 glucose residues per 1000 amino acid residues, the alcohol donor of the ester links in ichthyocol cannot originate entirely, if a t all, from the bound hexoses. These results also show that the hexoses of ichthyocol occur as single units and not as di- or polysaccharide units attached to the protein. TABLEXI11 Hexoses and NHzOHSensilive Links in Collagen of Various Agesajb

-~

Sample Ox collagen Calf collagen Calf cdlagen dissolved by pepsin and reprecipitatcd €'roc-ollagen a

Hexoses

NHZOH-Sensitive links

0.35 0.48 0.30

1.04 0.62 0.60

0.38

0 64

Data in moles/100 moles of amino acids. From Hormann (1962).

The question of the cross-links in collagen is, however, still not fully explicable. The oiily collagen sample studied by the above authors was ichthyocol which has no intermolecular cross-links and, as can be seen from Table 11, very few intramolecular links. Their data can therefore only show the state of the hexoses and ester groups before extensive cross-linking, and in this state the hexoses are bound a t a single position probably by an O-glycosidic link. It is of interest that acid-soluble collagen from calfskin appears t o have no more ester groups than ichthyocol (Gallop et aE., 1959) and yet has a much greater proportion of @-components (Table 11) and therefore more intramolecular cross-links. Possibly @-components can be formed from a-components by a transesterification process; such a process, without increasing the number of ester links, has been set out diagrammatically by Gallop (1964). He coiitiiiues the process to give intermolecular cross-links, but as insoluble collagen contains an increased number of ester links (Tables VI and XI) these are probably formed separately. An indication that hexoses are iiivolved in the intramolecular crosslinking is found in the data of' Hormann et al. (1959) on the action of periodic acid on acid-soluble collagen from calfskin (Table X). In this sample approximately 1 mole of HIO, is used per hexose unit present. If, homever, the hexose was linked only in the C-1 position as found in ichthyocwl (Blumenfeld et al., 1963b), then 2 moles of HIO, would be required. This supports the hypothesis that trarisesterification is involved in the formation of intramolecular cross-links. Such transesterification will

T H E UNUSUAL LINKS AND CROSS-LINKS O F COLLAGEN

I75

occur between two of the chains leaving the third unaffected. In this case calf procollagen should have two-thirds of the hexoses present linked in two positions and the remaining third liiikcd only in the C-I position. Its mean periodate uptake would therefore be 1.33 moles per mole of hexose. Referring again t o Table X it is clcar that if this periodate uptake is accepted instead of the figure of 1.0 mole per mole of hexose assumed by Hormann et al., the value for the periodate consumption in the last column becomes 0.79 moles/100 moles giving a total consumption of 2.80 moles/100 moles in exact agreement with the experimcntal figure. Thus, the hypothesis that intramolecular cross-linking takes place by the transfer of ester groups on to hexose residues seems reasonable. All the ester bonds present do not transfer in this way. The nature of the alcohol donor of these ester links aiid of all esters prior to cross-linking is not known. 7-Comporients could be formed from a-components by a similar mechanism. To fit in with the periodate consumption the hexose in the 0-components must be linked either in the 1-2 or 1 4 positions. Insoluble collagen is more difficult to study, but it appears (Table XI) that during intermolecular cross-linking new ester links are formed. These new ester bonds are equal or slightly greater in number than the hexoses present. Prior to intermolecular cross-linking it appears that one-third of the hexoses present are linked only in the C-1 position and two-thirds are linked either in the 1 4 or the 1-2 positions. If new bonds are to be introduced into the latter two-thirds that will still leave the hexose units open to periodate attack they will have to link to the C-6 positions. In the single chains present either one or two new ester links could be formed to each hexose unit. At maximum cross-linking there would be two ester links to each hexose unit leaving a single cis-glycol unit (2-3 or 3-4) available to periodate. A 2 : 1 ester to hexose ratio has been fourid in the cross-link enriched fraction of both trypsin and collagenase digests of mature collagen (Hormann, l96Ob, 1962). Before digestion the ratio is 3 : l (Table XI) accouiiting for the ester bonds that do not appear to be attached t o hexose. Involvement of esters in the intermolecular cross-links accounts for the dissolution of mature collagen by hydroxylamine, hydrazine or alkali in the presence of hydrogen-bond breakers. Similarly, the participation of hexoses accounts for the dissolution of collagen by periodic acid and a hydrogen-bond breaker. Recently, it has been shown that collagen can be completely dissolved by some proteolytic enzymes (see Section 11). These enzymes also cleave the intramolecular cross-links (Kuhn et al., 1963a; Rubiii et al., 1963). It appears therefore that either the crosslinks include a peptide chain or the chain close to the cross-links contains pepsin- and trypsin-sensitive bonds.

170 H. Discussion The ahovr hypothescs for the tlcvelopmeiit of intra- arid iiitermolecwlar c.ross-lirikiiig in collagrii apprar to ac.cwmit for the data kriowii a t preseiit . The figurcs used may, howcvrr, be somcwhat in error owiiig to the fa(*tors discussed in Sec%ioiisV aiid 171. The maiii problems arc the lack of spccific8ity of reagriits used aiid the impuritics in the collagen. The latter is especially difficult in the ('asp of insoluble collagen. The bulk of the work 011 the esters aiid hexoses has been carried out by trcating the whole protein with different specific' reagents. Unfortunately, thr spccificity rcquircmriits for a reagciit to study just a frw esters aiid hexoses, in the pirsciice of two or three hundred times as many pcptide links aiid teri times as many amides not t o meiitioii other groups, are very stringent. This caused the diffidties experienced with the lithium borohydride method arid with certain applications of the hydroxylamine arid periodic acid methods. The remaining experiments with hydroxylamiiie, hydraziiie, arid periodic acid may still be less accurate owing to such side reactions. I t would appear that a better may of approaching the problem is to first drcrease thc exwss of these other groups by a partial breakdown of thc collagen aiid isolation of the moieties containing esters and hexosc as has already beeii done by Fraiizblau (1'362) and by Hormann (1957, 1!)60b, 1!162). These materials should then form the basis of further study to determine the exact nature of the linkages involved. A similar approach has heeii follmed by Partridge et nl. (1963) to examine the cross-links of elastin. Which of these two basic, approaches is most useful in any particular (base will clearly depend on the ratio of thc amourit of extraneous material to the number of liiiks preseiit to be studied. Where this ratio is large the extraiicous material will have to be partly eliminated before direct chemical study is possible. This point is exemplified very well by the work cbarried out to study the proteiii-carbohydrate links of glycoproteins. The lattrr falls iiito two maiii classes dcpeiidirig on the number of carbohydrate uiiits present. This caii be illustrated by coiisideration of the ratio of thc iiumber of peptide links to thc riumber of carbohydrate units. In most glycoproteins this ratio is very high : egg albumin with one carbohydrate unit per molecvlar weight of 45,COO gives a ratio of approximately 450; likewise, the ratio for 7-globuliii is 1600. Oviiie submaxillary gland m u ( w protein with 800 carbohydrate units per molecular weight of lo6 yields the very low ratio of 12.5. In the latter case therefore, little iiiterfereiiw from peptide liriks arid amino acid side-chains is to be expected if the undegraded material is suhjectcd to direct chcmical attack by specific reagents, and this approach has been very profitably employed for this mucoprotein. Most glycoproteins, however, would not be ameriable to such an approach,

THE UNUSUAL LINKS AND CROSS-LINKS O F COLLAGEN

177

and in these cases the mass of the protein has been largely removed prior to chemical study. Collagen, having relatively few ester links or carbohydrate units (ca. 5 esters per lo6 gm) in a very large protein, falls clearly into this class of glycoprotein and it is this general approach that will probably prove to be more profitable in this instance. A further relevance of glycoproteins to collagen is the nature of the protein-carbohydrate link found. Blumenfeld and Gallop (1962b) have shown that aspartic acid is involved in the ester-like links of collagen which may be binding hexose molecules. Aspartic acid also constitutes the point of attachment of carbohydrate in most glycoproteins studied to date. It has been shown that 80-90 % of the prosthetic groups of ovirie and bovine submaxillary gland mucoproteins can be split off by alkali or lithium borohydride (Gottschalk, 1960; Gottschalk and Murphy, 1961; Murphy and Gottschalk, 1961) or by hydroxylamine (Gottschalk et aE., 1962; Graham et al., 1963). This demonstrates that ester linkages are involved in the protein-carbohydrate bonds. The products after reductive cleavage indicate that the side-chain carboxyl groups of both aspartic acid and glutamic acids are involved in these linkages (Murphy and Gottschalk, 1961). The other glycoproteins studied have had too few carbohydrate units to make a similar approach possible. For these the alternative approach has been adopted (a) extensive breakdown of the peptide chain, almost always by enzymes, followed by isolation and characterization of glycopeptides from the digest mixtures; (6) further breakdown, normally using aminopeptidase and carboxypeptidase, to yield a fragment containing all the carbohydrate but only one amino acid, usually aspartic acid; ( c ) acid hydrolysis of this fragment to degrade the carbohydrate portion resulting in an aspartic acid-glucosamine fragment in those cases where it has been applied; (d) this fragment can then be subjected to very thorough study to determine its precise structure and, by inference, the nature of the protein-carbohydrate linkage. Stage (a) has been carried out for ovalbumin (Cunningham et at., 1957; Johansen et aE., 1958; Jevons, 1958; Nuenke and Cunningham, 1961; Johansen et al., 1961; Lee and Montgomery, 1962), several y-globulins (Rosevear and Smith, 1958, 1961; Nolan and Smith, 1961, 1962a,b), al-glycoprotein (Eylar, 1962; Izumi et al., 1962; Kamiyama and Schmid, 1962a; Bourrillon et aE., 1963), fetuin (Spiro, 1961, 1962), ovomucoid (Hartley and Jevons, 1962; Montgomery and Wu, 1963; Neuberger and Papkoff, 1963), Taka-amylase A (Tsugita and Akabori, 1959), a glycopeptide from pig stomach mucus (Masamune, 1956), a chondroitin sulfate complex (Muir, 1958; Anderson et al., 1963), and a heparan sulfate complex (Jacobs and Muir, 1963). Stage (b) has been

178

JOHN J. HARDING

reached in the following cases : ovalbumin (Kaverzneva and Bogdanov, 1961, 1962; Yamashina and Makino, 1962; Marks et al., 1962b; Fletcher ei, al., 1963; Fletcher et al., 1963b), human y-globulin (Hosevear and Smith, 1961), rabbit y-globulin (Nolan and Smith, 1961, 1962a), ovomucoid (Montgomery and Wu, 1963), al-glycoprotein (Eylar, 1862; Kamiyama and Schmid, 1962b), fetuin (Spiro, 1961, 1962), and Taka-amylase A (Tsugita and Akabori, 1959). In the latter case the fragment contained serine as the only amino acid, but in all the others it was aspartic acid. Stage (c) has been reached only for ovalbumin (Rogdanov et al., 1962; Yamashina and Makino, 1962; Marks et al., 1963) and bovine y-globulin (Nolan and Smith, 1962b). Rothfus and Smith (1963) isolated a fragment from human y-globulin containing aspartic and glutamic acids as the only amino acids. Stage (d) has been reached only with this fragment (Rothfus and Smith, 1963) and with ovalbumin (Bogdanov et al., 1962; Yamashina and Makino, 1962; Fletcher et al., 1963a,b; Marks et al., 1963; Marks and Neuberger, 1961). A recent review by Bourrillon and Got (1963) discussed the structures of many glycopeptides produced as the breakdown products of glycoproteins as well as those found naturally. More general reviews have also appeared (Jeanloz, 1963; Spiro, 1963).

1. Collagen Any study of collagen by similar procedures is hampered by several drawbacks. The most difficult problem is that of purity of the original protein. With insoluble collagen, especially, it is very difficult to prepare a sample free from impurities. As the impurities include mucoproteins and elastin, which have carbohydrate components, it will be essential to avoid confusion with these possible contaminants. The nature of the protein-carbohydrate link in collagen seems to resemble that of most glycoproteins in that it involves aspartic acid. Here, however, the similarity ends because, in ovalbumin, y-globulin, and salivary mucoproteins a t least, glucosamine seems to be at the carbohydrate end of the linkage, whereas in Section VI it was established that collagen contains no bound hexosamine. If the collagen-hexose link is a n ester, this also differentiates collagen from the most fully studied glycoproteins except the salivary mucoproteins. In a very good general review on protein-carbohydrate complexes Bettelheim-Jevons (1958) suggested that hexosamine might be involved in the protein-carbohydrate links. This has been borne out in several of the studies summarized above.

VIII. OTHER UNUSUALLINKSAND CROSS-LINKS IN COLLAGEN There is a limited amount of evidence in the literature supporting the possibility of unusual links and cross-links in collagen other than those

THE U N U S U A L LINKS A N D CROSS-LINKS OF COLLAGEN

179

already discussed. These include aldehyde groups, arginine cross-links, peroxide cross-links, and phosphate-mediated cross-links. 1. Aldehyde Links

Landucci et al. (1958) demonstrated the presence of aldehydes in several proteins including gelatins and egg albumin. Part of the aldehyde seemed readily accessible to thiobarbituric acid, but the remainder would not react until severe degradation of the protein had occurred. They suggested that the labile aldehydes were present at the ends of the chains. They also suggested that the aldehydes were bound preferentially to proline and/or hydroxyproline in sequences of the type : -amino acid-aldehyde-imino acid- which constituted a labile link in the peptide chain, breaking to leave the NHP-terminal amino acid masked by the aldehyde. Blumenfeld and Gallop (1962b) also reported the presence of a few aldehyde groups in ichthyocol gelatin. Milch (1963) has demonstrated that a number of aldehyde metabolites produce changes in collagen very similar to those taking place during aging. Levene (1962), while studying lathyrism, has proposed that aldehydes are involved in the cross-linking of collagen. Lathyrogens prevent such cross-linking, but carbonyl compounds can inhibit this effect. Levene (1962) therefore suggested that the lathyrogens block the carbonyl groups of collagen required for cross-linking. This was supported by his finding that lathyritic collagen, unlike normal collagen, will not bind 2,4-dinitrophenylhydrazine. It is possible that this work is related to the carbohydrate moieties of collagen. Alternative mechanisms for the action of lathyrogens have, however, been proposed, e.g., Dasler et al. (1961). Very recently, Gallop (1964) has reported evidence that the moiety reacting with 2,4-dinitrophenylhydrazineis an a-keto acid, probably pyruvic acid. 2. Arginine Cross-Links

Gustavson (1955b,c) suggested that guanidinyl-carboxyl cross-links might exist in collagen on the basis of the alkali-binding capacity of collagen and gelatin and the fact that only about 40 % ’ of the guanidino groups in collagen are destroyed by HOCl (Bowes and Kenten, 1949). Bose and Joseph (1958) found that HOCl only destroyed 50 % of the arginine of collagen, but they achieved 70 % destruction using HOBr (Bose and Joseph, 1958; Joseph and Bose, 1960). They also found that only 70 % ’ of the arginine in gelatin was released as the NHrterminal amino acid by the action of trypsin. On this evidence they suggested that the remaining arginine might be linked to the y-carboxyl groups of glutamic acid residues. Later Joseph and Bose (1962) demonstrated that the availability

180

JOHN J. HARDING

of arginine in collagen to destruction by HOBr decreased from 88 to 65 % during the lifetime of rats. De la Rurde et al. (1963) have investigated the reaction of collagen with hydrazine. They found that the curves for percentage deguanidination and for percentage solubilization of collagen against hydrazine concentration followed the same course. They therefore suggested that arginine might take part in covalent cross-links, possibly to an €-amino group by way of a methylene bridge. The main increase in solubility and deguanidination, however, occurred only in 70 yo hydrazine, i.e., under extremely basic conditions. The relation of this to guanidino cross-links seems much less likely in the light of work of Hormaiin and Klenk (see Hormann, 1962) showing that 90 yo of hide collagen went into solution under the action of 2.4 % hydrazine in the presence of 2 M KCNS in 11 days. At this concentration de la Burde et al. could observe negligible deguanidination. TitIration curve data constitute the most satisfactory method for the study of masked groups. Unfortunately, guanidino groups can only be determined indirectly by this method. Even so, there is no evidence for masking of these groups in gelatin (Kenchington and Ward, 1954), although it is arguable that they may be released during the conversion of collagen to gelatin. There is, therefore, very little evidence to prove the presence of crosslinks in collagen involving arginine. It is, however, quite possible that a few links of this type may be present. 3 . Peroxide Cross-Links

Recently, Deasy (1962), having identified 3-chlorotyrosine in hydrolyzates of hide collagen, postulated that this compound originated from tyrosine cross-links. 3-Chlorotyrosine residues involved in peroxide (-0-0-) is known t o be produced by oxidation of tyrosine (Bidmead and Ley, 1958) and indeed is formed from tyrosine under conditions used for protein hydrolysis (Sanger and Thompson, 1963). This probably explains its presence in collagen hydrolyzates.

4. Phosphate-Mediated Cross-Links Veis and Schlueter (1963) have studied the solubility and swelling properties of dentine collagen after decalcification by ethylenediaminetetraacetie acid (EDTA). The dentine collagen behaved as if it were more extensively cross-linked than corium collagen. Having noted that the decalcified dentine collagen still contained some phosphate that was not in combination with calcium and could not be removed by EDTA,

THE UNUSUAL LINKS AND CROSS-LINKS OF COLLAGEN

181

they suggested that the uiiusual properties of this collagen were the result of cross-linking by way of phosphate residues.

ACKNOWLEIGMENTS This review was carried out during the tenure of a grant from the Medical Research Council t o the Gelatine and Glue Research Association. The author thanks Dr. P. Gallop and his colleagues a t Yeshiva University for the opportunity to see their manuscripts prior to publication. Thanks are due to the following authors and publishers for permission to reproduce certain tables and figures in the text: Dr. Gallop and the American Chemical Society for Table VII, Professor Grassman and Blackwell Scientific Publications Ltd. for Table IX, Dr. Hormann and Walter de Gruyter and Co. for Table X, Dr. Hormann and the editor of Beitruge zur Sililcosejorschung for Tables I and XI and Fig. XII, Dr. Hormann and the publishers of Das Leder for Table XIII. The author is grateful to Dr. D. A. Sutton for suggesting this study. Useful discussions have been held with Dr. A. A. Leach and Dr. A. W. Kenchington. The helpful criticisms and suggestions of the staff of the Gelatine and Glue Research Association and especially Dr. A. A. Leach are also gratefully acknowledged.

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THE CHEMISTRY OF KERATINS By W

. G. CREWTHER. R . D. B. FRASER. F. G. LENNOX.

AND H. LINDLEY

Division of Protein Chemistry. C.S.I.R.O. Wool Research Laboratories. Melbourne. Australia

I. Introduction . . . . . . . . . . . . A . Scope . . . . . . . . . . . . . B. Outline of Keratin Fiber Histology . . . . . . I1. Isolation and Characterization of Proteins . . . . . A . Breaking Disulfide Bonds . . . . . . . . B . Hydrolyzing Peptide Bonds . . . . . . . C . Origin of Extracted Proteins . . . . . . . D. Some Properties of Wool Protein Fractions . . . E . Protein Derivatives from Feathers . . . . . . I11. Composition of Keratins . . . . . . . . . A . Analytical Methods . . . . . . . . . B . Amino Acid Composition . . . . . . . . C . End Groups of Keratins . . . . . . . . D . Sequence Studies . . . . . . . . . IV . Chemical Reactivity of Keratins . . . . . . A . The Reactivity of the Cystine in Keratin . . . . B . The Reaction of Keratins with Ions . . . . . C . Photochemical Reactivity . . . . . . . . V. Molecular Structure of Keratins . . . . . . . A . The Structure of a-Keratin . . . . . . . B . The Structure of p-Keratin . . . . . . . C . The Structure of Feather Keratin . . . . . . VI . Relationship between the Physical Properties and Chemical Structure of Keratin . . . . . . . . . A . Introduction . . . . . . . . . . . B. Physical Properties of Animal Fibers . . . . . C . Effects of Chemical Modification on Physical Properties of Keratin Fibers . . . . . . . . . . References . . . . . . . . . . . .

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I . INTRODUCTION

A . Scope In the interval since the chemistry of keratins was reviewed by Ward and Lundgren (1954) in this series a considerable effort has been made to obtain a better understanding of the exceedingly complex chemical composi191

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G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

tion and molecular architecture found in these tissues. The foremost obstacle to applying the methods of protein chemistry to keratins is their natural insolubility. Thus, part of this review is concerned with efforts to prepare pure keratin derivatives for composition, sequence, and endgroup studies and to obtain monodisperse solutions of these derivatives for physicochemical characterization. Owing to the economic importance of wool most investigators have used this material as a convenient source of a-keratin. When parallel studies have been made on hairs from other animals and on nails, claws, hoofs, and quills it has been found that conclusions reached by studying wool proteins apply, with only minor qualifications, to other keratinized tissues. Feathers are only of slight economic value and correspondingly less attention has been devoted to their chemistry, despite the fact that feather proteins are more readily solubilized and purified and that feather rachis yields X-ray diffraction patterns of excellent quality. The contents of the present review reflect the emphasis on wool a s a source of a-keratin and are largely confined to a description of progress in the study of this material during the past 10 years. Many references to the histological components of keratin fibers are to be found in the text, and although it is beyond the scope of this contribution to review progress in this field, a brief summary of current knowledge is given in the following section. References to the original publications leading to this picture have been collected recently by Lundgren and Ward (1962).

B. Outline of Keratin Fiber Histology Animal hairs develop by the proliferation of layers of the skin in specialized structures known progress up the follicle toward the skin surface ferentiate and fuse together to give the principal

cells from the germinal as follicles. As the cells three types of cells difcomponents of the fiber.

1. T h e Cuticle

The fiber surface is bounded by a thin membrane 100 A thick called the epicuticle. T h e cuticle is a scaly, tubular layer and consists of flattened cells which overlap to give a rachet-like profile to the fiber. Each scale cell contains two distinct layers, a keratinous outer layer termed the exocuticle and a nonkeratinous inner layer that appears to be derived from cytoplasmic debris and is termed the endocuticle. There is some evidence that the exocuticle itself is complex with an outer cystine-rich layer termed exocuticle a. I n coarse fibers the cuticle may be many scale-cells thick and where the cells overlap they are separated by an intercellular layer formed during biosynthesis by the deposition of nonkeratinous protein between the cell membranes. This layer is sometimes referred to as intercellular cement.

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2. The Cortex The cortex forms the bulk of fine animal hairs and is derived from highly differentiated spindle-shaped cells that are densely packed with keratinous proteins. The long axes of the cortical cells are oriented parallel to the fiber length, and elongated cavities near the center of the cells are similarly oriented. These cavities are derived from the nuclei of the developing cells and contain debris usually referred to as nuclear remnants. Between cortical cells there is a layer 250-300 A in thickness which is similar to that found between cuticle cells; this also is referred to sometimes as intercellular cement. Many nonkeratinous inclusions are found within the cortical cells and these are believed to be cytoplasmic debris. The bulk of each cortical cell consists of closely packed microfibrils -75 A in diameter and embedded in an osmiophilic matrix. It is widely believed that the microfibril is a highly organized assembly of fibrous molecules and that the matrix is an amorphous cystine-rich cement or ground substance. In curly or highly crimped wool two types of cortical cells are present and they are grouped to form a paracortex which follows the concave surface of the crimp wave and an orthocortex which follows the convex surface of the crimp wave and thus is longer than the paracortex. In the paracortex large numbers of microfibrils are collected into bundles in quasi-hexagonal arrays; these bundles are termed macrofibrils. In contrast the macrofibrils in the orthocortex are much smaller; cytoplasmic debris is scattered throughout the cell and appears more abundant. In addition the microfibrils within each macrofibril are arranged in concentric sheets giving the appearance of a spiral or “whorl” in cross section. There is no evidence from electron micrographs of differences between the microfibrils in orthoand paracortex, but there appears to be a significantly higher proportion of matrix in the paracortex. The fine structures of microfibrils and the matrix are discussed in Section V, A . S. The Medulla

In fine animal fibers, e.g., Merino wool, the structure develops solely from cuticle and cortical cells, but with increasing diameter a third type of cell becomes more prominent in the follicle. The medulla is formed from an axial stream of cells which do not become densely packed with protein or develop highly asymmetric shapes. By contrast the cell contents shrivel up during dehydration leaving a series of vacuoles along the fiber axis.

11. ISOLATION AND CHARACTERIZATION OF PROTEINS The range of methods for characterizing keratins is greatly extended if the proteins are extracted and converted to a soluble and stable form. It

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then becomes possible to separate them from one another and to determine the molecular size and shape in solution, the peptide chain conformation, the identity of the amino and carboxyl end groups, and the amino acid sequence. Some of the early steps in the extraction and characterization of keratin derivatives are described in this section. Ultimately, it should be possible to extract particular proteins selectively from the tissue or fiber and, in conjunction with electron microscopy and related methods, to determine their location within the original structure. Extraction of wool with water or cold dilute acid or alkali yields remarkably little peptide or protein. Boiling in water for 12 hr, for example, extracts only 1.6 % of the fiber (Zahn aiid Meienhofer, 1956). Rupture of covalent bonds appears to be essential for any appreciable dissolution of wool protein. The acid hydrolysis of peptide bonds has been employed, but the action of acids is largely nonspecific. Proteins prepared by acid extraction or partial acid hydrolysis followed by alkali extraction may yield information which is complementary to that obtained by splitting the disulfide bonds. Although proteases effect more specific attack on the peptide bonds, trypsin dissolves only 10 yo of the fiber if it has been previously swollen for 20 hr at 20°C in buffer at pH 11, or 17 % if previously soaked in caprate at the same pH value (Crewther, 1956). Pronase, which has a broader range of peptide bond specificities, digests 10-20 yo of the wool during incubation for 6 days a t pH 8 and 28°C without prior swelling (Springell, 1963). Alkali solutions at sufficient concentration or a t sufficiently high temperature to extract protein from wool produce yellow discoloration owing to attack on the disulfide bonds and they convert much of the cystine to lanthionine (Section IV, A , 5). The most satisfactory methods of extracting wool proteins are those which depend on rupturing the disulfide bonds. Reduction methods presumably restore the proteins to a state similar to that in which they exist in the wool follicle immediately before keratinization. Reduction, oxidation, sulfitolysis, and oxidative sulfitolysis have all been used to dissolve wool. Whatever method is used to rupture disulfide bonds the proteins extracted can be fractionated into two groups. One fraction contains less sulfur than the parent wool and is believed to originate in the microfibrils. The other contains more sulfur than wool and probably originatm in the matrix. Nevertheless, splitting the disulfide bonds alone is insufficient to make proteins from the wool fiber soluble. I n addition it is necessary to increase or decrease the pH value of the solution, e.g., by adding alkali to confer negative charges on the ionizable acid groups and produce electrostatic repulsion between them or by incorporating a hydrogen-bond breaking agent, such as urea, in the extractant. Both the lowsulfur aiid high-sulfur fractions have been further fractionated and certain

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components obtained in a relatively pure state. I n general, however, the yields are low, even though they are believed to be major components of the fiber. Collectively, the low-sulfur proteins probably represent 50-60 % ’ and the high-sulfur proteins 20-30 % ’ of the wool fiber.

A . Breaking Disuljide Bonds 1. Reduction

a. Reduction in Alkaline Solution. The preparation of soluble derivatives of keratin by thioglycolate reduction, alkylation, and extraction under alkaline conditions, as described by Goddard and Michaelis (1934-1936) , was re-examined by Gillespie and Lennox (1953, 1955a,b). Following the nomenclature of Goddard and Michaelis, the term “kerateines” was adopted to describe the reduced form of keratin and the letters A and B were added to distinguish the low- and high-sulfur groups of proteins, respectively. For convenience “kerateine” has been contracted to the symbol “K” ; “KA” represents the reduced proteins containing less sulfur than the parent keratin and “KB” represents those containing more sulfur. A numeral suf€ix has been added to denote a purified or partly purified fraction of these main groups. Some of the kerateines have been studied in the thiol state, and it has been shown that the unfractionated kerateines are better resolved by electrophoresisin this form than after alkylation (Gillespie, 1960). Alkylation yields more stable derivatives, however, and the reduced wool proteins can be converted to the S-ethylamino-, S-benzyl-, S-carboxyphenylmercuri-, S-methylmercuri-, S-carbamidomethyl-, or S-carboxymethyl derivatives (Gillespie, unpublished data, 1958). The S-(carboxymethy1)kerateines (SCMK) obtained by reaction of kerateines with iodoacetate have proved to be the most suitable derivatives for purification and characterization. The SCMK derivatives containing less sulfur than the parent wool are termed “SCMKA” and those containing more sulfur are termed “SCMKB.” The SCMKA and SCMKB fractions include a small group of proteins that contain various amounts of sulfur and large amounts of glycine and tyrosine. For convenience these are described as the “highglycine” proteins. b. The Low-Sulfur Wool Protein Derivatives SCMKA. The essential steps in preparing a fraction termed SCMKAB (Gillespie and Lennox, 1953, 1955a) are summarized in Fig. 1. During successive extractions with pH 10.5 potassium thioglycolate most of the high-sulfur proteins and the high-glycine proteins are removed from the fiber and subsequent extraction at pH 11.3 yields a solution containing mostly low-sulfur proteins and showing only one peak in the moving-boundary electrophoresis apparatus.

1%

W. G . CREWTHER, R. D. B. FRASER, F. G . LENNOX, AND H. LINDLEY Preparation of Low-S Wool Protein. SCMKA 2 30 gm Merino wool top

Extract 5 times for 20 min at 50°C with 900 ml 0 . 1 M thioglycolate at pH 10. 5

I

I

Residue Extract with 900 ml 0.1 M thioglycolate at pH 12.3 falling to pH 11.3 Residue Discard

Add Zn acetate at pH 6.0 to bring concentration to 0.02 M , centrifuge, discard supernatant, dissolve precipitate in 0.1 A4 citrate and dialyze in running tap water. Repeat Zn precipitation twice

Precipitate SCMKA 2

A

Extracts contain low-S and high-S proteins (electrophoresis in glycine- NaOH containing 0.05 M thioglycolate). Discard

Extract 2001 to 20°C, add to each liter 40 gm iodoacetic acid in 150 rnl water adjusted to pH 7 to give pH 9 in mixture; stand until nitroprusside test is negative. Add sufficient thioglycolate to give positive nitroprusside reaction. Dialyze against tap water. Then either Or add acetate thffer at pH 4 . 4 to bring the ionic strength to 0 . 5 when 85 - 90% of the protein is precipitated. Dissolve in saturated Na borate and repeat precipitation with acetate at pH 4 . 4 twice

Supernatant contains traces of h i g h 4 proteins

__L

FIG. 1. Fractionation of wool proteins at 50°C to give low-sulfur fraction SCMKAQ (Gillespie and Lennox, 1953, 1955a; Gillespie, 1957). All moving boundary electrophoresis runs were carried out a t an ionic strength of 0.1 except in the presence of thioglycolate where the ionic strength was 0.2. The electrophoresis diagrams rcfer to runs in glycine-NaOH buffer at pH 11.0 except where otherwise stated.

This preparation can be freed of traces of high-sulfur proteins by precipitation at pH 6.0 with zinc acetate (Gillespie, 1957) or by acidification t o pH 4.4in the presence of 0.3-0.5 M KCl (Gillespie et al., 1962). The zinc ions can be removed by dialysis against citrate (Gillespie and Springell, 1957). Although SCMKA2 exhibited a single peak on moving boundary electrophoresis, it was shown to be heterogeneous by a constant solvent solubility test (Gillespie, 1957), by fractional precipitation with (NH&S04, and by

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197

fractional elutioii of the precipitate from a L)EA~~-:-cellulose columii followed I)y amiiio :wid aiialysis of the fractioiis (Gillespie, 196%). Similarly, rhromatography of a tryptic digest of SCMKA2 011 a Dowcx 50-X2 coliimii produced a 1 t q c number of unresolved overlapping peaks (Gillrspir et ul., 1960). ICveii mow striking evidence of protein heterogeneity in SCRlKX2 and othcr low-sulfur keratin derivatives has been obtained 4rccc~itlyby Thompson aiid O’Doiiiiell (1964) using starch-gel elcc+trophoresis. As show11 iii Fig. 2 at least five slow-moving balds are formed in additioii to

A

B

C

FIG.2. Starch-gel elcrtmphorcsis liattc~rnsof wool protrin drrivntivrs in pH 8.6 l d f r r containing 8 M urea. A, SCMKA; B, SCMKAB; C, SCMKB (Thompson and O’Donnell, 1964).

1‘38

\V. G. CHE:\V‘I’HEX, It. D. B. FItASEII, F. 0. LENNOX, AND H . LINDLEY

SCMKR chomponelits migrate much more rapidly thaii the SCMKA components. Another is the similarity between the starch-gel patteriis of SCMKA and SCMKAB, although some componeiits have been largely removed during the preparation of SCMKA2 from SCMKA. Some progress has been made by 0’I)onnell atid Thonipson (1964) in isolating thc caotiiponents respoiisiblc for the major baiids by using buffers containing 8 M urea to elute the proteins froiii DEAE-cellulose arid Sephadex G-200 colunlw. Another low-sulfur protein preparation termed SCMKAl was obtaiiicd by extracting wool with thioglycolate a t pII 10.5 for a longer period than was used for the preparation of SCMKAB. The low-sulfur protein in thc extract was repeatedly precipitated at pH 4.4 atid redissolved a t pH 8.5. It was finally precipitated with acetone to separate it from the high-sulfur and high-glycine compoiients (Gillespie, 1058, 1!)60). SCMKAl closely resembles SCMKA2 it1 terms of electrophoretic, mobility, sedimentatioii constant, and amino avid composition, but it is more easily salted out with ammonium sulfate or sodium acetate and requires a lower pH valuc for precipitation. It seems likely that the SCMKAl contains a spectrum of closely related protein compoiieiits similar to but not identicd with that in SCMKAB. c. The High-Sulfur M.’ool Protein Derivaiives SCMKH. The solubility properties of various derivatives of the uiifractionated high-sulfur proteins are shown in Table I. The S-ethylamino KB derivative was very solnhle over a wide pH range, but its preparation by rcwtion of the KB fractioii TABLEI SolubLldy Properties of High-Sulfur Wool Protein Derivatiuesu

Itragent

Side chain

Soluble abovc pH of:

:t

--CHaSCH?CH&H 11 -CH2SCHzCH(OH)CIIa 11 -CH$CH,CH (OH)CH,OH 13 Glycidol -CHZSCH&ONHz 9 Iotloacetamidc 11 -CH 2SCH 3 Methyl iodide --CH28CH*CHaCHzCHa 11 n-Butyl iodide -CHzSCH(CHa)CHzCH3 11 scc-Hutyl iodide -CH&H&H&N 12 ,\crylonitrile Ihtirc pH range (slight t l t w c w v i3-Rromoct,hyl:tIIliiio --CH2SCH&HjNH, pH 9-10) Entire pH range (minimum solu-CHzSCHzCOOH Iodoaretic arid ijility :it 2.1)) -CHySOjH Ehtirc pH ratigr Iutiocthanol Propylcrie oxide

~~

Gillespie (1963b).

199

T H E CHEMISTRY OF KERATINS

with bromoethylamine was very slow. The faster reaction of SH groups in proteins with ethylenimine (Raftery and Cole, 1963) may be worth studying in this connection. Moving boundary electrophoresis and chromatography on DEAE-cellulose indicate that SCMKB contains a large number of proteins-at least eight being detectable (Gillespie, 1959, 1963b). These components differ in charge owing to differences in sulfur content and, consequently, in concentration of introduced S-carboxymethyl groups; they also differ in size and amino acid composition. Preparation of the high-sulfur derivative SCMKB1 (Gillespie, 1962c) is summarized in Fig. 3. A second high-sulfur derivative SCMKB2 is “yellow recovered by dissolving the fraction described as precipitate oil” in Fig. 3 in 1 yoNaHC03 and fractionating as shown in Fig. 4. d. Eflect of Temperature and Ionic Strength on Extraction of Wool Proteins. To minimize changes in conformation during extraction, the reduced highand low-sulfur proteins may be extracted separately from wool at a low temperature as outlined in Fig. 5. The high ionic strength achieved by the use of 0.8 M thioglycolate allows preferential extraction of the highsulfur proteins by decreasing the solubility of the low-sulfur group (Gillespie, 1962a). The fraction of low-sulfur proteins is then recovered from the residual wool by urea extraction if changes in conformation are unimportant, or by plasmolysis (Harrap and Gillespie, 1963). The protein extracted in 0.8 M thioglycolate at 0°C accounts for 17 % by weight of the wool; at 40°C under similar conditions 26 % of the fiber is dissolved. The products obtained a t these temperatures are indistinguishable by physical methods. The total protein extracted with 0.8 M thioglycolate followed by urea extraction or plasmolysis corresponds to 70 % of the wool. The amount of protein extracted from wool with 0.1 M thioglycolate is greatly increased by raising the temperature from 40” to 50°C (Gillespie and Lennox, 1955a). Temperature presumably affects the extraction by changing the state of aggregation or conformation of the wool proteins, especially the low-sulfur group, or by affecting the extent of reduction or the membrane permeability in the fiber. The amount of protein extracted by solvents such as thioglycolate is greatly decreased by preheating the wool (Lennox, 1956) especially in alkaline solution (Lees and Elsworth, 1956). e. T h e E j e c t of Urea and Sulfuric Acid Pretreatment. Urea has frequently been added to reducing agents and bisulfite solutions to iiicrease the solubility of keratins. The addition of 8 M urea t o 0.2 M thioglycolate a t pH 9 has an effect similar to raising the pH value of the thioglycolate solution to 11 when extracting a t 50°C. Both extractants remove approximately 85 yo by weight of protein from the fiber (Harrap and Gillespie, 1963).

+

200

W. G . CREWTHER, R. D. B. FRASER, F. G . LENNOX, AND H. LINDLEY

Preparation of High-S Wool Protein, SCMKB 1 - Low Ternuerature Method

Extract for 18 hr at 0°C with 1500 ml 0.8 M thioglycolate at pH 1 0 . 2

Adjust to pH 5 - 6 with 50 ml glacial acetic acid

SCMKA may be prepared from this as described in a separate chart

I Supernatant Discard

I Precipitate Dissolve by homogenizing in 1 liter 0.1 M thioglycolate at pH 9.0, alkylate with 40 gm iodoacetic acid and add A a slight excess of thioglycolate. Adjust to pH 6 . 0 - 6 . 2 with acetic acid. Add (NH,),SO, to concentration 1. 6 M. Centrifuge for 1 h r at 40,000 g. I

I

Precipitate + "yellow oil" Supernatant contains SCMKB 2 and minor Adjust pH to 4.0 and components. Fractionation increase SO, in a separate concentration to

A ck?A?;-ibed -

Prec pitate Dissolve in 1%NaHCO,, dialyze, chromatograph on DEAEcellulose column a t pH 4.5 in 0.01 acetate with NaCl gradient 0 to 0. 65 M, collect eluate between 0.45 M and 0.54 M NaC1, dialyze to remove salt and freeze-dry

n

2.0 M

I

Supernatant Discard

SCMKB 1

FIQ. 3. Fractionation of wool proteins at 0°C to give high-sulfur fraction SCMKBl (Gillespie, 1962~). The electrophoresis diagrams refer to runs in acetic acid-sodium acetate bufTer at pH 4.5.

20 1

T H E CHEMISTRY O F KE R A TI N S Preparation of High-S Wool Protein, SCMKB2 - Low Temperature Method Precipitate

+ "yellow

oil" p o r n SCMKB 1 preparation Dissolve in 1%NaHCO,, adjust pH to 7.8, add (NH,),so, to concentration

-

in glycine and tyrosine (electrophoresis in P-alanine-NaOH buffer a t pH 11)

Adjust pH to 6 .0 - 6.2 and adjust (NH,)$O, concentration to 1.6 M

1

Ibcipitate

-

Supernatant

Dissolve in 1% NaHCO, and reprecipitate by adjusting (N&),SO, concentration to 1 . 6 M , dissolve in 1% NaHCO,, dialyze against tap and distilled water, dilute to 0 . 5 % protein concentration, cool to O"C, add equal volume 0. 1 M H,PO, a t O"C, hold for 4 h r at 0°C

Discard

Dissolve in I% NaHCOJ, dialyze against tap water, reprecipitate with equal volume 0 . 1 M H,PO, a t 0°C

minor high-S components

A __L

I

I

I

1

Dissolve in 1%NaHCO, and recover the fraction precipitating between 1 . 4 5 and Repeat this precipita1.8 M (NH,),SO,. tion I

I

Precipitate SCMKB 2 which may be further purified by chromatography on DEAE-cellulose

Discard

I

Supernatants Discard

FIG. 4. Fractionation of wool proteins a t 0°C to give high-sulfur fraction SCMKB2 (Gillespie, 1963a). The electrophoresis diagrams refer to runs in acetic acid-sodium acetate buffer a t pH 4.5 except where otherwise indicated.

The aniourit of protein extracted from wool or hair with thioglycolate a t pH 10 can be increased by pretreatment with concentrated sulfuric acid (Lustig and Kondritzer, 1945). Some fractionation of the protein extracted from the cortex was achieved by Lustig et al. (1945). f. Reduction in Neutral Solution. Several methods have been described for extracting protein from keratins following reduction in neutral solution. Urea-thioglycolate was used for this purpose by Jones and Mecham (19431944) when comparing the proteins from a variety of keratins. Thompson and O'Donnell (1962b) reduced wool proteins in neutral solution and then

202

W. G . CREWTHER, R. D. B. FRASER, F. G. LENNOX, A N D 13. LINDLEY

Preparation of Mixed Low-S Wool Proteins, SCMKA - Low Temoerature Method

2 gm Merino wool top Extract for 18 h r at 0°C with 60 m l 0 . 8 M thioglycolate at pH 1 0 . 2 I Residue

I

Homogenize a t 0 ° C in 19 nil water containing 30 gm u r e a in micro stainless Waring blendor. Akylate by adding 2 gm iodoacetic acid adjusted to pH 9, add slight e x c e s s thioglycolatc and dialyze

E x t r a c t SCMKB repres e n t s 17% of w o i , can b e used for preparation Or plasmolyze by t r a n s of SCMKB 1 and SCMKB 2 ferring to 400 ml disas described in s e p a r a t e charts tilled water a t 2"C, homogenize and alkylate as in u r e a method

1

I I

1

Residue

Supernatant Adjust pH to 4 . 4 and

Discard

to 0 . 5 with acetic acid

- sodium acetate

Precipitate SCMKA

Urea extraction niethod

Superiratant contains t r a c e s of h i g h 4 proteins. Discard

(Electrophoresis i n acetic acid - sodium acetate buffer a t pH 4. 5)

Plasmolysis method

Dissolve in 30% acetic acid and p a s s through Sephadex G-75 column

I

F i r s t fraction, purified SCMKA

I

Second fraction contains proteins r i c h in glycine and tyrosine

FIQ.5. F r a c t i o n a t i o n of wool proteins at 0°C to give low-sulfur fraction SCMKA and Gillespie, 1963). The electrophoresis d i a g r a m s refer to runs at pH 11 in p-alanine-Na0H buffer unless otherwise indicated. (Harrap

T H E CHEMISTRY O F KERATINS

203

alkylated and extracted them a t p H 11. The protein extracted in this way was equivalent to about 75 % by weight of the original wool. Separation of the low-sulfur from the high-sulfur protein fractions was effected by repeated precipitation of the SCMKA a t pH 4.4 in the presence of 0.5 M KCl (Gillespie et al., 1962; O’Donnell et al., 1962) using 5 N acetic acid or concentrated citrate buffer and finally dissolving a t p H 9 in alkali buffered with dilute borate solution. Gradient elution and stepwise elution of the SCMKA preparation on DEAE-cellulose in 8 M urea-tris buffer a t pH 7.4 revealed heterogeneity similar to that described previously for another low-sulfur wool protein preparation (O’Donnell and Thompson, 1961). In a study of the reducing action of various thiols at 0.1 M concentration in aqueous solutions of alcohols (Maclaren, 1962), benzyl mercaptan was found to be the most effective. Maximum reduction exceeding 90 yo was obtained in 20 % propanol solution, and under these conditions the wool remained intact. If 5 M NaI was incorporated in the solution approximately 65 yo of the protein was extracted in 48 hr a t 20°C under essentially neutral conditions (Maclaren, unpublished observations, 1962). This protein could be alkylated with iodoacetate and fractioned into lowsulfur and high-sulfur fractions using the methods of Gillespie. I n aqueous solution phosphines reduce the disulfide bonds of wool. Almost complete reduction can be obtained in a single treatment at pH values above 4 using solutions of tris(hydroxymethy1)phosphine or tris(diethylaminomethy1)phosphine (De Deunvaerder et al., 1964). Following reduction with substituted phosphines SCM groups can be conveniently introduced into wool by shaking with 0.1 M iodoacetate a t p H 8. Extensive reduction and extraction of wool proteins can also bc obtained using phosphines dissolved in fornianiide. Partial acid hydrolysis using 6 N HCI a t room teiiiperature has been used to release cortical cells as the initial step in the preparation of wool proteins. The product has been subsequently reduced and extracted with 0.5 M mercaptoethanol4.2 M NaCl a t p H 8 (Ward and Bartulovich, 1956) or reduced with 0.5 M niercaptoethanol and then alkylated in an inert atmosphere using acrylonitrile to give the S-cyanoethyl derivative (Tomimatsu el al., 1950; Bartulovich et al., 1960). The alkylation method gave a protein fraction of molecular weight 30,000 that comprised 20 yo by weight of the wool. Like the SCMKA proteins and a-keratose, it contained less sulfur than the original wool or the cortical cell residues. The high-sulfur fraction was apparently lost by dialysis or through failure to precipitate a t pH 4.8-5.1. Cortical cells released from wool by digestion with pepsin have also becw used as a source of SCMKA proteins (Biscrte and Moschetto, 1962). The proteins were separated into two fractions

204 w.

G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

by passing a solution in 8.3 M urea and 0.035 M thioglycolate through a Sephadex G-75 column. g. Reduction in Acid Solution. Savige (1960) has shown that acid solutions of thiols a t low ionic strength extract proteins from wool. Almost complete solution of the wool occurred when treated with 9 M thioglycolic acid for 24 hr a t 60°C. No free amino acids were detected in the acid thiol extracts, nor was any significant increase in the concentrations of free amino groups observed in the extracted proteins or in the residual wool; however, further evidence is needed to exclude all possibility of peptide bond hydrolysis. h. h’lectrolytic Reduction. Recently, Leach et al. (unpublished observations, 1961) showed that the disulfide bonds of wool can be reduced electrolytically in 0.075M mercaptoethanol at p H values 7-10. The mercury cathode was maintained a t - 1.3 volts using a n automatic potentiostat (Wood, unpublished data, 1961), and the progress of reduction was followed by amperometric titration. Complete reduction was achieved at pH 7 in the absence of denaturants. i. Reduction with S o d i u m Borohydride. Because sodium borohydride contains no sulfur, it is unlikely to form addition compounds with wool and would be expected to be highly satisfactory as a reducing agent. When this reagent was applied to either wool or bovine plasma albumin (Gillespie, 1959), however, about 30 % of the proteins became dialyzable, indicating peptide bond hydrolysis (Gillespie et al., 1960). A similar conclusion was reached from studies of the reduction of ribonuclease and insulin with sodium borohydride (Crestfield et al., 1963). j . Extraction of Proteins from Wool Roots. Protein fractions similar to those extracted from the fully keratinized wool fiber have been recovered from wool roots harvested by the wax-sheet technique (Ellis, 1948). I n a modification of the original method the short fibers were allowed to remain in the wax and the exposed roots scrubbed with a toothbrush saturated with the extractant. The proteins were extracted from the shorn roots with 8 M urea solution and alkylated with iodoacetate; SCMKA was precipitated from solutions of alkylated wool root proteins by dialyzing against 0.25 M acetate buffer to adjust the pH value to 5.1. The precipitated protein corresponding to most of the extracted protein contained 1.7 % ’ sulfur. The remaining protein was precipitated by adjusting the pH to 3.5 with HC1 to yield a high-sulfur wool root protein fraction SCMKB containiiig 4.1 % sulfur (Rogers, 1959~). Films cast from the root SCMKA gave an a-diagram when examined by X-ray diffraction (MacRae and Rogers, unpublished observations, l M O ) , whereas the root SCMKB pattern itidicated poorly oriented cross-P material (Rogers, 1964). Like the low- and high-sulfur SCMK derivatives

T H E CHEMISTRY O F KERATINS

205

from wool, the wool root SCMKA and SCMKB fractions were heterogeneous by starch-gel electrophoresis (Thompson and O’Donnell, 1964). k . High-Sulfur Protein from Various Keratins. The electrophoretic patterns of the high-sulfur proteins obtained from a variety of different keratins have been measured and compared (Gillespie, unpublished observations, 1964). It was found that the patterns varied markedly for different keratins, e.g., horn or hair, of a particular animal, but were the same

Mobility

1 2 3 4 5 6 7

Dorsel Horn

Merino

Soay

Rornney Marsh

Border Leicesler

Lincoln

Southdown

FIG.6. Moving boundary electrophoresis patterns at p H 4.5 of high-sulfur fractions SCMKB from wools of various breeds of sheep (Gillespie, unpublished observations, 1964).

206 W.

G. CREWTIIER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

for corresponding keratins from different animals of the same txeed. Each pattern contained a characteristic set of components of differing mobility; in wool the proportions of the components were found to vary with genetic history and diet (Gillespie, 1964; Gillespie et al., 1964). When the patterns of the high-sulfur wool proteins from different breeds of sheep were compared (Fig. 6) the major components were found to differ considerably in mobility, although the patterns were similar otherwise. Patterns obtained from a similar structure, e.g., hair, from different species of animals showed little resemblance to one another in most instances. The electrophoresis patterns of the SCMKB proteins from cashmere and mohair were similar to those from wool. The SCMKB proteins from hair, porcupine quills, whale baleen, and finger nails, however, gave much simpler patterns than wool, generally consisting of one major component with varying amounts of several others having similar mobilities (Gillespie, unpublished data, 1964).

6. Oxidation a. Peracetic Acid. Preparation of the sulfonic acid derivatives of wool proteins, known as keratoses, by oxidation of the disulfide bonds with peracetic acid and extraction with ammonia solution was first described by Alexander and Earland (1950). The low-sulfur fraction, a-keratose, is precipitated by acidification leaving the high-sulfur fraction, y-keratose, in solution. The insoluble residue is termed P-keratose. The a-keratose ’ of the fiber contains 1.9 yosulfur (Corfield fraction representing about 60 % et al., 1958) and thus corresponds to the SCMKA proteins described in Section II,A,l,b. The y-keratose protein represents approximately 30 % of the fiber and contains 5.8 yo sulfur corresponding to the SCMKB fraction. 0-Keratose proteins are equivalent to 10 % by weight of the wool (Alexander et al., 1951a,b) and contain 2.1 yosulfur. Wool oxidized with peracetic acid contains 14 % less histidine, 6 % less phenylalanine, and 1 yoless tyrosine than the original wool (Corfield et al., 1958), and large amounts of dialyzable nitrogen have been detected in y-keratose (Alexander and Smith, 1956). Thompson and O’Donnell (1959a) found that peracetic acid not only ruptures peptide bonds in the wool proteins, but also fails to oxidize all the disulfide bonds. Performic acid suffers neither of these disadvantages and is therefore preferable. b. Performic Acid. Blackburn and Lowther (1951) used performic acid to oxidize completely the disulfide bonds of wool and showed that some protein dissolves in the reagent. Thonipson and O’Donnell (1959a) showed that ammonia extraction of the oxidized wool yielded 75-82 yo of the weight as a- and y-keratoses compared with 74 % for wool oxidized

THE CHEMISTRY OF KERATINS

207

with peracetic acid. Using chromatography on DEAE-cellulose columns in buffers containing 8 M urea, O’Donnell and Thompson (1961) showed that a-keratose and y-keratose from wool oxidized with performic acid were heterogeneous. Stepwise elution of a-keratose with 0.1 M , 0.2 M , and 0.5 M KCl gave three arbitrary fractions which showed significant differences in their contents of cysteic acid, basic amino acids, and amino acids absorbing strongly at 276 mp. These differences were confirmed by more detailed amino acid analysis (Thompson and O’Donnell, 1962a). Peptide maps obtained by two-dimensional high-voltage electrophoresis of enzyme digests of the arbitrary fractions, however, failed to reveal any differences. The peptide maps of the enzyme digests of a-keratoses from Lincoln and Merino wools and of a low-sulfur protein preparation from wool roots were also remarkably similar. Heterogeneity in y-keratose prepared by performic acid oxidation and extraction with pH 6 buffer was demonstrated by .Haylett et al. (1963) on both a charge and a molecular size basis using moving boundary electrophoresis, column chromatography, and gel filtration. The heterogeneity of a-keratose prepared by the peracetic acid oxidation method has been revealed by solubility curves and chromatography on hydroxylapatite columns and attributed to peptide bond fission during preparation (Corfield, 1962, 1963). Separation of a-keratose from y-keratose following oxidation with performic acid was facilitated if the proteins were treated with warm formic acid solution, trichloroacetic acid, or alkali at pH 11 (O’Donnell and Thompson, 1962). It was suggested that contamination of a-keratose, prepared by the performic acid method, with a high-sulfur protein, termed z-keratose, was partly responsible for the chromatographic heterogeneity and variation in amino acid composition of the low-sulfur proteins. These two types of protein may be bound together by secondary valence bonds. The product obtained by treating a-keratose for removal of x-keratose has been designated (a-z)-keratose. x-Keratose so obtained is rich in glycine and tyrosine and may therefore correspond to one of the highglycine protein fractions separated in the studies of the reduced protein derivatives. It was not possible to remove all the z-keratose with trichloroacetic acid. Like the SCMKA and SCMKA2 preparations, the oxidized and purified low-sulfur protein fraction (a-x)-keratose is resolved into a number of bands by starch-gel electrophoresis in 8 M urea. 3. Methods Employing Bisulfite a. Urea-BisuEfite. This reagent has long been used for the preparation of protein extracts from wool. The amounts of protein extracted from various keratins with 0.3 M NaHSOr10 M urea solution during 18 hr at 40°C were 80 yo for chicken feathers, 50 yo for wool, and only 5 yo

208 w.

G. CREWTHER, R. D. B. FRASER, F. G . LENNOX, AND H. LINDLEY

for ovokeratin (Jones and Mecham, 1943-1944). A concentrated aqueous solution of urea containing 5 yo sodium bisulfite, adjusted to p H 8, extracted about 20 % of the protein from wool during 24 hr a t 50°C (Mercer and Olofsson, 1951). Extraction of wool with 8 M urea and 4 % sodium bisulfite for 2 days at 50°C also removed about 20 % of the protein (Woods, 1952). Asymmetrical fission of the disulfide bonds in wool, as with bisulfite, adds a n unwanted degree of complexity to the products. b. Oxidative Suljitolysis. Symnietrical fission of the disulfide bonds is achieved if the action of sulfite is supplemented with some oxidizing agent such as cupric ions (Swan, 1957b), tetrathionate (Bailey, 1957), or atmospheric oxygen (Leach and Swan, 1962, 1963). Under these conditions each SS bond yields two SS0,- groups. The S-sulpho derivatives of the wool proteins formed by the use of either cupric ions or tetrathionate can be extracted with slightly alkaline solution. If the copper oxidation method is used however, it is necessary to remove all traces of this strongly bound metal from the extracts before fractionation and this is difficult. The electrophoretic properties of the S-sulphokerateine A fraction have been studied by Woods (1959). The SS0,- groups produced by oxidative sulfitolysis are interconvertible with other groups such as SH and SCN.

B. Hydrolyzing Peptide Bonds The method of extracting protein material from wool by splitting the peptide bonds while leaving the disulfide bonds intact has attracted some attention. Such a procedure might retain the original conformation of the amino acid chains if disulfide interchange during hydrolytic extraction could be avoided. Biserte and Pigache (1951, 1952) found that partial hydrolysis of wool with sulfuric acid released aspartic acid preferentially together with high molecular weight compounds. The sulfur content of the residue increased as hydrolysis proceeded. In a recent application of the acid hydrolysis method (Blackburn, 1959, 1962) Merino wool was heated for several hours a t 65°C in 2 N HC1. The product was filtered and washed with distilled water, and the protein extracted a t p H 10.5. After filtering off the insoluble matter the alkaline extract was acidified with acetic acid to yield Component 1. After dialyzing the suspension for 24 hr Component 1 was recovered by centrifuging. The supernatant was concentrated in wcuo and Component I11 precipitated by the addition of ethanol. Amino acid analysis showed close resemblance between Component 1and the lowsulfur proteins, a-keratose and SCMKA2, and between Component 111 and the high-sulfur proteins, y-keratose and SCMKB1. Moreover, oxidation converted Component 1 t o a product resembling a-keratose together with a very small amount of material resembling y-keratose, whereas the oxida-

T H E CHEMISTRY OF KERATINS

209

tion product of Component 111 resembled y-keratose. This work recalls the experiments of Lindley (1947) who treated wool with 0.05M cetyl sulfonic acid for 6 days a t 65°C to hydrolyze the wool and extracted the fiber with dilute alkali. Approximately 70 yo of the fiber was extracted and portion of this was precipitated with acid. The residue from the extraction was very rich in cystine compared with the original fiber. Drawing on previous experience of conditions leading to specific release of aspartyl residues from proteins (Leach, 1956a), Leach et aE. (1964a) extracted Merino wool with boiling hydrochloric acid a t pH 2 for four consecutive 24-hr periods to give protein extracts containing disulfide bonds. After digestion for 72 h r approximately 75 % by weight of the fiber was removed; 50 yowas of sufficient molecular size to be nondialyzable. Protein fractions obtained from this extract were very similar in amino acid composition to a-keratose from the same wool. The amino acid composition of the nondialyzable material was similar to that of the dialyzable material from each fraction.

C . Origin of Extracted Proteins The various methods that have been used to extract soluble derivatives from wool all lead to the conclusion that wool is heterogeneous and contains two main groups of proteins which differ markedly in chemical composition and physical properties. Structural studies also indicate a heterogeneous texture in which microfibrils are embedded in an amorphous matrix, and it is generally believed that the low-sulfur group is derived from the microfibril and the high-sulfur group from the matrix. This view is supported by the observation that the low-sulfur proteins are fibrous and yield an a-pattern when oriented films or fibers are examined by X-ray diffraction, whereas the high-sulfur proteins yield only diffuse halos indicating a n amorphous structure. Rogers (1959a) was able to demonstrate that the high-sulfur proteins extracted from oxidized wool by neutral buffer originated, a t least in part, in the matrix. In addition there is a considerable amount of indirect evidence, which has been summarized by Mercer (1956), Rogers (1959b), Filshie and Rogers (1962), and Lundgren and Ward (1962), to support this hypothesis, although much of it could be explained in other ways. Perhaps the best evidence for associating the low- and high-sulfur proteins, respectively, with the microfibrils and matrix is that wools from sheep receiving cystine supplements through an abomasal fistula contained up t o 50 yo more high-sulfur protein than wools from sheep in a control group, and a large increase in the amount of matrix and in the intensity of matrix staining could be seen with the electron microscope (Gillespie et al., 1964). The more recent studies of microfibril and matrix structure described

210

W. G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

in Section V indicate the complexity of both components arid emphasize the need for further electron microscopy studies of extraction products. It would be unwise to assume, for example, that the microfibril is entirely composed of low-sulfur protein without direct evidence. A common finding in studies of the origin of the extracted proteins has been a marked difference in the rate or degree of extraction between the orthocortex and paracortex. In most instances the orthocortex was more readily attacked than the paracortex. Studies using the light microscope played an important part in detecting these differences, but the detailed conclusions were often highly speculative. More recent studies using the electron niicroscope to examine the wool fiber at various stages during treatment with boiling acid (Leach et al., 1964a) showed that a major part of the extracted material originated in the ortho segment. Extraction was most apparent at the center of each macrofibril, confirming the observation of Kassenbeck (1961). The residual paracortex could be fractionated into a- and y-keratose coniponents in good yields. These paracortical keratoses were shown to be very similar in amino acid composition t o the a- and y-keratoses from whole wool. Blackburn (1962) suggested that acid extraction of wool to yield protein having amino acid composition similar to the low-sulfur proteins SCMKA and a-keratose may indicate that the low-sulfur proteins and high-sulfur proteins in the intact orthocortex are not linked together through disulfide bonds. This is based on the assumption that disulfide interchange does not occur during acid treatment or extraction. No evidence for this has been provided yet. It has been shown by De Deurwaerder et al. (1964) that a protein fraction rich in glycine, tyrosine, tryptophan, and phenylalariine can be extracted from wool with a solution of tris(diethylaminomethy1)phosphine in formamide. Examination with the electron microscope showed that this material was derived mainly from the membrane complex between the cuticular cells; the microfibrillar texture of the cortex appeared to be intact.

D. Some Properties of Wool Protein Fractions 1. Molecular Weight

The wide range of molecular weights reported for derived wool proteins reflects varying degrees of degradation during extraction from the fiber, difficulty in separating individual protein species from other proteins in the extracts, especially in the low-sulfur protein group, and varying degrees of aggregation. The amount of aggregation is markedly affected by the pH value, the presence or absence of disaggregating agents, such as urea, dodecyl sodium sulfate, and formamide, and whether the protein is a t the surface or in the bulk solution. The mixed high- and low-sulfur proteins extracted from wool with

T H E CHEMISTRY OF K E R A T I N S

211

sodium sulfite solution (Olofsson and GralBn, 1947) or following oxidation with chlorine dioxide (Das and Speakman, 1948) may have suffered peptide bond degradation to give a mean molecular weight of about 8000. Extraction with urea-bisulfite solution gave protein of molecular weight 84,000 a t pH 8, but this value fell to about 8000 when the pH value was increased to 12 (Mercer and Olofsson, 1951). Using the osmotic pressure method, urea-bisulfite extracts of wool were found to have a number-average molecular weight of 12,000 to 16,000 (Friend and O’Donnell, 1953). I n molecular weight studies on the low-sulfur mixed protein preparation, SCMKA, values of 200,000 (O’Donnell and Woods, 195Ga) and 1,000,000 (Harrap and Woods, 1958) were observed for aqueous solutions by sedimentation and light-scattering techniques, respectively, indicating considerable aggregation. Urea or dodecyl sodium sulfate were used to disaggregate SCMKA2 and a-keratose, giving values of 45,000 to 50,000 as calculated from Sedimentation velocity, viscosity, and diffusion measurements (O’Donnell and Woods, 1956a,b). A similar value was reported independently for a-keratose by Peacock and O’Callaghaii (1959). From experiments in which 60 % formamide was used as a disaggregating solvent, sedimentation equilibrium studies showed the molecular weight of the stable polymer of SCMKA in this solvent to be 70,000. This polynier disaggregates on dilution and, although it has not been possible to determine the molecular weight of the subunit in this solvent, an upper limit of 35,000 can be set (De Deurwaerder and Harrap, 1964). None of these values approaches the estimate of 9000 to 10,000 reported for the molecular weight of the low-sulfur proteins from measurements with the surface balance (Harrap, 1955-1957). Spreading a t the air-water interface apparently causes the molecules to unfold, thereby permitting further disaggregation. The molecular weights of the high-sulfur proteins are much easier to determine; these proteins do not aggregate readily and are soluble over a much wider pH range. Sedimentation measurements in the ultracentrifuge, using the Archibald method, and the light-scattering method agree in placing the molecular weight of SCMKBl a t 27,000 (Harrap, 1962) and SCMKB2 at 22,000 (Gillespie and Harrap, 1963). Since the values obtained by the light-scattering method are very sensitive to the presence of large aggregates, these high-sulfur protein preparations are presumably free of such material. There is evidence that the molecular weight of SCMKB proteins increases with increase in sulfur content (Gillespie, 1963b, Haylett et al., 1963). Human (1956) has shown that the ease of forming filaments from protein fractions may be related to the mean molecular weight (Harrap, 1955). Some molecular weights and other properties of SCM kerateine fractions are summarized in Table 11.

TABLEI1 Some Properties of SCM Kerakine Fractions ~

Property Molecular weight Electrophoretic mobility, I 70 (cmz volt-' sec-1) Extinction coefficient El1% cm, 276 mp Helix content (70)" pH of minimum solubility Sulfur content (%) Acetyl groups (approx. mole/mole) Lys (approx. mole/mole) His (approx. mole/mole) Determined Determined Determined d Determined Determined a

Wool SCMKA 24, OOOe

Woo1 SChIKA2

Wool SCMKBl

Wool SChIKB2

Feather rachis SCMK

10,000b 50 ,0ooc - 7 . 3 x 10-5 pH 11.0 S . 6 (approx.)

27 ,OOOd

"2, OOOd

10,100e

- 6 . 7 x 10-5, pH 4 . 5 5.5

-6.1 X 10-5, pH 4 . 5 5.9

Heterogeneous pH 4 . 0 - 6 . 0 7.0

50 4.4 1.5-2.0 1

50 4.4 1.5-2.0 -

Nil 2.9 6.7

Xi1 2.3-3.3 5.3 2

Nil 2.8 2.2

-

-

Sil Nil

0.1 Trace

-

-

by optical rotatory dispersion. from surface balance measurements. from hydrodynamic data. from light-scattering data and the Archibald method. from sedimentation equilibrium data.

-

1 1

1

THE CHEMISTRY OF KERATINS

213

2. Conformation

Optical rotatory dispersion measurements on aqueous solutions of SCMKAl and SCMKA2 gave values indicating that in both cases approximately 50 % of the material is in the form of a n a-helix. The percentage a-helix was calculated from the expression, % a-helix = -bo X 100/630, where bo is the Moffitt-Yang (1956) parameter. When the proteins were dissolved in 2-chloroethanol the helix content increased to about 60 %. The high helix content is consistent with the assumption that these proteins originate in the microfibril. On heating to 70°C or dissolving sufficient urea in the aqueous solution of SCMKA to bring its concentration to 8 M the helix content was reduced to zero, but this change was completely reversible (Harrap, 1963). Similarly, as the concentration of formainide is increased in an aqueous solution of SCMKA, there is a decrease in both the sedimentation coefficient and the helical content up to 50 % formamide concentration. Both of these changes also are reversible. When the low-sulfur protein fractions SCMKAl and SCMKA2 are digested with trypsiii or Pronase, two phases in the reaction can be distinguished corresponding, it has been suggested, to attack on the random coil and on the helical portions of the structure. This view is supported by the observation that the acid-precipitated residue recovered after completion of the first stage of digestion, which represents about 20 yoof the SCMKA, is about 85 yo helical (Crewther and Harrap, 1965). Unlike the low-sulfur proteins the high-sulfur proteins SCMKB1 and SCMKB2 behave as random coils in aqueous solution and very little a-helix structure is formed even when they are dissolved in the helix-favoring solvent 2-chloroethanol (Gillespie, 196213; Gillespie and Harrap, 1963). Absence of a-helix structure in the high-sulfur proteins is consistent with their location in the amorphous matrix of the wool fiber. One of the protein fractions obtained from wool by partial hydrolysis was nonhelical in aqueous solution a t p H 3, 9, or 10.5 judging from optical rotatory dispersion measurements, but in 2-chloroethanol the helix content rose to about 30 yo (Leach, unpublished observations, 1963). Also, proteins extracted from wool by aqueous urea containing reducing agents may be precipitated and drawn into fibers which show an a-type X-ray pattern (Mercer, 1949). Thus, the diffraction pattern and optical rotatory dispersion data show that intact disulfide bonds are not essential to the reformation of trhe a-structure. 3. End Groups

Although a t least seven different amino acids are represented among the N-terminal groups and also among the C-terminal groups detected in

214 w.

G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

the keratin protein fractions, the combined concentrations are insufficient to account for all the expected end groups assuming molecular weights in the range 10,000 to 50,000. The situation is thus similar to that described for the end groups in whole wool (Section 111,C). The existence of cyclic molecules in the protein preparations was tentatively proposed as a n explanatioii (Woodin, 1956; Thompson, 1957). Fractionation of the solubilized keratin, however, into proteins of very different properties was not accompanied by segregation of the end groups into individual fractions (Alexander and Smith, 1956; Thompson, 1957). Because the amino acids occurring as end groups in wool and wool proteins were the same as those created during the conversion of collagen to gelatin (Courts, 1954), it was suggested that they may be formed either by “weathering” on the sheep’s back or during preparation of the derivatives. The N-terminal groups in keratin proteins have been estimated using the dinitrophenylation technique of Sanger (1945). Thompson (unpublished observations, 1963) used the method of Steuerle and Hille (1959) arid Hille (1960) to deterniirie the N-terminal residues of the three keratose preparations included in Table 111. Results similar to those reported in TABLE111 Termznal Amino Acids in Proleins from Feathers and Woola

N-Terminal groups

C-Terminal groupsd

Wool keratoses prepared by performic acid oxidationc

Amino arid

(a-x)Feather keratoseb a-Keratosee a-Keratosef Keratoscg

Wool

Wool SCMKA2

Nil 2 2 3 3 3

Nil 3 4 Nil 3 Nil

~~

Alanine Aspartic acid Glutamic acid Glycine Serine Threonine Yaline Total

1.1 4.5 0.6 1.1 0.6 1.5 1.1

0.1 0.1 0.2 0.3 0.5 1.5

-

0.2 0.2 0.3 0.4 0. 9 1.8 -

Trace 0.2 0.4 0.4 1.0 2.3 0.1

10.5

2.7

3.8

4.4

-

-

Values given in pmole per gram protein. Woodin (1956). Values corrected for destruction of DNP-amino acids during hydrolysis. Values uncorrected. c From Thompson (iinpnblished observations, 1962). d From Bradbury (1958). 8 Extracted at pH 8 and precipitated a t pH 4 with acetate. Extrartcd at pH 1 1 and precipitated a t pH 4 with acetate. 0 Extracted at pH 11 for 24 hr and precipitated with trichloroacetic acid. a

b

THE CHEMISTRY OF KERATINS

215

the table for the (a-x)-keratose were obtained for a keratose fraction prepared by repeated precipitation of a-keratose with trichloroacetic acid. It is apparent from Table I11 that few additional N-terminal groups are formed by increasing the period of extraction a t p H 11 from 3 to 24 hr. Removal of 2-keratose apparently does not involve rupture of peptide bonds (O’Donnell and Thompson, 1962). Gillespie and Harrap (1963) reported that SCMKB appears to contain only one N-terminal amino residue per molecule and that is arginine. Following the suggestion by Thompson (1959) that terminal amino groups in wool might occur as N-acetyl derivatives, O’Donnell et al. (1962) showed that acetic acid in amounts equivalent to 1 mole/20,000 gm wool was present in wool hydrolyzates. The conditions necessary to liberate the acetic acid suggested the occurrence of N- rather than 0-acetyl groups and, since the €-amino groups of the combined lysine of wool are all readily accessible to l-fluoro-2,4-dinitrobenzene, it would appear likely that the acetyl groups are N-terminal. N-Acetyl groups were also found in extracted wool proteins, much higher amounts being found in the high-sulfur proteins. This is in line with physicochemical evidence that the high-sulfur proteins have a lower molecular weight than the low-sulfur proteins of wool. Application to wool and the wool protein SCMKA2 of the method of hydrazinolysis for estimating C-terminal groups also indicated the presence of several end groups but again in very small amounts (Bradbury, 1958). As shown in Table 111, the values for wool and a wool protein fraction are similar, and this suggests that no appreciable peptide chain breakdown occurs during preparation of the protein. This does not support the suggestion of Corfield (1962, 1963) that peptide bonds are ruptured during the preparation of SCM lcerateines and keratoses. The presence of many different C- and N-terminal groups in low concentration in wool protein preparations may be due to the presence of many protein components, the rupture of different peptide bonds in a small number of different protein molecular species, the adsorption of amino acids or peptides on the proteins, or a combination of these possibilities.

4. Arnino Acid Composition The amino acid analyses of the a-, 0-, and y-keratoses, shown in Table IV indicate that y-keratose contains more cysteic acid, proline, serine, and threonine and less alanine, aspartic acid, glutamic acid, leucine, lysine, phenylalanine, and tyrosine than is found in oxidized wool (Corfield et al., 1958). a-Keratose in contrast contains less cysteic acid, proline, serine, and threonine, and more of the other amino acids which are present in low concentration in y-keratose. The amino acid composition of P-keratose is similar to that of wool.

21G w. c.

CIZEWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

TABLEIV llmino Acid Composition of

01-,

y-,

and p-k'eratoseslk

Peracetic: acidb Amino acid Alanine Ammonia Arginine Aspartic arid Cysteir acid Glutamic acid Glycine Histidine Isoleucine Lenrine Lysine Phenylalanine Proline Swine Threonine Tyrosine Valine 11

__

a-Keratose y-Keratose p-Keratose or-Keratose y-Keratose @-Eierat,ose 4 83 10 25 20 8 6 25 :3 72 10 9 5 10 1 24 2 49 7 30 4 60 1 94 2 69 0 70 3 45 2 44 3 '38

2 58 11.05 19.0

1.79 14.5 5.87 4.!)7 I .57 2.14 2.55 I .03 1.15 9.85 9.70 7 46 1 41 4.15

4.84 8.61 17.7 4.75 4.40 7.32 6.53 2.64 2.85 6.17 6 .43 2.35 4.57 7.99 4.40 2.25 4.41

4.44 7.24 20,66 5.48 5.3 9.16 ti.70 1.49 2.02 6.47 4.52 2.2!1 2.88 6.26 3.05 3.17 3.38

Prep:tred from Merino 64's wool. Amino acid N as (1958). From Gillespie et al. (1960).

* From Corfield et al. c

Performic acid?

2.81 6.56 21.62 2.42 14.2 6.37 5.25 1.8ti 2.22 3.21 0.96 1.56 9.66 10.06 7.27 I 50 3.94

4.39 ti.22 25.13 1.13 ti.45 7.91 5.03 3.Iti 1.98 5.24 7.84 1.45 4.40 7.08 3.24 1.17 2.35

yo total N.

The predominantly acidic and basic character of the low-sulfur and high-sulfur proteins of wool, respectively (Crewther and Dowling, 1960a; Gillespie and Simmonds, 1960; Table V), could contribute to strong binding between these two groups in the intact fiber (Gillespie et al., 1960). It has been suggested that the disulfide bonds ill wool are mainly involved in linking together the low-sulfur and high-sulfur proteins (Gillespie and Springell, 1961). This caonclusioii is contrary to that of Blackburii (1962) (Section I1,C). The disulfide bonds inay be concentrated in particular regions since pcracetic acid oxidation has yielded dialyzable peptides rirher in sulfur than the nondialyzable residue (Alexander and Smith, 1'356). The SCM-cysteine content of the SCMKB proteins ranges between that of wool itself and double this value. Compared with wool these proteins contain high concentrations of proline, serine, and threonine, but low concentrations of aspartic acid, glutamic acid, histidine, lysine, and leucine. In general, reverse trends are shown by the aiiiino acid analyses for SCMKA fracations. SCMKB2 and SCM feather rachis are unique in that they contain virtually no histidine or lysine. In this respect they resemble

SCMKA4* Amino acid dlanine Ammonia drginine Aspartic acid Half-cystineg Glutamic acid Glycine Histidine Isoleucine Leucine Lysine ILIethionine Phenylalanine Proline Serine Threonine Tyrosine Valine

TABLEV Amino Acid Comvosition of Fractions from Wool SCMKa SCMKAQC SCh4KBld SCMKB2d

High glycine group

Ic

IIf

I

I1

I

I1

I

I1

I

I1

4.32 8.54 19.53 5.46 4.55 9.49 5.91 1.33 2.46 6.89 5.44 0.37 2.02 2.85 4.90 2.95 2.88 3.98

518 1021 585 655 546 1138 i0Y 53 295 826 3‘26 44 2$3 3.a 588 354 345 47’7

4.93 8.97 21.10 6.37 4.32 10.76 4.52 1.23 2.53 7 . 13 5.34 0.34 1. i 8 2.33 5.89 3.29 2.26 4.32 __ 16.8

59 1 1076 632 764 578 1290 542 49 303 855 320 41 213 279 706 394 27 1 578

2.30 9.33 15.81 0.58 17.80 7.46 4.82 1.35 2.08 1.39 0.i2 Xi1 0.57 9.35 10.40 8.22 1.58 3.19

238 888 398 60 1859 772 497 45 215 144 38 Xi1 59 969 1163 893 164 33 1

2.60 10.28 9.36 0.78 16.41 8.56 6.64 0.04 3.12 1.43 0.03

275 1086 248 82 1734 905 702 1 330 151

1.43 6.91 13.60 2.85 8.11 2.52 18.76 0.99 0.88 4.28 0.44

158 764 376 315 897 279 2075 36 97 473 24

Nil 0.98 8.07 10.41 7.87 1.43 3.00 __ 14.5

Nil 103 853 1100 832 151 317

Nil 3.73 4.39 8.34 3.40 8.89 2.19 __ 15.5

Nil 413 486 922 376 984 242

~

~

1

Nitrogen (yo) 16.8 14 5 Determined on 24 hr hydrolyzates. Prepared from single Merino fleece MW 118 following reduction with mercaptoethanol (Thompson and O’Donnell, 196213). Prepared from top wool MW 148 from Wintoc flock, 1961 shearing (Harrap and Gillespie, 1963). The SCMKA2 previously analyzed (Gillespie et al., 1960) was obtained from an earlier shearing of the Wintoc flock, and the method of preparation did not include the zinc acetate purification step. Prepared from top wool MW 12i from Wintoc flock, 1959 shearing (Gillespie, 1962c, 1963a). e Throughout the table values designated in column I are given as N as yo total N. f Throughout the table values in column I1 are given as rmole per gram. Cystine values reported include SCM-cysteine and a small quantity of cystine formed during hydrolysis.

4 B

E! ;; 2* 0 9

! i

?-

5

5

218 w.

G. CREWTHER, R. D. B. FRASEK, F. G. LENNOX, AND H . LINDLEY

certain protamines (Gillespie, 1963a; Harrap and Woods, 1964a). The high-sulfur proteins extracted with 0.8 M thioglycolate from mutant Merino wool and horsehair contain more SCM-cysteine than nornial wool. The amino acid composition of the corresponding protein from mohair resembles the SCMKB mixture from wool, but those from porcupine quill and whale baleen contain more proline. High-sulfur protein from quill contains less arginine than that from wool. The high-sulfur proteins from human fingernails and hair are richer in SCM-cysteine than are those from wools, but in other respects they are similar (Gillespie and Inglis, unpublished observations, 1963). When the low-sulfur and high-sulfur proteins are removed from the thioglycolate extract of wool a t pH 10.5, a fraction remains which contains a very high concentration of glycine, equivalent to one residue in every four, high concentrations of tyrosine and phenylalanine, and low concentrations of lysine and glutainic acid (Gillespie, 1960; Harrap and Gillespie, 1963, and Table V). Norinal and “copper-deficient” wools, which had been oxidized with performic acid and extracted for 7 hr a t 30°C with pyridine-acetate buffer a t pH 6, yielded soluble arid insoluble protein fractions differing in their content of cysteic acid, asparagine, proline, leucine, arid lysine (Burley, 1960; Rurley and Horden, 1960). The soluble fraction, y-keratose, when prepared from copper-deficient wool contained slightly less cysteic acid than when prepared from normal wool, but the insoluble fractions from the two types of wool were indistinguishable in amino acid composition. I n more recent studies, however, it was concluded that copper deficiency causes a shift in synthesis of high-sulfur wool proteins to components of lower sulfur content than normal but containing high concentrations of aspartic acid, leucine, and phenylalanine (Gillespie, 1964). Because wool grown on sheep which had received cystine supplements via an abomasal fistula showed an opposite trend in protein synthesis, it was suggested that the changes in electrophoresis pattern of wools from copper-deficient aninials may be due to a shortage of cystine in the developing follicle (Gillespie et al., 1964). As shown in Table VI, aniino acid analysis of the wool root SCMKA and SCMKB proteins has shown that they are similar to the corresponding low- and high-sulfur proteins from wool (Rogers, 1964). Although wool root SCMKA contains higher proportions of the avidic and basic amino acids than the wool root SCMKB fraction, the values for aspartic acid, glutainic arid, and lysirie do not differ as greatly for the wool root protciiis as for the corresponding fractions derived from the wool fiber. The cystine content of the wool root SCMKB was more than five times that of wool root SCMKA, a greater ratio than in the corresponding proteins from wool,

219

T H E CHEMISTRY OF KERATINS

but the actual concentration per gram of protein was less. The relatively low cystine concentration in SCMKB from roots compared with SCMKB from wool is consistent with the introduction of cystine into the fiber above the bulb region in the follicle (Downes et al., 1963). Both root fractions, and the high-sulfur fraction in particular, contained high concentrations of phosphorus in the form of ribonucleic acid contaminant. TABLE VI Amino Acid Composition of Wool Root Proteinsa

Amino acid

SCMKA

SCMKB

Alanine Ammonia Arginine Aspartic acid Citrulline Cysteic acid Half-cystined Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Methionine sulfoxide Phenylalanine Proline Serine Threonine Tyrosine Valine

560 1050 560 800

400 1040 440 510

280 1330 440 80 310 770 420 80

1480 920 510 70 270 550 240 30 160 550 740 520 190 460

-

-

190 260 530 570 250 470

-

Free amino acids in wool rootsb

Inner root sheath protein

42.9

440 1200 250 540 280

-

93.3 25.7 38.0 8.1"

296 144 38.4 36.9 63.8 76.2 37.7 50.0 26. 2c 37.7 49.8

-

Trace 1550 460 90 230 700 700 90 60 2 10 240 450 250 170 310

Values given in pmole per gram. Rogers (1964). Ako six unknown peaks (Rogers, 195%). c Incompletely resolved from other amino acids. d Includes SCM-cysteine. 5

b

Inner root sheath proteins have been obtained froin tissue dissected from the roots of rat vibrissae and porcupine quills and then extracted with 8 M urea containing iodoacetate and pH 8 buffer. The inner root sheath proteins (Table VI) and those isolated from the medulla of hair or porcupine quill are unusual in containing citrulline in protein combination (Rogers, 1958, 1962; Allen et al., 1964).

220 w.

G. C I ~ E W T I I E I Zit. , D. B. F R A S E I ~ ,F. Ci. IJCNNOX, A N D H. L I N D L I C Y

6 h t y qf ~acr~rtlizntiott oj 11rru‘/io Acids duririg I<.elraction

It is possible that raceniization of some of the amiiio acids, siwh as cystiiie, seriiie, aiid threotiitie, occurs during extrartioti aiid awouiits for soiii(’of t h c cwinplexity of wool protein frac.tions (Lindley, uripublished obswvatiotis, 1962). Perforniiv a d , however, used iti the preparation of the keratoses did iiot produce racemizatioii iii proteins (Hill arid Sniith, 1957). I t has not proved possible to solve unequivocally the problem of whethcr or iiot the reduction arid alkylatiori procedures used in the preparation of SCM kcratciiics vause raceiiiizatioii. 1,iiidlcy (uiipublished, 1961) has showti that S-c.arboxynictliy1 cystciiie isolated from acid hydrolyzatcs of SCM kcrateines is partially raceiiiizcd as iiicasurcd tmth by direct optical rotation procedures and also by the usc of a C-S lyase cwzytiie whicah is spcc4ir for the L-forin (Sehwitiiiiier arid Kjaer, 1960). Cotitrol cxpcriiiicnts showcd, howcver, that L-S-(,ar~oxyt1iCthylcysteiiie itsdf is partially racciiiizcd on wfiuxing with 5 N acid, arid when allowatire was made for this it appeared that the amount of raceniization attributable to the reduction and alkylatioii procedures was siiiall (less than 5 yo)even whcn the iiiost drastic c~onditioris(pH 12.5 and 50°C) were used to prepare the SCM kcratciiics. Since Scarboxyniethyl cystcirie in pcptidc conibiriation may well racmiizc iiiore readily on acid hydrolysis than does the free aiiiino acid, even this niay be an over-cstimatc, arid it would seem urilikely therefore that raceiiiization is a serious problem in SCM kcrateines as presently prepared. 6.Interaction between Proteins Extracted f r o m Keratin

Coliii (1922) described the fortilation of saltlike complexes between proteins of opposite net charge. This was encountered in the studies by Gillespie in attempting to separate SCMKA (isoelertric point (+a.pH 4.4) froiii SCMKR (isoelcctric point ca. pH 2.9) by precipitating at pH 4. lTiiderthese (.anditions partial coprecipitatioii of these two fractions oc(~irs. It can be eliiiiinated by increasing the pH to 4.4 and the ionic strength to 0 . 3 4 . 5 or by precipitation with zinc acetate a t pH 6, which is outside the interisoelertric region (Gillespie et al., 1962). This coprecipitation cffert has riot t)ceti reported in studies of the separation of a- aiid y-keratoses. Cosoliition, however, has been observed with these derivatives, but if the ionic strength is high, as in the original method of Alexander arid Earland (1050), a-keratose is conipletcly precipitated (O’Donnell arid Thompson, 1959).

E. Protein Derivatives f r o m Feathers Keratin derivatives can be extracted from feathers more easily than from mool. Heating in bisrilfite-dodecylbciizene sodium sulfonate, for c~xaniple,

T H E CHEMISTRY O F KERATINS

221

has been used t o extract 80-90 yoof the protein (Ward et al., 1946). Solutions prepared by extraction with urea-bisulfite and subsequently characterized either in this state or following oxidation with performir acid were shown t o have a inolecular weight of 10,000 (Woodin, 1954; Rougvie, 1954). The concentration of N-terminal end groups, deteriniried by the dinitropheiiylatioii niethod, corresponds to only one-tenth equivalent per mole of soluble feather keratin, arid it was assumed that cyclic structures were present (Woodin, 1956). Studies of the protein derivatives prepared froiii the various inorphological parts of feathers by thioglycolate reduction aiid alkylation with iodoacetate, oxidation with performic acid, and oxidative (a)

-

FIG.7. Elec*troplioresis diagram of SCM derivatives of proteins extracted from feather (a) rachis, (b) calamus, (c) barbs, and (d) medulla (Harrap and Woods, 1964a).

222

\V. G. CREWTHER, R. D. B. FHASER, F. G. LENNOX, AND H. LINDLEY

sulfitolysishave roiifirmed the earlier estimates of molecular weight (Harrap and Woods, 1964a,b). Extracts containing the SCM proteins correspond’ of feather rachis, for example, gave results indicating ing to 85-90 % uniformly sized units of molecular weight 10,400 in the parerit rachis. The presence of 8 M urea failed to produce disaggregation. As with the wool proteins the absence of expected concentrations of N-terminal amino groups was shown to be due to masking by N-acyl groups, so that it is unnecessary to postulate the presence of cyclic molecules. Optical rotatory dispersion measurements have shown that feather rachis protein has a random coil structure in aqueous solution (Westover et al., 1962; Harrap and Woods, 1964b). The hydrodynamic behavior of the protein is also consistent with a random conformation in aqueous solution; however, it is partly converted to the cu-helix form by the addition of certain organic solvents such as 2-chloroethanol (Harrap and Woods, 1964b). SCM proteiiis from the feather rachis, calanius, barbs, and medulla have all been shown to be electrophoretically heterogeneous (Fig. 7) and TABLEVII Amino Acid Composition of Fractions jrom SCMK from Feather Rachisa ~

Amino acid

~~

Unfractionated

PI (5 %

PZ(12.5 %

SCM protein

ethanol precipitate)

ethanol precipitate)

793 957 334 504 762 630 1285 Trace 283 724 13 Trace 302 970 1392 365 66 107 743

596 1014 405 55!) 530 717 I066 50 270 589 115 32 203 765 1051 366 27 1 614

690 1104 370 553 710 70 1 1163 Nil 285 603 6 Nil 344 926 1154 336

SP (unprecipitated Insoluble fraction) residue -

~~

Alanine Ammonia Argininc Aspartic acid Half-cystine Glutamic acid Glyrine Histidine Isolencine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valinc

Proportion of rachis proteins (yo)100

7

124 724

916 967 313 441 688 41-8 1436 Nil 281 887 10 Nil 273 1048 1211 395 94 731

43

50

-

Values given in rmole per gram. Prom Harrap and Woods (1964a).

584 881 444 677 Nil 842 811 120 366 710 450 114 227 428 542 369 232 585

THE CHEMISTRY OF KERATINS

223

to contain a t least five components (Harrap and Woods, 1964a). Ethanol fractionation of the SCM rachis proteins in the presence of zinc acetate yielded fractions of widely differing amino acid composition (Table VII). Two of the fractions, called Pz and Sz, resembled SCMKB2, the highsulfur protein from wool, in containing virtually no histidine, lysine, or methionine. Most of these residues were retained in the insoluble residue. It is noteworthy that while electron microscope studies reveal a microfibrilmatrix structure in feather keratin (Filshie and Rogers, 1962) it has not proved possible to effect separation of the feather proteins into low-sulfur and high-sulfur protein fractions.

111. COMPOSITION OF KERATINS A. Analytical Methods Most of the analytical procedures applicable to keratins have been developed for general protein studies or, if developed specifically for the study of keratin, they are equally applicable to proteins generally. In this section only methods of particular significance in the study of keratins will be discussed.

i. Cystine or DisuZJide Determination Most methods for determining cystine in proteins involve preliminary hydrolysis of the fiber. Under certain conditions of hydrolysis cystine is destroyed (Lewis et al., 1960) or reformed from its derivatives (Maclaren et al., 1960). The development of a method for determining cystine in the intact fiber is therefore of some importance. Leach (1956b. 1959, 1960) devised such a method based on the amperometric methods of Stricks et al. (1954) for thiol determination. Sulfitolysis of the disulfide bonds in the intact fiber is carried to completion by a simultaneous reaction of the thiol groups with mercurial coinpounds, preferably methylmercuric iodide; the excess mercurial is determined polarographically. The method has been used to demonstrate that after oxidation wool and other proteins contain intermediate oxidation products of the disulfide bonds such as the S-oxide and the S,S-dioxide (Maclaren et al., 1959; Crewther and Dowling, 1961a). It has been shown also that during hydrolysis of S-(carboxymethy1)kerateines by refluxing with HCI, S-(carboxyniethy1)cysteine may be extensively degraded with the production of some disulfide groups (Gillespie, 1963a). Crestfield et aZ. (1963) report a similar finding with the 8-carboxymethyl derivative of ribonuclease and show that S- (carboxymethy1)cysteine remains undegraded if all oxygen is removed during hydrolysis.

221 W. G .

CltIC\?"l'HE>R, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

111 iisiiig thcl aiiipclrotiietric method it is essential to eiisure that reaction of the disulfide t)oiid is not limited hy diffusioii and that the protc'>1n ('011-

taitis 110 groups other than thiol capable of reacting with iiiercwials. Fletcher ef ul. (1!,63) have reported that uptake of pheilylniercirric hydroxidc does not twch a definite end poiiit, but continues very slowly for prolonged periods arid Zahn ct a1. (1962) also consider this possibility. 1,cac.h (1960) has not experienced this difficulty with niethylniercuric iodide and statcs that mercuric chloride is also specific in its reaction with thiols if sufficient chloride ion is present to convert Hg++largely to complex ioiis such as HgC13- aiid HgClh. Wronski (1961) arid Nakaniura arid Netiioto (1961) have also developed niethods for rystirie aiialysis depending 011 rcduc%ionto the thiol and reaction with a niercurial. Hereiitly, 1,eaclh el al. (1964b) have developed a iiiicroiiiethod for the deterniiiiatioii of cystiiie based 011 the reaction of thiols with nicrcurial reagents. The insoluble protein is equilibrated with a solution containing 5 radioac6v.e tiicrcwial coinpoutid such as H~;~~~-phenylmercuric acetate or Cl~-iiicttiyliiiercuriciodide in a solution coritairiirig NaHS03 and urea. Thc caoriceiitration of residual tilercurial roinpourid is then deterniined by autoniatic liquid scintillation couiitirig. The iiiethod can be used with as little as 1 iiig of wool and large nutnbers of routine determiriatioris can be performed quickly. 2. Cystetrie or Thiol Determi~ialion

The polarographic method used by Leach (1'360) for cystiiie deteriiiiiiatioii is also applicable in the deterniiiiatioii of cysteine in intact keratin. The reagent coiitaiiis a swelling agent and a iiierwrial, the excess of the latter being deterinined atiiperoriietrically. Burley (1956a,b) has also proposed a method for detrrmining thiol groups in iiitart wool, using a coloriiiietrir procedure. The values obtained by these iiiethods are not always iii close accord, and neither method (mi be considered to give prec*isedata. This limy bc due to the small aiiiourit of thiol preseiit in rioriiial wool saniplcs. Any side reactions of the reagent then assume great relative iiiiportaiicc Zubcr et al. (1'356) determined cysteiiie by rea(*tiiigwool with 1-fl iioro2,4-di1iitroberizeiie, hydrolyzing, separating the diiiitropheiiyl (DNP) derivatives b y high-voltage electrophoresis, eluting the 8-DNP rysteiiie, aiid estiiiiatiiig it caolorinietrically. There are losses during hydrolysis and recovery of the S-DNP c'ysteine which ricccssitate the use of correction factors in methods using dinitrophenylation. In an alternative method the S-DNP cysteirie is separated on a column of nylon powder arid detcrmined caolorimetrirally (Hille, 1960). Also, radioactive l-fluoro-2,4-dini-

THE CHEMISTRY OF KERATINS

225

trobenzerie can be used arid the S-DNP cysteine detcrmiried by a suitable counting method (Zahn et al., 1961b). Zahn et al. (1962) have also devised a method for thiol analysis in which the wool is hydrolyzed for 2-4 hr and the hydrolyzate titrated with phenylmercuric hydroxide. In general, the use of hydrolyzates for cysteine analysis has proved uiisatisfactory (nowling arid Maclaren, unpublished observations, 1963). The method of’ Leach et al. (1964b) described above for disulfide estimation, which is also applicable to thiol determination, iiiay prove satisfactory for dcterriiiriing thiol in intact wool. This method, like all niethods which rely on reactions with niercwrial reagents, does riot distinguish between thiol groups aiid thiolsulfiiiate (disulfidc monoxide) groups (Savige and Maclareri, 1964). It is therefore unsuitable for analyzing oxidized protein material. There is a need for further coniparisoil of available nicthods arid selection or developnieiit of a precise means of detcriiiiniiig thiol groups in the intact fiber. Leavh (1964) has discussed in detail the merits of various niethods for deteriniriiiig disulfide arid thiol groups in protcins. 3. Lnnthaonzne Delermination Sullivan and Rlijai (1959) have described a specific coloriiiietric method for the deterniinatioii of lanthionine based on its reaction with cyanogen bromide. Although the experimental data provided by the authors indicates that the method gives adequate precision with pure lanthionine, the method of Sullivan and Folk (1952), which is based on the same reaction, is reported (Loiiw, 1963) to be unreliable wheii applied to analysis of wool. The niechariisrii of the reaction of lanthioiiirie with cyanogen bromide has not been eluridated. Because cyariogen bromide also reacts with nirthioiiiric (Gross and Witkop, 1962), cystine, and cysteine (Sullivan aiid Mijal, 1959), a careful appraisal is needed. Blackburn and Lee (19564 have deterniiried lanthioiiine in wool hydrolyzates by chroiuatographir separation on Dowex 50 and developiiieiit of color with Chiiiard’s (1952) acid iiiiihydriri reagrnt. The riiethod has disadvaiitagcs. It is time corisutiiing, lanthioiiine appears as two overlapping peaks owirig to the presence of two diastereoisomers, arid the assumption is made that other aniirio acids do not interfere with color development. This assumption is not correct for all amino acids (Chinard, 1952; Dowliiig arid Crewther, 1!)64c). 1)ecroix at id Rlaziiigue (1958) oxidizrd latithioiiiiio to the N-oxide by rractioii with I120a, separated the oxide oil paper by developing with butaiiol :acetic arid :water for 4 days, eluted, and determined the lanthioiiiiie %oxide c.oloririietric~allyusing Chinard’s reagent. The method is

226 w.

G. CREWTHER,

R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

not precise (Dowling and Crewther, 1964c) because the extent of oxidation is affected by the presence of metal ions, contact with 0 2 while on the paper, and the presenc‘e of other amino acids. I n addition, the prolonged chromatography required for separation of the S-oxide is inconvenient. Dowling and Crewther (1964,) separated the lanthionine on paper without prior oxidation by developing for 24 hr with pheno1:acetic acid : ethanol :water. After elution the lanthionine was determined colorimetrically with Chinard’s reagent. The coefficient of variation was f 3 yo for amounts of larithionine exceeding 40 pniole per gram protein and increased to about f 1 2 yofor 5 pniole per gram protein.

4. Tryptophan Tryptophan analyses for proteins are difficult because this amino acid is easily destroyed during hydrolysis. The development of color with p(diniethy1amino)benzaldehyde in the presence of nitrite under carefully controlled conditions provides a satisfactory means of determining the free amino acid in an hydrolyzate. The conditions used by Veldsman (1959), Graham and Statham (1960) and Inglis (unpublished observations, 1964) for hydrolyzing the wool were 2-3 hr a t 65°C in 85 % HzS04,7 days in 18 N H2S04a t room temperature, and 1.5 hr at 65°C in 18 N H2S04, respectively. In Inglis’ method the wool is powdered to facilitate hydrolysis. In Miro’s (1962) method the wool is hydrolyzed in concentrated HCI for 2 hr a t 70°C in a sealed tube. The results from these methods are comparable in precision. 5. A m i n o E n d Groups Steuerle and Hille (1959) and Hille (1960) developed a method for the quantitative determinatioii of the N-terminal residues normally present in wool. After treatment with l-fluoro-2,4-dinitrobenzene the wool is hydrolyzed and the ether-soluble DNP derivatives applied to a column of nylon 66 powder and developed with phosphate buffer a t 60°C. The D N P derivatives of aspartic acid, glutamic acid, serine, threonine, glycine, alanine, and valine separate cleanly and can be readily determined in the eluates. Hence, it is well suited to the determination of N-terminal residues in normal animal fibers. In its present form, however, it is not suitable for general use with proteins or modified wool fibers as some DNP derivatives, such as those incorporating two D N P groups, are bound so strongly by the nylon that they cannot be eluted. 6. Ionized Busic Groups

Maclarcn (1960) has used the observation (Steinhardt and Harris, 1940a,b; Speakman and Elliott, 1946) that the maximum uptake of acids is practirally unaltered by the nature of the anion, to develop a method

THE CHEMISTRY O F KERATINS

227

for determining the content of ionized basic groups in wool. He used formic acid as solvent for the dye Orange I1 in order to hasten the attainment of a n equilibrium and determined the residual dye in solution colorimetricalIy. It is noteworthy that whereas Orange I1 is adsorbed from concentrated aqueous solutions in amounts greater than the number of equivalents of basic groups (Goodall and Hobday, 1939) this does not occur with formic acid as the solvent.

B. A m i n o Acid Composition During the last decade manual and automatic column chromatography (Spackman et al., 1958; Simmonds, 1958a; Woods and Engle, 1960; Corfield and Robson, 1962; Inglis, 1964), supplemented in some instances by special methods for certain amino acids, have been extensively used for determining the amino acid composition of keratin hydrolyzates. These techniques, together with microbiological and paper chromatographic methods, have shown that animal fibers vary in composition as a result of genetic, nutritional, and environmental differences. Some insight into the changes in the structure of the fiber owing to these differences has been afforded by analysis of the constituent proteins and by assessment of their proportions in the fiber (see Section II,D,4). Hydrolyzates of feathers and their constituent parts have also been analyzed, but there are fewer comparative data for this material. Skin keratin, horny materials, and the hard keratins of birds and reptiles have received little exact analytical investigation, although several studies of skin proteins have been reported. Difficulties in isolating the various skin structures and in defining the material studied have not encouraged precise analysis. Keratinized tissues contain several types of cells and these cells in turn contain many protein constituents. It is possible therefore for keratins to differ in amino acid composition either because the constituent proteins differ or because of differences in the relative amounts of identical proteins. It is rarely possible therefore to relate differences in over-all amino acid composition to differences in character or content of a particular protein constituent. 1. Animal Fibers

a. Whole Wool, There is good agreement, in general, between the amino acid analyses of hydrolyzates of wool obtained by column chromatography and by microbiological assay (Table VIII). Differences in the results obtained by Simmonds (1954) and Corfield and Robson (1956), both using manual column chromatography, and O’Donnell and Thompson (1962) using a n automatic analyzer may not be due solely to differ-

A vimo

‘I’\BLE V I I I Corripositzon of WOOF

Actd

hustralian Merino 64’sh 0’l)onncll and ‘l’hoinpsorr hrlllno :tcld

Ahnine Ammonia Arginirit: Aspartic arid Cgstine Glutarnic acid Glgcinc, Hist idinr Isolcuciiic~ J,f~uclnc

Lysinc Methioiiiiic~ Ph~nylitlniiiric Proline Stxrine Thrriitiinc~ Tryptophnii ‘I’yrosirrv Vrilinv

(IWL?)r

4.20 (7.0%) 20.16 4.!)4

(8.67) 8.59 (i. ti!)

I .S7 2.(il 5.!J2

4.55 0 .:39 2 , ‘LO 4.61 (7 . 3 3 ) (4.63) ( 3 . 12) 4.49

3.51

(7.46) 20 . 3 4.21 7,03 8.58 5 . so 1 .1ti 1 ,!)7 1.!JO :3.25 0 . YO

1.75 5 .: 3 3 7.25 4.61 1.73 2 . !)T 3.57

[L

Vslurs given as N as

1 ti . (i2

It(,flI1S

RoHiis

2 &hr

I6 hr

-

4.38

21.1 4.7

7 .:30

!) !)

8.48

0 .2

6.2!) 1 .!)I 2.44 5.85 :3,!)2 0.32

-

.

I .s :3 , o 5 .:< :3

-

-

Ward et al. (1955)“ 3 .5 5.8 18.9 4.3 7.1 8 0 (i. :3 1 .(i 2 .6

[8.68]

-

[5.12] (0.82) 2 . fi2

4.8

5.2 :I.8 0.1 1.8 5.0 6.2 4.9

2.7 4.2

2.0 4.5

0.4 2.1 (i. I

p.191

5.05

4.16

-

1.18

17.05

-

4.12 6.73’ 19.1

0.71 R I r t h o r l of‘ Hgtlrolysis

__

Corfirld and Graliain Sirninoutls Itohson et al. (1054)({ (1!)55,195!i)d (lOIO)p

Mrans of eight wod samples

Itc.fliis 24 hr

Yfit,ot:tl N.

Itr~firis ti N HCI, 3 1 hr in st.alr4 8 N HCI tutw at X hr

-

1 18°C:

Niimtiors i n parcntlirses indicate that, the valiies were ol)taint:d by extrapolation t,o m’ro tirnt, of hytlrolysis; niimbcrs i n square tirac*kctsindicate that a cwrrection was :Lpplied fc ir thst riict ion during hydrolysis. c

d 0

1)etr.rinind I,y :tiitonintic an:tlyzc.r. 1 )c~tc~rrniiic~l tiy msnual column chromst,ogr:ti,hy. I)ctrrininotl 1)g r~iicrol)iolo~ic~:11 ass:ty. Hytlrolyzrtl 5 hr iii 2 .I HCI ’ i i i i d e r rcflus.

229

THE CHEMISTRY OF KERATINS

eiices in the wool analyzed. The different methods used to correct for dcstruc*t,ioii of serine and threoiiine duriug hydrolysis iiiay be partly responsible for differences in the values reported for these aniiiio acids. The aninioiiia coiiteiits for all five analyses reported in Table VIII are somewhat higher than the values for aniide content obtained by Leach and Parkhill (1956) by hydrolysis under carefully controlled conditions (Table IX). TABLEIX Amzde Content of WOOF

a

WOO1

‘I’otal Nb

hmide content’

Lincoln 36’s Crosshrrd 50’s Corrirdale 56’s Merino 64’s Merino 70’s Merino 88’s

16.58 16.52 16.60 16.57

6.84 6.43

16.33 16.39

6.10

6.16 6.30

6.3s

From Leach and Parkhill (1956).

* Value given as yo dry weight; mean 05 % confidence limit 0.30. c

Value given as N as

70 total

N; mean 95 % confidence limit 0.26.

b. The Sulfur Balance for Wool. Siminonds (1956) obtained a satisfactory sulfur balance for wool when cystiiie was determined as cysteic acid by the method of Schram et al. (1954) and both “hydriodic acidreducible” sulfur and tnethioiiine were included in the sulfur balance. A micromethod for cystirie determination based on the Shinohara (1935, 1936) method, however, gave values for total sulfur in amino acids 17 yo higher than the results of sulfur analysis or1 wool. Determination of cystine as such after elution froni a chroniatogi-aphit. colunin (Sininionds 1054) gave values about 28 lower than the results of the micro-Shinohara method. Corfield and ltobsoii (1956), on the other hand, obtained a satisfactory sulfur balance by combining analyses for inethionirie with the results of cystiiie analysis by the Shinohara method, but considered this to be suspect in view of the known degradation of cystirie during hydrolysis. Earlier, Cuthbertsori and Phillips (1945) had reported a similar result for analyses on wool, whereas Lindley (1948), using similar methods, found major discrepancies for other keratins such as calf hair and cow hair. Wibaux et al. (1960) list numerous reports of discrepancies in sulfur balances for wool. They determined sulfur in untreated and chemically modified wool samples by three methods with satisfactory agreement. To obtain a sulfur balance, cystine was determined by the Shinohara (1936) method, cysteic acid by the electrophoretic method of Bauters and

230 w.

G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

van Overbeke (1959), lanthionine by the method of Decroix and Mazingue (1958), total sulfate by the method of Hille et al. (1958), free sulfuric acid by the method of Barritt (1935), and bromine-oxidizable sulfur by the method of Blumenthal and Clarke (1935). The methionine value of Simmonds (1956) was assumed to be correct. If the value for “bromineoxidizable sulfur” was excluded, satisfactory balances were obtained with untreated wool but not with modified wool samples in which the cystine content had been changed. This was attributed to the formation of unknown sulfur-containing products by degradation of cystine. La France et al. (1960) have in fact detected acid sulfates of seririe and threonine in carbonized wool, one class of damaged wool used by Wibaux et al. Wibaux et al. (1960) have thus confirmed the conclusion of Corfield and Robson that if the Shinohara method is acceptable for determining cystine in wool a satisfactory sulfur balance is obtained. Earland (1961) arrived a t a similar conclusion using the Schoniger (1955, 1956) method for a total sulfur determination. If this is so, the inclusion of “hydriodic acidreducible” sulfur in the balance (Simmonds, 1956) was in error, and the method of Schrarn et a1 (1954) gave low values for cystine. This implies that the deconiposition of cystine during acid hydrolysis (Stein and Moore, 1949; Simmonds, 1954; Corfield and Robson, 1956; Leach, 1956b) gives products that yield as much color with the Shinohara reagent as the original cystine. Lewis et al. (1960) report that cystine is degraded during the acid hydrolysis of wool with the appearance of seven new sulfur-containing compounds, one of which is cysteine. These compounds produce color in the Shiiiohara reagent, although the color yield differs from that of cystine. Fletcher and Robson ( 1962a, 1963) have isolated another of these compounds and identified it as bis(P-amino-/3-carboxyethy1)trisulfide. There was evidence also for the presence of the corresponding tetrasulfide. Fletcher and Robson (1962b) found that S3%ystine incorporated in the 5.7 N HC1 used for hydrolyzing wool samples underwent exchange reactions with the cystine in the fiber and that all the labeled products had the same specific activity. This provided a means for determining cystine in the original wool. A small known amount of radioactive cystine was added to the HCl before hydrolysis, and after elution of the cystine from a suitable column the specific activity was determined. The content of (cystine S cysteine S) was then obtained from the ratio of the total activity before hydrolysis to the specific activity of isolated cystine. The method was unsuited to routine work, but provided an excellent check on other determinations of cysthe. The method gave slightly higher values for (cystine S cysteine S) in wool than a polarographic method using phenylniercuric hydroxide for titrating SH groups in the presence of sulfite

+

+

THE CHEMISTRY OF KERATINS

23 1

(Fletcher et al., 1963). This in turn gave slightly higher values than the Shinohara (1935) method. Excellent agreement was obtained by Fletcher et al. between the total S content of wool obtained by three methods (mean 3.65 yo)and the sulfur content predicted from the analyses for cystine, cysteine, and methionine (total 3.57 %) using chromatographic data for methionine and the radioactive dilution data for (cystine S cysteine s). This work suggests that problems of obtaining a sulfur balance have been due partly to destruction of cystine during acid hydrolysis and partly to uncertainties in the determination of cystine in the hydrolyzates. c. Fibers from Different Strains, Breeds, and Species. Siminonds (1956) used the manual chromatographic method to analyze hydrolyzates of Merino wool derived from different animals and strains of animals. Two animals were used from each of two strains: one strain gave relatively coarse wool (28-30 p ) , the other gave fine wool (15-17 p ) . No significant differences could be demonstrated between analyses of wool from different sites on the same animal. Values for aspartic acid, cystine, glycine, phenylalanine, and threonine differed significantly between wools from individual sheep of the same strain. Tyrosine was the only amino acid showing significant variation between strains. Simmonds (1958b) also investigated the amino acid composition of two Merino wools having similar diameters but differing widely in the number of crimps per inch. Proline, which was determined very precisely, was the only amino acid showing statistically significant differences between the two types of wool, and it was not possible therefore from amino acid analysis to infer differing proportions of para- and orthocortex in the two wools. Synman (1963), on the other hand, reports that high crimp wools contain a greater proportion of paracortex and a higher sulfur content than low crimp wools. I n Table X a comparison is made of amino acid analyses for fibers from different breeds of sheep and different species of animal. With the exception of Lincoln, which was analyzed using the Spinco automatic analyzer (Thompson and O’Donnell, unpublished observations, 1963), the analyses were carried out by Simmonds (1955, 195813) using the manual method. Although the results suggest differences between breeds and species, they cannot be regarded as statistically significant in some instances owing t o the high standard error. It is to be expected that further replication or more precise analyses will disclose significant differences other than those demonstrated by Simmonds (1955, 195813). Human hair shows the greatest variations from mean values having less alanine, leucine, tyrosine, phenylalanine, glutamic and aspartic acids, lysine, and arginine than the other fibers and being richer in cystine and proline. These differences probably reflect a higher proportion of high-sulfur proteins, as it

+

3 0 1-

c3 c1

a m

I-

?A 0

-r c-?

a

-

I.

0

1 0 3

-

I0 c.?

10 3

-

c.? I.

. I

01

m. -I-. ?c. N. c:. m . a,. +. s. '". 0 1. m. .I . m . 01 . c3 _.I .0 .m _ 'C 3

C c 1.0

54

t

15

tm 0

m I.

+

N

m

2

0

.-

2 L n

TIIE C H E M I R T l t Y O F I
233

is kiiowii that hunian hair has a higher niatrix c.oiiteiit than iiiost wool fibers. They would also be in accord with the suggestion of Iluscnbury and Menkart (1956) that huinaii hair caoiitains oiily paracortical c~lls. Rogcrs and Filshie (1963) find a higher proportion of matrix iii para- thaii in orthocorticd cells (Section I,B,2). Sininioiids coninieiits (1958b) that the ready uptake of’ dyes by mohair, which Duseiibury arid Meiikart class as orthocortex, may be due to the presence of a high content of ionizable side-chain groups. As remarked earlier, however, the correlation of differences in atniizo composition with niicrofibril/matrix and ortho/para ratios is complicated by the fact that both the low- and high-sulfur fractions are complex groups of proteins with variable aver-all amino acid conipositioi~s(see Section II,D,4) d. Morphological Components of Wool and the Wool Follzcl~. The papcr chroinatographic. data of Golden et al. (1955) rctiiain thc solc direct analyses of epicuticle arid cortical cell niei~ibranesfroiii wool. Their analyses (Tablc XI) for paracortex arid epicuticle iiiay be compared with the values of Siminonds arid Bartulovich (1958) for “heavy” and “light” cortical cells separated by a density gradient in chloral hydrate solution. The materials analyzed were so iiiipure arid subject to such modification during preparation that only major differeiiccs in analysis are important. The light cortical cell fraction contains relatively greater amounts of glutaniic acid, leucine, lysine, glycine, and alaniiie, but less cystine, proline, and seriiie than the heavy fraction. These differences suggest that the heavy fraction contains a greater proportion of high-sulfur protein than the light fraction (cf. Hogers and Filshie, 1963). Leveau (1959a) has observed siniilar but smaller differencm in dense and less dense cells prepared froin hleriiio wool. Haly and Iiiglis (1964), oii the other hand, have found that the ortho segment prepared by abrading Lincoln fibers is almost identical with whole wool with respect to amino acid analysis. Table XI also compares the aiiiirio acid arialyses of Merino 64’s wool with that of cuticle obtained by subjectiiig the wool to ultrasonic irradiation in 98 yo forniic acid (Bradbury and Chapman, 1964). The cuticle contaiiied less arginine, aspartic acid, glutaiiiic acid, leucine, aiid phenylalanine than whole wool, but inore cystine, proline, serine, and valitie. Cysteic acid is probably formed during the ultrasonic treatment. These analyses cannot be accounted for quantitatively on the basis of a simple combination of low-sulfur and high-sulfur protein fractions, but the data suggest that the cuticle would not contain as much material in the cY-heliLd conforriiation as the original fiber. The values obtained by Bradbury (1960) in an earlier exaiiiination of cuticle-rich inaterial obtained by a iiiechanical descaling technique are in reasonable agreement with thc values in Tablc XI.

234 w.

G . CKEWTHER, R. D. B. FILASER, F. G. LENNOX, AND H. LINDLEY

TABLEX I A wino Acid Compositions of Components of Wool Fibers” Cuticled Original Heavy Light Merino Merino cortical cortical 64’s 64’s Epicuticleb cellsc cell@ wool woold

Paracortex

+

Amino acid

Epicuticlcb

Alanine Ammonia Arginine Aspartic acid Cysteic acid Half-cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine Total accounted for (yo)

34

-

184 1856

-

1124 1909 93

-

146 897 377

-

-

-

1417 1017 377 794 268 542 246

1163 1273 467

-

-

107 867 923 590 198 454

144 678 797 525 226 422

90

66

-

18 217 551 168 179 72

74

-

550 489 640 553

1425 952 359 663 115 1487 999 293 205

61 96

417 547 626 579

81 278 698 296

541

-

469

-

422 600 344 560 64.7 7.2 1426 922 796 1049 852 757 80.2 81.7 222 275 541 676 265 269 28.2 43.5 164 257 886 522 1289 902 478 572 268 349 629 486 96

97

Values given in pmoles per gram. From Golden et al. (1955). c From Simmonds and Bartulovich ( 1958). d From Bradbury et al. (1965).

Table XI1 presents a comparison (Rogers and Simmonds, 1958; Rogers, 195%) of the amino acid composition of hair, hair root, and the inner root sheath from the vibrissae follicles of the rat. The nitrogen recoveries in these analyses were not good, but there is a suggestion that cystine, serine, and threonine were less abundant in the root than in the hair. The inner root sheath on the other hand is unusual in that it contains a large amount of the amino acid citrulline. Its high content of glutamic acid, aspartic acid, and leucirie suggests that the fibrillar material in its structure (Rogers, 1959~)may be largely a-helical. e. E$ects of Nutrition and Weathering. Early studies by Lightbody and Lewis (1929) showed that rats fed on a cystine-deficient diet reverted to the “puppy coat” characteristic of young rats. This hair was shown to contain less cystine and total sulfur than the norma1 coat. On the other hand Barritt et al. (1930) and Barritt and Rimington (1931) found

235

T H E CHEMISTRY O F KERATINS

TABLEXI1 Amino Acid Analyses for Hair, Hair Roots, and Root Sheaths from Vibrissae Follicles" Amino acid Alanine Ammonia Arginine Aspartic acid Citrulline Half-cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Serine Threonine Tyrosine Valine Total accounted for (%)

Hair 393 707 448 435 1139 789 674 84 145 418 212

Hair root 359 707 442 458 613 788 560 90 153 457 219

-

-

163 816 386 243 264

193 638 327 226 248

83.9

74.6

Root sheath 347 236 293 533 349 828 1279 387 65 122 541 191 7 163 371 207 116 154 70.0

a Data given in pmoles per gram, determined by column chromatography (Rogers and Simmonds, 1958).

that addition of cystine to the diet of rabbits had no significant effect on the sulfur or cystine content of the fur. There have been many reports that the diet of sheep affects the diameter and cystine content of the wool produced. Bonsma (1931), for example, found that the sulfur content of wool of veldt-fed sheep varied along the staple, the root sections which were produced under good grazing conditions being higher in sulfur than the middle sections which were produced during lean grazing. Maize-fed sheep, on the other hand, showed no such differences in sulfur content. Louw (1960), who also worked with South African fleeces, found that in normal fibers and in steely fibers from copperdeficient sheep the tip wool contained considerably less cystine and tryptophan, but more tyrosine and cysteic acid, than the middle and root sections of the fiber. His data suggest a general decrease in the sulfur content of the staple from the root to the tip. The steely wool was richer in thiol groups and the tip of this material contained greater amounts of cysteic acid than the corresponding portion of a normal staple. Richards and Speakman (1 955, 1956) observed a similar decrease in sulfur content along the staple, and Human (1958) demonstrated a major decrease in the con-

230 w. G.

CKEWTHER, R. D. B. FI~ASEH, F.

c.

LENNOX, AND H . LINDLEY

tcwt of disulfide plus thiol groups in single fibers on passing from root to tip. Both Louw (1960) arid Richards and Speakman (1955, 1956) attributed their results to weathering of the tip. Nevertheless, Richards arid Speakinan observed a decrease in tyrosine along the staple which conflicts with the results preseritcd by Louw. Unlike 13oiisiiia, Ross (1961) found that wool grown in New Zealarid during periods of high production contained less sulfur than that grown during the lcan graziiig period. He suggested that weathering does riot alter thc total sulfur content of the fiber, although some cystine is ronvcrtcd to vysteic arid as a result of weathering. He also suggested that wool productioii is riot limited by sulfur availability and lists the possible niitritioiial influrncw on thc sulfur content of the wool. The (*onflirtingcaoiiclusioiis froiii these experiments emphasize the difficwlties inhei-eiit in field stitdies. The usc of controlled diets is more satisfactory in nutritional studies. Makar (1961), for example, observed that by supplr~iirlitiiig t,hc diet of sheep with sodium sulfate the cystine, tyrosiiir, arid phenylalanine coritcnts of the wool were increased arid the glutmiir acid, aspartic arid, valirie, leucine, and isoleucine contents were decreased. The wool produced 011 this sulfur-rich diet was longer, stronger, and of greater diameter than wool from the control group of sheep. Burley arid Hordrri (1960) fourid that copper deficiency in the diet of the sheep led to a decrrase in thc cystine caontent of the wool, but no significant diffcreiices were ohservcd in the contents of other amino acids. The variation in rystitic content was iiot reflected in the coniposition of the lowsulfur group of proteins, but was restricted to the high-sulfur proteins. Heis and Schinckel (1963) denionstrated that the introduction of cystine directly in the abomasuni of the sheep caused a dramatic increase in wool produvtion arid in sulfur ronteiit of the wool. Introduction of niethionirie or casriii solutions produccd like rffccts, whereas the administration of mixtures of glutanlic acid and glyriiic caused a slight but delayed increasc in wool growth, hut had no effect on sulfur content. I t has now hcen shown that caonipared with wool produced on a iiornial diet wool produced during the addition of rystine to the aboiiiasurii contains a greater proportion of niatrix arid high-sulfur proteins (Gillespie et al., 1964), particularly those with very high cystiiie content. It Tvoiild appear that the follide rells produve a range of high-sulfur protcins that, vary in relativc amount ac+cordingto the availability of vystiiic aiid possibly othcr aniiiio avids. 2. Other K c r ~ l i t i i z e dStructurxs n. Fealher Keratin. The aiiiiiio acid coniposition of various parts of fcxtlicrs froiii thrcc species of birds (Schroeder et al., 1955; Harrap and

TABLE XI11 Amino Acid Compositions of Component Parts of Turkey, Leghorn Hen, and Goose Feathersa

Turkeybjc Amino acid llanine Ammonia Arginine Aspartic acid Half-cystinee Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Tryptophan Threonine Tyrosine Valine

N accounted for (70) S accounted for (yc) a

Rachis

Calamiis

860 (754) 355 557 707 601 1350 22 298 616 60 26 348 952 (1340) 378 162 738

799 (724) 384 533 689 594 1278 38 300 875 67 23 348 953 (1437) 398 219 719

96 91

93 86

Leghorn4

Barbs

Gooseb

Medulla

Rachis

Calamus

Barbs

Medulla

Barbs

Down

450 (937) 371 492 723 618 965 25 380 554 84 24 300 912 (1228)

652 (683) 377 527 675 585 1184 50 302 713 90 30 338 943 (1176)

446 858 384 519 769 686 970 17 383 605 71 15 296 1056 1236 22 437 118 705

655 965 389 537 691 684 1156 24 313 720 65 18 330 891 1363 75 366 147 684

444 (1112) 375 542 942 616 967 21 363 590 96 22 230 852 (1178)

365 211 733

729 857 371 534 709 698 1171 20 280 664 52 13 319 885 1299 77 345 143 673

460 (1123) 347 512 895 612 1113 28 350 587 89 17 245 874 (1192)

394 128 735

840 963 370 547 761 671 1326 23 308 802 62 8 30 1 948 1365 72 40 1 139 756

373 247 627

454 204 673

91 96

90 90

101 95

95 97

93 84

95 92

88 89

93 92

-

-

-

e ~

M d

II

B

1 2 0 Y

R % .

55

Values given in pmde per gram. From Schroeder et aZ. (1955). Analyzed by the manual method of Itloore and Stein (1948, 1951). Numbers in parentheses indicate extrapolation to zero time of hydrolysis. Other values for turkey and goose are mean values for 24

and 72 hr hydrolyzates. From Harrap and Woods (1964a). Analyzed by the method of Spackman et aZ. (1958) using a Spinco Autoanalyzer and hydrolyzing 24 hr in 6 N HC1 under reflux. Ward et al. (1955) obtained similar though somewhat lower values by microbiological assay. 6 Values for Leghorn feather obtained by the method of Leach (1960).

N W

--I

238 w.

G. CREWTHER, R. D. B. FIIASER, F. G . LENNOX, AND H . LINDLEY

Woods, 1964a) are compared in Table XIII. In all structures alanine, cystine, glycine, leucine, proline, serine, and valine, are present in considerable amounts, whereas compared with animal fibers the values for arginine, lysine, and tyrosine are small. In general the amino acids with acidic side chains greatly exceed those with basic side chains, but as with animal fibers the content of amide groups accounts almost quantitatively for this diff erenee. Differences are apparent between the amino acid compositions of the component parts, but the values for barbs show greatest dissimilarities particularly in alanine, glycine, isoleucine, and the aromatic amino acids. Goose barbs and goose down show less marked differences in composition. Although there are significant differences between similar components derived from different species, the patterns of amino acid composition are very similar, suggesting selection of a particular protein structure suited to the function performed. Schroeder et al. (1955) comment on the high proline content of feathers and suggest that neither a-helical forms nor p-pleated-sheet forms could be accommodated if the distribution of proline were approximately random. b. Horn and Quill Keratin. No complete analyses of horn are available, nor has column chromatography been applied to hydrolyzates of this material. Table XIV lists the data of Graham et al. (1949), who used microbiological assay, and of Marecek (1959), who used paper chromatography. The two sets of data for horn are largely complementary, and it will be seen that the amino acid analyses of horn and wool obtained by microbiological assay are very similar. The data for porcupine quill (Table XIV) obtained by manual chromatography (Fraser et al., 1957) are also remarkable in their similarity to the data for wool. The most notable features are the rather low value for proline, which is in accord with the relatively high crystallinity of quill, and high values for glycine and tyrosine as compared with other &-keratins. c. Bgg Shell Keratin. Baker and Balch (1962) combined ionophoresis and paper chromatography in analyzing the double membrane lining the shell of the hen’s egg, the protein material of the matrix, and the outer cuticle. The membranes have a considerable cystine content (Table XV) and a high proportion of aspartic and glutamic acids. The cuticle is characterized by a smaller cystine and a higher glycine content than the nienibrarie; it also contains some hexosamiiie and other sugars. The matrix contains considerable amounts of nonprotein organic matter and it could not be classified as a typical keratin structure. d. Skin Keratin. The complexity of skin and difficulties encountered in separating its components have retarded the achievement of a detailed picture of amino acid distribution in those components. Furthermore,

239

THE CHEMISTRY O F KERATINS

TABLEXIV The Amino Acid Compositions of H o r n a n d Quill@ Hornb

Amino acid Alanine Ammonia Arginine Aspartic acid Half-cystine Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Pheny lalanine Proline Serine Threonine Tryptophan Tyrosine Valine ~

Microbiological assayC 681 663 1172 1060 72 377 727 279 41 216 836 59 7 341 549

Quill

Paper chromatographyd

Column chromatographye

1622

641 1250 634 739 062 1310 1057 60 328 808 282

-

947

-

-

1498 401

285 585 948 512 412 585

~~

Values given as pmole per gram. b The reliability of values obtained by Graham et al. (1949) by microbiological assay can be judged from a comparison of their values forVwool in relation to the values obtained by column chromatography (Table VIII). Maracek’s values for alanine, glycine, and serine appear to be high. c From Graham el al. (1949) From Maricek (1959). From Fraser et al. (1957). (1

many skin structures contain free amino acids or peptides, so that cleaning treatments affect their over-all composition. The thickened epidermis found in horse burr and cow’s nose differ from human skin both histologically and chemically. For example, epidermin, the fibrous protein froni epidermis of cow’s nose, cannot be extracted froni human skin (Matoltsy and Herbst, 1956; Roe, 1956), whereas the fibrous protein, tonofibrin, can be isolated only from the cellular layers of human epidermis (Roe, 1956; Flesch, 1958). Both materials give a n X-ray pattern resembling a-keratin and on stretching yield an oriented 0-pattern. Neither protein contains significant amounts of sulfur. Table XVI compares the analytical data for human epidermis with analyses of horny material from human skin or skin lesions. Possibly, the

240

IV. G . CREWTI-IER,

n.

D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

‘~‘IBLE XV Airtino Acid Composition of Shell Keratins from Hens’ Eggs ~~

~

Cuticle

Shell mernbranes . h i n o nc%l

111

2 7

(1

1)

I1 412

255 167

1254 53 352 3 31 365

4.7

522

5.3

604

2.1 5.2 4.7 3.7 3.3

233 578 522 412 367

I .6 3.3

182 377 536 502 592

6.4 6.2 9. 1 4.5 4 .0 I .o 2.3 3.0

(x)

:MI

I 3 6 12.7 6.1 3.1 7.8 11.1 1.4 3.1 2.9 6.4

9 .6

7‘ot:tl N (Ih,) Htbxosaminc. N ’I’otal N ac.cwimted for

II*

15 54 0 II 73

267 ill 688 1012 500 181 111

4.!1

4.4 5.2

362

696 353 689

15 94 0 24 8’2

Throughout tahlct values in column I are given as N as % total N. Throughout table values in column I1 are given as pmole N per gram.

two most important dif‘fereiicesbetween the analyses for whole epidermis and riorinal callus are the differences in cystine content and the lack of hydroxyproliiir i n the horny layer (Muting et al., 1955). Both play an important role in the process of keratinization in the outer layers of skin. It is noteworthy that methiorline accounts for a largr proportion of the total sulfur of both structures. Siuce Giroud and Lablond (1951) reviewed the question of keratiriization iii cpidcrinal tissues and Hudall (1952) reviewed the proteins of epidermis, it has hceii shown that the horny layer contains some residual thiol groups (Ziiigshcim, 1952; Van Scott arid Flesch, 1954; Magnus, 1956). It has also been possihlc by suitable staining techniques to demonstrate the presence of a “keratogmioiis” zone in epidermis (Montagna, 1956; Matoltsy and Sinesi, 1957). Iplesch (1958) suggests that thiol conipounds and enzymes niay accumulate in this zone for the final consolidation of the keratin and that in view of the greater total content of sulfur-containing amino acids in the horny layers as compared with the Malpighian layer that there may

24 1

THE CHEMISTRY O F KERATINS

RE ABLE XVI Aniino Acad Composition of Epidermis and H o m y Layers of H u m a n Skina

Amino acid

Keratin B from Exfoliate Normal normal Psoriasis dermatitis lCpidermisbbr Epidermis”,e rallusc rallus’ sc-ales@ s c d c ~ ~ ’ ~

Alanine Arginine Aspartic acid Half-cystine Glutamic, acid Glycine Histidine Hydroxyproline Isoleucine Leucine Lysine Methioninc Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Weight acronnted for yo

730 207 413 399 1052 1040 155 393 282 687 397 127 303 426 922 444 108 202 563 88.32

385 -

1 I7

-

1 (j8

-

404 107 29 1 -

__

49 238 -

775 247 353 757 087 498 1x7 Nil 160 777 542 134 121 573 970 503 122 22 1 572

89.4

830 299 722 140 1248 1372 116 Nil

381 732 303 141 200 677 56 1 378 57 1 496 I00

337 454 632 2!)1 I 190 :w9 122

Nil 289 ti02 487 67 176 287 1617 293 25 297 462 89.30

a Values given as pmole per gram. Since this article was written, Crounse (l!)(iX) has published amino acid analyses for hydrolyzates of human callus, stratum corneum, and fractionq obtained from human epidermis. Automatic ion exchange chromatography was used. The values for callus bear little resemtilance to the values of Muting et al. (1955) for normal callus. Thus glycine and aspartic acid contents were much greater and proline and cystine contents much less than the valiies given above. The differences appear to be too great to attribute to the different techniques uscd and probably indicate major differences in the callus samples anslysed. On the other hand, the analyses for epidermis are in fair agreement. * Separated by heating. Det,ermined by paper chromatography (Miiting et d., 1055). d Separated with 0.1 N acetic acid (Mardashev, 1947). Determined by various procedures. J Extracted with 0.2 N NaOH, 0.2 N Na& or 50% urea in 0.2 N NaOH. Cystine determined by the Block and Bolling method (Matoltsy and Balsamo, 1955). 0 Analysis of material insoluble in sodium lauryl sulfate solution. The soluble material was similar in composition; determined by column chromatography (Liss and Lever, 1963). *From Block (1951).

242

W. G. CREWTHEH, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

be a considerable uptake of sulfur-containing amino acids during consolidation of the keratin in normal skin. Certain pathological conditions, particularly psoriasis, produce horny material with very low cystine content (1,iss arid Levcr, 1963) (Table XVI). Certain of the data in Table XVI were obtained from proteins extracted from the horny material by solutions of alkali or reducing agents, and complete analyses of both insoluble and soluble fractions were not performed. The low cystine content of the soluble components may therefore reflect a high cystine content in the residue rather than a low total cystine content of the horny layer.

C. find Groups of Keratins Middlebrook (1951) established that wool contains at least seven different N-terminal residues : those of alanine, aspartic acid, glutamic acid, glycine, serine, threonine, and valine, but they were not present in simple molecular proportions (Table XVII). More recently, Thompson (1959) found that half-cystine is also an N-terminal residue and is the second most abundant. The most recent quantitative data relating to N-terminal groups in worsted fabric (Zahn et al., 1963) are similar to the results of Steuerle and Hille (1959) for Merino top wool (Table XVII). TABLEXVII N-Terminal Amino Acids" ~

Merino woolJ

Amino acid Aspartic acid Glutamic acid Scrine Threonine Glycine Alanine Valine Total

Lincoln fleeceb 0.6 1.2 1.2 4.8 5.2 1.2 2.4 16.4

Merino 64's fleecec

Merino 64's yarnd.0

Merino Cu-deficient Normal 64's region region tope*@ of staple of staple

(0.3) (0.7) (1.2) (4.4) (1.1) (0.8) (1.0)

0.5 0.9 1.0 3.2 3.8 1.1 1.0

0.3 0.5 1.3 4.3 1.4 0.6 0.7

-

19.9 (9.5)

ll.G

9.1

0.5 1.1 1.7 5.6 7.8 1.5 1.7

-

Values given as pmole per gram of dry wool. From Middleblebrook (1951). From Thompson (1857). Uncorrected values in brackets. d From Alexander and Smith (1956). a From Steuerle and Hille (1959). f From Burley and de Kock (1957). 0 These values are uncorrected for hydrolytic losses.

0

b

0.7 1.2 2.4 3.6 0.5 1.0

0.4 0.7 2.5 1.1 0.3 0.8

T H E CHEMISTRY O F KERATINS

243

Kerr and Godin (1959) showed similar end groups to be present in human hair and horsehair, and Hahnel (1959) found the same end groups in hair, callus, nails, and psoriasis scales. An unexplained finding in this latter work is that the total amount of N-terminal amino acids in psoriasis scales is at least ten times greater than for other keratins. Burley and de Kock (1957) found an increase in the number of end groups, mainly glycine and alanine, in wool from sheep suffering from copper deficiency. This increase was much smaller than in the case of psoriasis scales and could be revealed only after pretreatment of the steely wool with phenol solutions. Control experiments showed this pretreatment to be without effect on the end groups of normal wool. Woodin (1956) investigated feather keratin and found the same seven amino acids to be N-terminal and in amounts roughly similar to those for wool. Blackburn and Lee (1954) using hydrazinolysis showed qualitatively that alanine, glycine, serine, and threonine were present as C-terminal amino acids in wool, and Kerr and Godin (1959) obtained similar results from human hair and horsehair. A more thorough study by Bradbury (1958) added aspartic and glutamic acids as C-terminal for wool and gave rough quantitative figures. Woodin (1956) reported on the amino acids released by carboxypeptidase from solubilized feather keratin, but there is no guarantee that these were all necessarily C-terminal. While there has been reasonable agreement on the results of end-group analyses on wool, there has been much divergence of opinion as to the significance of the results. Middlebrook (1951) used his results to propose a polychain model for wool, but quantitative agreement with molecular weights determined on solubiiized keratins was poor. Difficulties of this kind led to the suggestion that cyclic peptides may be present in wool (Thompson, 1957). The more recent demonstration (O’Donnell et al., 1962), however, that wool contains acetyl groups with properties indicating their substitution on nitrogen atoms has provided a more satisfactory explanation for the analytical data. Since the eamino groups of lysine side chains react almost quantitatively with fluorodinitrobenzene (Middlebrook, 1951), it is probable that these acetyl groups mask terminal amino groups. Although arginine has been identified as an N-terminal residue in SCMKB2 (Gillespie, 1963a), it has not yet been demonstrated in whole wool. This is almost certainly due to the difficulty of detecting and estimating small amounts of a-DNP arginine in the presence of large amounts of eDNP lysine. Summarizing, it would appear that wool contains both N-acetyl and a variety of amino N-terminal groups, and these latter are not entirely artifact in origin. The comparative paucity of terminal earboxyl groups

244 w.

G. CREWTHER, 11. D. B. FRASER, F. G. LENNOX, AND II. LINDLEY

detected suggests that these may be masked largely as amide groups, but no evidence for this is available. The discovery of N-acetyl groups, however, in comparatively high amounts has removed the necessity for postulating cyclic chains as a feature of keratin structure. Recent work by Harrap and Woods (1964a) suggests that a similar situation may exist in feather keratin, which they have shown to contain 1.3 acetyl groups per mole assuming a molecular weight of 10,000.

D. Sequence Studies Fifteen years ago it could be claimed that more was known about amino acid sequences in wool than in any other protein, but comparatively little has been added to our knowledge since the pioneering investigations of Consden and colleagues (Consden et al., 1948; Consden, 1949; Consden and Gordon, 1950). Table XVIII gives a list of the dipeptides found in wool hydrolyzates during this work; higher peptides were also found, but were not well characterized. In addition to this list cysteicylcysteic acid was shown t o be present in a partial hydrolyzate of oxidized wool by Sanger et al. (1956), and subsequent evidence (Crewther and Dowling, 1960a) indicated that this sequence occurred in the low-sulfur rather than in the high-sulfur protein fraction. TABLEX V I I I Dipeptides Isolated from Partial Acid Hydrolyzate of Woola Aspartic peptides ~

Glutamic peptides

Cysteic pepticlesb

G1u-G1u Glu-Gly Glu-Ala Glu-Tyr Glu-Leu Glu-Cys Ser-Glu Gly-Glu A1a-G1u Tp-Glu Va1-G1u Leu-Glu CYS-GIU

Asp-Cya Glu-Cya Ser-Cya Gly-Cya Thr-Cya Ala-Cya Leu-Cya Cya-Gly Cya-Thr Cya-Ala Cya-Va1 Cya-Leu Cya-Phe

~

Asp-Glu Asp-Val Asp-Leu Gly-Asp, Leu-Asp Glu-Asp

b

From Consden et al. (1948). Cya is used as an abbreviation for cysteic acid.

The only other keratin to be similarly investigated is turkey feather calamus (Schroeder et al., 1957). These results are shown in Tables XIX and XX.

245

THE CHEMISTRY O F KERATINS

TABLEXIX Peptides Isolated from a Partial Acid Hydrolyzate o j Unoxidized White Turkeu Feather Calamus Peptide5

Amount isolated'

Dipeptides Ala-Ala Ala-Asp Ala-GI y Ala-Leu Asp-Gly Asp-Leu Glu-Asp Glu-Gly Glu-Pro Gly-Gly Gly-Leu Gly-Phe Gly-Pro Leu-Ala Leu-Glu Leu-Gly Leu-Leu Phe-Gly Pro-Glu Ser-Ala Ser-Glu Ser-Gly Ser-Leu Ser-Phe Ser-Pro Ser-Ser Ser-Thr Thr-Ala Thr-Leu Thr-Ser Tyr-Gly Val-Gly

5.1 14.2 7.5 7.1 7.1 1.2 9.9 3.9 3.9 17.3 30.3 35.4 41 .O 4.7 3.1 29. I 2.4 34.6 3.2 50.8 15.4 55.0 26.0 4.3 2.4 36.2 1.2 48.9 10.6 2.0 3.5 49.3

4

Peptiden

Amount isolatedb

Tripeptides Ala-(Gly, Val) GIy-(Gly, Leu) Gly-(Leu, Pro) Leu-(Ala, Ser) Phe-(Glu, Pro) Pro-Leu-Leu Ser-(Ala, Leu) Ser-(Gly, Leu) Thr-(Ala, Val) Thr-Pro-Leu Thr-(Leu, Pro) Thr-Val-Val Val-Ala-Leu Val-(Leu, Val)

38.2 7.5 7.8 3.1 2.0 18.9 11 .o 5.5 9.5 14.2 6.3 8.7 13.8 5.5

Tetrapeptides Ser-(Ala, Gly, Val) Ser-(Glu, Phe, Pro) Ser-(Gly-Leu, Pro) Thr-(Ala, Leu, Pro) Thr-(Ala, Gly, Val) Thr-(Gly,'Leu, Pro)

5.1 11 . 0 4.3 13.8 22.4 10.2

Pentapeptides Ser-(Asp, Glu, Phe, 'Pro)

3.1

Leucine and isoleucine are nowhere differentiated.

* Values given in wmoles per gram.

Fell et al. (1960) and Blackburn and Lee (1963) are studying the amino acid sequences in a-keratose. Although this fraction is heterogeneous and therefore is not an ideal starting material, it is preferable to the complex mixture of proteins in the original fiber. A preliminary note has appeared regarding the work of Blackburn and Lee (1963), but this gives no results. Fell et al. (1960) have published an account of their investigations of basic peptides isolated from a tryptic digest of a-keratose. The peptides

246

W. G;. CREWTHER, R. D. B. FRASER, F. G . LENNOX, AND H. LINDLEY

TABLEXX Cvsteic Acid Peptides from Hydrolyzate of Oxidized White l’urkey Feather Cahmus Peptiden

Approx. amountb

-

Cya, Cya, Cya, Cya, Cya, Cya, a

b

Pro, Ser Glu, Pro Ser Glu, Pro Glu Glu

18 19

50 12 10 7

Cya is used as abbreviation for cysteic acid. Values given in pmole per gram.

isolated and characterized by them are shown in Table XXI. Bearing in mind the specificity requirements of trypsin the results show unequivocally that the basic amino acids tend to occur as clusters rather than evenly distributed along the peptide chain. TABLEX X I Basic Peptides Isolated From a Tryptic Digest of a-Keratosea (Arg, Glu(NH2), Glu(NH2), Val, 1leu)-Arg Ala-(Thr, Val, 1leu)-Arg Ser-Ser-Arg Phe-Arg Gly-Ser-Arg Ser-Arg Ser-Lys Gly-Arg Ala-Lys Ala-Arg Lys-Lys a

Free lysine and arginine were also detected.

In studying acidic residues, glutamylglutamic acid was the most abundant peptide detected by Consden et al. (1948), but since hydrolysis by strong acid was used it is impossible to say whether the residues were originally glutamit, acid or glutamine. More recent work by Fell (unpublished observations, 1963) gives some results on both neutral and acidic peptides. Table XXII lists the peptides characterized. The very large number of peptides so far isolated from a-keratose reinforces doubts as to the homogeneity of the starting material and, indeed, the paper contains reference to fractionation of the a-keratose into two fractions of widely differing amino acid composition.

THE CHEMISTRY OF KERATINS

247

TABLEXXII Neutral a d Acid Peptides Isolated from a-Keratosea Ser (His, Aspz, Tyr, Ileu) Arg Ala (Glys, Glu, Ileu) Arg Ser (Asp, Glu, Leu) Arg Gly (Cya, Gly, Ser, Ala) Arg (Asp, Val) Arg Leu (Ser, Glu, Ala, Tyr, Leu) Lys Ala (Asp, Glu, Ileu, Thr, Phe) Lys (Asp, Glu, Leu, Ileu) Lys Phe (Asp, Glup, Leu) Lys Leu (Thr, Glu, Leu) Lys Glu, -1leu-Lys (Glu, ( C m ) ) Asp (Cya, Asp, Glu, Ma, Val, Leu) Arg Glu (Cya, Asp, Serl-s, Gly, Glun-, Alas) Arg a

Cya is used as the abbreviation for cysteic acid.

IV. CHEMICAL REACTIVITY OF KERATIKS

A . T h e Reactivity of the Cystine in Keratin

I. Introduction Cystine chemistry must obviously be a matter of some importance in keratins which, as proteins, are characterized by their high-sulfur content. An early review of keratin sulfur chemistry is given by Alexander and Hudson (1954), while the more recent review by Cecil and McPhee (1959) includes extensive reference to work on keratins. Some treatment of the topic is also to be found in articles in “Sulfur in Proteins” (Benesch et al., 1959). I n this section attention will be directed to material where the special properties of keratins or the rather unusual interests of the keratin chemist have given a different viewpoint to the subject. 2. Reduction

Reduction of the disulfide bonds of keratin has been much studied not only because of its use for preparation of soluble derivatives (Section II,A,l), but also because it is the basis of some “setting” methods (Section VI,A,2). To the chemist interested in solubilizing keratins for fractionation studies the major concern is t o achieve complete and specific reduction of the cystine. Early work on the reduction of wool suggested that complete reduction of the cystine could only be obtained under conditions where the wool was solubilized, i.e., high p H or in the presence of a high concentration of urea.

248 w.

G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

Techniques have now been evolved which circumvent this, and wool fibers with few residual disulfide groups have been prepared. This has been achieved by (a) reduction with 4 M mercaptoethanol (Thompson and O’Donnell, 196ll1962b), (b) using 0.1 M benzyl mercaptan in ethanol-water mixture (Maclaren, 1962), (c) electrolytic reduction a t a constant cathode potential, using catalytic concentrations of thioglycolate maintained in the SH form continuously (Leach et al., unpublished observations, 1963)’ and (d) repeated reduction and alkylation of the SH groups of the wool (O’Donnell, 1954). The use of mass action effects to achieve almost complete reduction and alkylation suggests that the reaction of wool with thiols can be formulated most simply as an equilibrium between two redox systems. Nevertheless, the multiplicity of proteins present must be taken into account. A study of the problem has been initiated by Gillespie and Springell (1961) and Springell et al. (1964). These workers reduced wool t o varying extents using redistilled thioglycolic acid and alkylated the thiol groups of the wool with iodoacetate-2-C14. The wool was subsequently treated by standard reduction and alkylation procedures, using unlabeled iodoacetate, to isolate high- and low-sulfur proteins. These were then hydrolyzed and the specific activity of the S-(carboxymethy1)cysteine in the hydrolyzate determined. The rather surprising result was that the high- and low-sulfur proteins had similar reactivities toward mercaptoacetate. By a similar technique they showed that on reacting extensively reduced wool with limited amounts of iodoacetate-2-C14 the proportions of thiol groups alkylated in the high-sulfur and low-sulfur fractions were equal. Similar results were obtained whether the reactions were carried out a t p H 5 or 9. Using fP-labeled mercaptoacetate Human and Springell (1959) obtained evidence for the formation of mixed disulfide between wool and thioglycolate. It seems probable, however, that some of the bound S35found by these workers originated from the acylation of free amino groups by impurities in the thioglycolate as shown by Schoberl (1948) and White (1959). The amount of residual mixed disulfide is very dependent on reaction conditions.

s. Sulfitolysis Interest in the reaction between wool and sulfite stems from the bleaching of wool by sulfur stoving or by the application of bisulfite solutions, the use of sulfite solutions as “antichlors” after treatment to make wool unshrinkable and, more recently, the use of sulfite solutions for the setting of wool fabrics. Early work established that the reaction of sulfite with wool followed a similar pathway (I) to that found earlier by Clarke (1932) for cystine. R--SS-R

+ SOa-

~

R-S-

+ R--SSOa-

(1)

249

THE CHEMISTRY OF KERATINS

This work was concerned mainly with the extent of reaction and conditions affecting its reversibility and led Phillips and co-workers to postulate fractions of cystine of differing reactivity; the significance of this in the light of recent work will be discussed in Section IV,A,6. More recent work has been concerned with techniques to cause complete fission of the cystine bonds as a means of solubilizing keratins and of estimating cystine. Complete reaction has been achieved in two ways: (1) By use of mixtures of sulfite and a mercurial to form a mercaptide with the thiol (Leach, 1960). The reaction can be considered as displacing the equilibrium by renioving the thiol as soon as it is formed or less probably as due to a sulfite-mercurial reactant of increased nucleophilic character (Milligan and Swan, 1962). ( 2 ) B y oxidative sulfitolysis. Originally this method was conceived as involving (i) fission of disulfide by sulfite to produce thiol and S-sulfocysteine, and (ii) oxidation of this thiol to disulfide which again reacts with sulfite as in (i). The over-all reaction is the conversion of disulfide to S-sulfocysteine. On this basis the reactions can be formulated as in reaction (11). R-S-S-R

+ SO-:

A

R - S-

-k

R- S-SO;

(11)

--

oxidant

Suitable oxidants for proteins are cupric ions (Swan, 1957b, 1961), iodosobenzoate, or tetrathionate (Bailey, 1957; Bailey and Cole, 1959). Literal application of this mechanism to the case of complete oxidative sulfitolysis of wool under conditions which do not lead to solubilization of the fiber requires that the thiol groups so formed, although derived from different disulfide bonds, are suitably placed to form a new disulfide bond. Difficulty in visualizing this has led t o suggestions of more complex reaction mechanisms that do not involve protein disulfide bonds. Thus Milligan and Swan (1962) postulate the following mechanism for oxidative sulfitolysis using tetrathionate R-S-S-R

+ SO:-=

R-S- + - O $ - S - S - S O , e R-S-S-SO,

+ SO:-=

R-S-

+ R-S-SO;

R-S-S-SO, R-S-S03-

+ S,O,2-

(111)

iS,O,2-

They also suggest mechanisms for the copper sulfite procedure that do not involve disulfide intermediates. The iodosobenzoate technique of Bailey (1957), since it involves adding the iodosobenzoate to the sulfite solution, may in fact be a variant of the tetrathionate procedure. The stoichiometry of the sulfite-merruric chloride reaction is less well explained. Leach (1959, 1960) observed a well-defined 2 : 1 disulfide :mer-

250

W. G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

curic ion stoichiometry in the reaction between wool and sulfite-mercuric chloride solutions. This is most simply explained by assuming that a mercury atom links two sulfur atoms that originally belong to different disulfide bonds as in reaction (IV). S

s

I

I-

Leach (1960) has suggested that it is more reasonable to forniulate the reaction as:

This is merely a formulation, however, and any plausible reaction mechanism seems to involve precisely the same type of difficulties as are associated with the simpler formulation (IV). For example, Milligan and Swan (1961) obtained some evidence that Bunte salts can undergo an exchange reaction with disulfide bonds. They suggest (Milligan and Swan, 1962) that such an exchange mechanism can lead to the reactions.

I SI S I

Hg Hg S

A-s- so; so; I

s-so,

s0;-

11

I + I -S038-Hg + 8S-S03S-Hg

TIIT-

For such an exchange to occur it would be necessary for the second disulfide bond to be accessible to the S-sulfocysteine residue. The accessi-

T H E CHEMISTRY O F KERATINS

25 1

bility of one group to the other would depend on the retention or nonretention of a n ordered structure in the fiber. If the polypeptide chains assumed a randoin conformation as disulfide bonds were ruptured, accessibility of one group to the other would be greatly increased. Leach points out that the steric problem is a genuine one, since the “natural” thiol groups of proteins do not exhibit this same stoichionietry toward mercuric ion. Observed ratios of sulfur to mercuric ion range from 1 to 2. Alternatively, disulfide bonds may be so positioned in the fiber that reactions such as formulation (IV) or interchange reactions are facilitated (cf. Crewther and Dowling, 1960a).

4. Oxidation Oxidation is a well-studied reaction in the case of wool keratin. Thus hydrogen peroxide is used as a bleaching agent and chlorine, bromine, potassium permanganate, and organic peracids have all been used to produce “nonfelting” wool, while the use of oxidation methods for the solubilization of keratin has already been described (Section II,A ,a). Each of these reagents can convert cystine to cysteic acid, and various intermediate oxidation products are theoretically possible (Savige and Maclaren, 1964). These intermediate oxidation products if formed would be expected to decompose on acid hydrolysis to give cystine and cysteic acid. Analyses on hydrolyzates of partially oxidized wool could therefore be quite misleading with regard to the extent of cystine modification in the intact protein. This has in fact been demonstrated by Maclaren et al. (1960) using Leach‘s (1960) method for estimation of cystine and cysteiiie on unhydrolyzed proteins. It is possible for most of the disulfide bonds of wool to be partially oxidized and yet for the hydrolyzate of such a wool to contain substantial amounts of cystine formed during the hydrolysis of the wool. Interesting though this result may be, however, it merely serves to delineate the problem. Possible intermediate oxidation products between disulfide and -S02-S-, -SO-SO--, -S02-SO-, sulfonic acid are -SO-S-, -S02-S02-, -SOH, and -S02H, and no method of even qualitative analysis is available to distinguish between them in the fiber. Thus there is no conclusive evidence to show whether the first step in the oxidation of a disulfide bond in wool gives two SOH groups or -SO-S-. Both are equivalent oxidation states, but one involves bond fission, whereas the other leaves the cross-linkage intact. The situation may be further complicated by reactions involving C-S fission in the cystine side chains. Thus cysteine sulfonic acid (CyS-SO,H)

252 w.

G . CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

can be obtained as a major product upon oxidation of cystine in alkaline solution with hydrogen peroxide plus catalytic concentrations of cupric or vanadate ions or with pernianganate, hypochlorite, or persulfate (Eager et al., 1964). Probably the precise oxidation pathway is a function of the nature of the oxidizing agent and the conditions under which it is used. Savige and Maclaren (1964) have fully discussed the oxidation of cystine and related compounds. The choice of a particular oxidizing agent for a specific end purpose in wool technology may well be ultimately determined by the required oxidation pathway, although at present the choice is necessarily based on a purely pragmatic approach. The ultimate unravelling of the details of oxidation mechanisms, however, could be a valuable contribution to wool technology apart from its importance in sulfur chemistry. 5. Lanthionine-Forming Reactions

Although Horn et al. (1941, 1942a,b) first isolated and correctly characterized lanthionine, it had been isolated earlier by Kuster and Irion (1929), but incorrectly characterized by them. A re-examination of their original preparation by Irion (1951) subsequently proved its identity with lanthionine. Horn et al. studied wool, hair, feathers, and lactalbumin, pretreated with Na2C03, NaOH, or NazS solutions. Lanthionine was isolated from the acid hydrolyzate of these modified proteins, but no attempt a t quantitative evaluation was made. These results were confirmed and extended by Schoberl (1942, 1943). Cuthbertson and Phillips (1945) added potassium cyanide to the list of agents that readily produce lanthionine in wool and attempted to estimate the amount present by a n over-all sulfur balance. They suggested that two reactions were occurring simultaneously I

I

co

co

I

co I

I

I

co I

CH- CH,- S -S -CH,-CH I I NH NH I I

rlrH

I

NH

I

THE CHEMISTRY OF KERATINS

253

At the time, model reactions for (VIII) were known, but no simple analogy for (VII) had been discovered. Schoberl and Wagner (1956), however, showed that the treatment of uncombined cystine with alkali leads to the formation of some lanthionine and, hence, the reaction with the protein was not unique. There has been considerable discussion about the extent to which the lanthionine reaction can account quantitatively for the alkaline degradation of the cystine of keratins and about the mechanism of the reaction. As mentioned above Cuthbertson and Phillips suggested that two reactions proceeded simultaneously and Lindley and Phillips (1945) extended their observations to a wider range of conditions This work, however, depended on very indirect estimates of lanthionine which necessarily were of a low order of accuracy. Blackburn and Lee (1956b) made a further study of the reaction using an ion-exchange chromatographic technique for the estimation of lanthionine and came to the conclusion that the reaction of wool with alkali could be adequately described by reaction (VII), i.e., only lanthionine was formed. Examination of their analytical data reveals some internal inconsistancies, and it now appears probable that their method of cystine analysis was unreliable (Fletcher et al., 1963), so that their conclusions are open to doubt. Crewther and Dowling (1965) also obtained a stoichiometric conversion of the cystine in wool t o lanthionine under mild reaction conditions, but with prolonged alkali treatment or more severe conditions the decrease in disulfide content greatly exceeded the lanthionine formed. In view of the fact that peptide-bonded lanthionine is unstable to alkali (Zahn and Kessler, 1958; Decroix et al., 1958), it seems likely that decomposition products in addition to lanthionine occur in alkali-treated wool, but the reaction pathway may well be more complex than that formulated by Cuthbertson and Phillips. One of the major problems in all this work has been the estimation of lanthionine (cf. Section 111,A13). Recent unpublished work by Dowling and Maclaren (1963) suggests that, just as for cystine analyses, the use of non hydrolytic methods of analysis may be necessary in certain circumstances. These workers have shown that considerable amounts of lanthionine can be detected in hydrolyzates of wool reduced with various reagents, but if the reduced wool is treated with some alkylating agent such as iodoacetate prior to hydrolysis no lanthionine can be found in the hydrolyzate. It is clear therefore that lanthionine can be an artifact formed during hydrolysis of reduced wools. This may have some bearing on the report by Savige (1960) that lanthionine was formed during the extraction of wool proteins with thiols in the presence of acid. The result also casts doubt on some conclusions which have been drawn regarding the effect of small amounts of lanthionine on the physical properties of the wool fiber

254 w.

G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

(Lees et al., 1960; Atkinson and Speakman, 1960; Zahn e l al., 1960; Swan, 1960). The mechanism of lanthionine formation has been the subject of many investigations. Three possibilities have been considered for the initial reaction of the disulfide bond with alkali. a. Hydrolytic Fission of the Disulfide Bond. This theory was accepted for many years as applying to the reaction of wool with alkali (IX) and is R--SS-R

+ OH- -+

+ R-S-

R-SOH

(IX)

associated especially with the names of Speakman (1933a, 1936a) and Schoberl (1936, 1940). Lanthionine was presumed to arise from reaction between the thiol and combined a-aminoacrylic acid formed by the decomposition of the sulfenic acid. These views were criticized by Rosenthal and Oster (1954) who proposed the mechanism in Section IV,A,5,b as the initial step. b. The a-Elimination Reaction. Rosenthal and Oster (1954) refer to reaction (X) as a @-eliminationreaction implying elimination of a hydrogen

+ OH- + R-CH=S + R-CHr-S- + HzO

R-CHg-SS-CHz-R

(X)

atom in a 0-position with respect to the point of fission. More recently, the term @-eliminationhas been used generally to imply elimination of the hydrogen atom ,B to a sulfur atom (via the mechanism in Section IV,A,5,c); using this nomenclature the significant hydrogen atom for Rosenthal and Oster’s mechanism would be in the a-position. The confusion is increased by the fact that the important hydrogen in what we are calling 0-elimination is the one the amino acid chemist would refer to as the &-hydrogen. The nomenclature used in this section of the review is shown in the acconipanying formula : I

I

co 0

co

I

CH-CH,-S-S-CH,-CH l

I

(3

(XI)

AH

NH

I

I

c. The @-Elimination Mechanism. This type of reaction (XII) was suggested by Tarbell and Harnish (1951) and has been strongly supported by Swan (1956, 1957a). R- S -S

? -Rl-

- CH,-CH

1

+ OH-

R2

I

R-S-S- -t. CH,’CH-RR,

+ H,O

255

T H E CHEMISTRY OF KERATINS

All these papers, however, have really been concerned with the use of model substances rather than proteins, and it is doubtful whethex extrapolation of the results is justified. Thus Zahn and Golsch (1962) studied the alkali decomposition of cystine, cysteine, cystine dihydantoin, lanthionine, and lanthionine dihydantoin. It was concluded that cystine decomposes either by mechanism given in Section IV,A,5,a or b, whereas the dithiohydantoin undergoes ,@-elimination. The mechanism of decomposition of lanthionine dihydantoin was apparently even more complex and depended on the acetate ion concentration. The mechanism of formation of lanthionine by the action of cyanide probably involves the following reactions W-&-S-W

+ CN-+

+ W-S-CN

-S-W

-+

W-S-CN W-S-W

+ -SW + CN-S-

(XIII)

(XIV)

Reaction (XlV) is visualized as a direct nucleophilic attack of the thiol anion on the carbon atom holding the SCN group (Swan, 1 9 5 7 ~ Parker ; and Kharasch, 1959). Earland and Raven (1960, 1961) draw the same conclusions from studies on model compounds. 6. The Problem of the Varying Reactivity of Cystine of Wool

Middlebrook and Phillips (1942) suggested a subdivision of the combined cystine of wool into four fractions of differing reactivity. The basis of differentiation was both gross differences in rates of reaction and also differences in the nature of the end products. The early work of Phillips and collaborators was reviewed by Lindley (1959), but a reassessment seems worthwhile. The major differentiation of the cystine suggested by Middlebrook and Phillips was into fractions designated as A B and C D. The A B cystine reacted with bisulfite solution and could be reduced by thioglycolate a t pH 5, whereas the C D fraction was supposedly completely inert under these conditions. If made to react under more drastic conditions (e.g., boiling bisulfite) the C D fraction reacted in a different and less wellunderstood fashion involving sulfur loss from the protein. Two types of evidence cast doubt on this subdivision of the cystine residues. First, all the cystine can be made to react a t p H 5 with reducing agents (Section IV,A,2) or sulfite (Section IV,A,3). It follows that these reactions are reversible and, while there may be a spectrum of differing reactivities of cystine, there is no clear division into reactive and nonreactive fractions. Second, recent work by Maclaren (unpublished observations, 1963) shows that there may be considerable losses of thiol during acid hydrolysis. Some of the apparent differences in chemical reactivity attributed t o the A B and C D fractions may therefore be due to

+

+ +

+

+

+

+

256 w.

G . CREWTHER, R. D. B. FRASER,

F. G . LENNOX, AND H. LINDLEY

destruction of thiol groups during acid hydrolysis rather than to differences in the initial reaction with bisulfite or thioglycolate. The rigid subdivision of the cystine into A B and C D fractions is therefore probably unjustified, but on the other hand much of the evidence reviewed by Lindley (1959) still remains t o be explained. A complete reinvestigation of the problem using nonhydrolytic methods for thiol and disulfide analysis would seem t o be needed. It is interesting that Gillespie and Springell (1961) and Springell et al. (1964) have shown that the varying reactivity of the combined cystine extends to both high- and low-sulfur proteins.

+

+

7 . Unsolved Problems in the Cystine Chemistry oj Keratin Two related hypotheses have been invoked to explain aspects of keratin chemistry that obviously require further investigation. These are the problem of S H S S interchange and the problem of interchain and intrachain disulfide bonds. Thiol-disulfide interchange is a well-established reaction in solution and is reported to play a part in the dough-forming property of flour in breadmaking (Frater et al., 1960, 1961). It has been used similarly to explain many of the effects of chemical changes on the physical properties of the wool fiber (Section VI). Similarly, the presence or formation of intrachain disulfide bonds has been postulated to account for physical and solubility properties of wool (Zahn and Kessler, 1958). On the other hand the results of Gillespie and Springell (1961) (Section IV,A,2) are explained most simply on the basis that most of the disulfide bonds of wool are interchain between the high- and low-sulfur proteins. An urgent requirement is for unequivocal evidence on these possibilities.

B. The Reaction of Keratins with Ions The reaction of wool with ions is of fundamental importance in the industrial processes of dyeing, shrinkproofing, and setting, but papers dealing specifically with industrial processes will not be considered here unless they impinge on the more general problems of ion absorption. Ionic interactions niay also cause conformational changes in the fiber proteins and are therefore of importance in physical and chemical research relating to wool and hair. Titration data for wool have been available for more than two decades (Speakman and Stott, 1934; Steinhardt and Harris, 1940a,b) and have been the subject of various quantitative theoretical treatments and extensive controversy. Other more qualitative theories have been proposed to account for the effects of salts on chemical and conformational changes in animal fibers immersed in aqueous electrolytes.

257

T H E CHEMISTRY OF KERATINS

1. The Isoelectric

Point of Wool

The amphoteric nature of wool was demonstrated in the early studies of Speakman and Hirst (1933), Elod (1933), and in particular by the complete acid-base titration curve obtained by Speakman and Stott (1934). Even earlier attempts had been made to determine the isoelectric point of wool by the methods indicated in Table XXIII. Some variation in the isoelectric point is to be expected because the pH a t which the net charge, including bound ions, is zero depends on the nature and concentrations of ions in the aqueous environment. For example, Sookne and Harris (1939) have shown that the early electrophoretic value of Harris (1932) was affected by the absorption of phthalate ions from the buffer solutions. With acetate buffer they obtained values of 4.2 and 4.5 for powdered wool and cortical cells, respectively. The isoelectric points listed in Table X X I I I are TABLEXXIII The “Isoelectric Point” of Wool Determined by Various Methods Reference

Method

Isoelectric point

Speakman (1925) Elod and Silva (1928) Meunier and Rey (1927) Marston (1928) Harris (1932) Speakman and Hirst (1933) Dumanskii and Dumanskii (1934)

Ferricyanide absorption Swelling minimum SwelIing minimum Acid absorption Microelectrophoresis Titration curve Electrophoresis

4.9 4.9 4.0 3.4 3.4 5.0 4.9

probably in error for various reasons. The limiting p H a t which measurable acid binding by well-washed wool can be demonstrated is invariably lower than the true isoelectric point; the pH of minimum swelling may be influenced by factors other than ionization of side-chain groups, e.g., by the stability of disulfide bonds; the value obtained by electrophoresis is largely determined by the surface charge and may not reflect the composition of the fiber as a whole. Speakman and Hirst (1933) and Speakman and Stott (1934) consider that a n isoelectric region rather than an isoelectric point should be recognized; similar results are observed with silk (Howitt, 1946). Lemin and Vickerstaff (1946) claini to have determined the isoionic point of wool by measurement of the p H a t which addition of salt does not affect the pH. They obtained a value of 6.2 which is approxiniately the midpoint of the “isoelectric region.” It is not possible to determine the isoionic point of a protein by measurements made, as in the present instance, a t constant salt concentration. The true value could be obtained by carrying out the

258 w.

G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

Lemin-Vickerstaff experiment a t various salt concentrations and extrapolating t o zero concentration. The value obtained by Lemin and Vickerstaff will be approximately correct if the affinities of both cation and anion for wool are very low or nearly equal. The isoelectric point of wool has limited significance because of the heterogeneous character of the wool fiber. 2. The Locution of Charged Groups in the Fiber

Despite ample data concerning the contents of the various ionizable side-chain groups in wool and its constituent proteins little, if any, direct information is available to indicate their respective positions in the fiber. Speakman and Hirst (1931, 1933) have concluded that chains carrying positively and negatively charged groups are so positioned in the fiber that the groups form salt linkages between the polypeptide chains. This view is based on their observation that the work required to extend wool fibers by 30 yo in water reaches a maximum in the isoelectric region and is considerably decreased in acid or alkaline solutions. The near equivalence of “acidic” and “basic” side-chain groups in wool and the decrease in work to stretch fibers 30 % following treatment with reagents capable of destroying side-chain amino or guanidino groups were quoted in support of this view (Speakman, 193313, 1941, 1947). Certain facts appear difficult to reconcile with the saIt-linkage hypothesis, Speakman and Elliott (1946) showed that the work required to stretch wool fibers 30% in acid solution varied with the anion present. They found that the greater the affinity of the anion for the fiber, the smaller was the decrease in work of extension for a fixed acid uptake by the fiber. The work to extend the fiber was actually increased by absorption of the free acid of the dye Orange 11. Speakman and Elliott suggested that weakening of the fiber owing to rupture of salt linkages was masked by a n increased cohesion between protein micelles or peptide chains arising from their affinity for the anions of the acid. These results could also be explained by supposing that the work required to extend a wool fiber is greatest when the net charge on the fiber is zero. A decrease in the net positive charge on the fiber owing to the binding of anions in acid solution would be expected to strengthen the fiber. The observation by Speakman and Stott (1938) that treatment of wool with nitrous acid lowers the pH a t which the fiber has greatest strength is readily explained by the latter postulate. The similarity, however, in the load-extension vurves obtained with untreated and deaminatcd fibers in 0.1 N HCl (Speakman, 1933, 1947) supports the salt-linkage hypothesis, but further information on the effects of nitrous acid treatment are needed twforc thcsc rcsults can be interpreted with certainty. For example,

T H E CHEMISTRY O F KERATINS

259

Sookne and Harris (1937) found that the disulfide bonds of wool are modified by treatment with nitrous acid. They considered the similarity of the load-extension curves for untreated and deaminated fibers in 0.1 N HC1 to be fortuitous. The work of Astbury and Dawson (1938), later confirmed by Speakman and Elliott (1946), showed that the X-ray diffraction pattern of wool was unaltered by saturating the fiber with an anionic dye. This requires that ionized carboxyl groups within the crystalline portions of the fiber reacted with hydrogen ions, while the bulky anions remained in the more accessible portions. This implies that ionic interactions in the fiber can be effective when the groups concerned are not in immediate proximity. A similar conclusion can be drawn from the absorption of equivalent amounts of monovalent and polyvalent acids by wool (Steinhardt et al., 1941, 1942a; Speakman and Elliott, 1946). Peters and Speakman (1949) suggest that the true pK, values for the carboxyl groups of wool lie near 4.6. If the ionized form of these groups was favored by what is equivalent to a zwitterion relationship with positively charged groups, a much lower pK, value would be expected, such as is observed with the a-carboxyl groups of free amino acids (Cohn and Edsall, 1943) or carboxyl groups adjacent to positively charged groups in polyampholytes (Mazur et al., 1959). Thus the presence of salt linkages in hydrated wool has not been established and this is in accord with the conclusion of Jacobsen and Linderstr@m-Lang(1949) that there are no salt linkages in proteins in solution. 3. Maximum Capacity of Wool for Acids and Alkalies

The maximum capacity of wool for acids, whether they be monovalent or polyvalent, small inorganic compounds or large organic compounds, is remarkably consistent. The capacity of wool for inorganic acids is usually considered to lie in the range 0.81 and 0.86 meq per gram (Speakman and Hirst, 1933; Steinhardt and Harris, 1940a,b; Olofsson, 1952). Examples for organic acids in milliequivalents per gram are as follows: 0.75 for the uptake of Crystal Ponceau (Pelet-Jolivet and Anderson, 1908), 0.85 for Orange I1 at pH 1.3 and 100°C (Smith and Harris, 1937), 0.87 for Metanil Yellow YK, and 0.94 for acid Orange I1 G (Speakman and Elliott, 1946). Goodall and Hobday (1939) recorded values for acid Orange I1 G (1.00), Orange G (l.lO), and the milling dye Polar Yellow R (1.10). Skinner and Vickerstaff (1945) obtained a value of 0.92 for Solway Blue B and of 2.10 at pH 1.2 for the milling dye Carbolan Blue B. In general, the milling dyes, which have a higher affinity for wool than leveling dyes, are more frequently observed to exceed the normal capacity for acids. Compounds of this type are considered to be bound as “a

260 w.

G . CREWTHER, R. D.

B. FRASER,F. G . LENNOX, AND H . LINDLEY

second layer” on the hydrophobic portions of the molecules bound initially. The constancy of the capacity of wool for different acids has led t o the view that the amount of anion bound is determined by the number of groups in the fiber capable of assuming a positive charge. Maclaren’s (1960) method for determining basic groups in wool depends upon this assumption (see Section 111,A15). As the groups capable of assuming a positive charge are approximately equal in number to the carboxyl groups, adherents of the salt linkage theory reason that the anions replace the carboxyl groups associated with positively charged groups when the carboxyl groups associate with hydrogen ions. Wright (1953) has shown that the acid-binding capacity of horn is almost identical with that of wool, but insufficient data are available, particularly with respect to amide content, to permit this value to be related to the amino acid composition of horn (Table XIV). Harrold and Pethica (1958) have observed that normal callus absorbs a little more than 1 meq of dodecyl sodium sulfate per gram in the absence of salt at pH 6.6 and almost 3 meq per gram a t a total ionic strength of 0.2. Binding continues to a maximum at a concentration above the critical micelle point of dodecyl sodium sulfate. There appears to be no relationship between the large absorption in the presence of salt and the amino acid composition of callus (Table XVI). If the amount of acid required for titration of the “acidic” side chains of wool is calculated from the amino acid composition of the fiber (Table X), the theoretical acid-binding capacity of the fiber is from 0.63 meq per gram for Merino 70’s to 0.69 meq per gram for Lincoln. The apparent discrepancy can be attributed largely to excessive values for the amide content of wool. These errors arise from the formation of ammonia by degradation of amino acids such as serine and threonine during hydrolysis. If the values of Leach and Parkhill (1956) for amide content are accepted (Table IX), the expected titration value becomes approximately 0.8 meq per gram. The amount of alkali bound by wool is less definite (Steinhardt and Harris, 1940a)b; Steinhardt and Zaiser, 1950; Horner, 1954) because (a) the high pH required for complete titration of the arginine residues causes extensive hydrolysis of aniide and peptide groups, even though a low temperature and long period of equilibration are used, and ( b ) in addition to hydrolysis of aniide and peptide groups the disulfide groups undergo hydrolytic and other reactions in alkaline solution. This adds greatly to the magnitude and uncertainty of the corrections which must be applied to the experimental results. After applying the appropriate corrections the amount of H+ removed from wool a t pH 14.29 was 0.83 mmole per gram (Steinhardt and Harris, 1940a,b). The aniount expected for coni-

26 1

T H E CHEMISTRY O F KERATINS

plete removal of protons from ionized histidine, lysine and arginine side chains, and the tyrosine side chains, is approximately 1.18 meq per gram (Table VIII). For determining directly the alkali-binding capacity of wool McPhee (1958a) has devised a method based on his observation that high salt concentrations protect the wool from alkali degradation without preventing reaction with ionizing side-chain groups. He obtained a value of 1.2 meq per gram for the alkali capacity.

4. The Titration of Wool The effects of salt concentration on the acid titration curve for wool are illustrated in Fig. 8. The broad isoelectric region observed in the absence of salt becomes less apparent with increasing salt concentration,

O N l C STRENGTH

0.8

0

1.0 0.5

B

0.2 0.1 0 0.04 8 0.02 e 0.01 0.005 o N O SALT 0

c \

>i3

0.6

-I

0

I

3

i

0.4

3 m E

s

-*

I

0.2

0

I

3

7

PH

FIQ.8. Combination of acid with wool as a function of the p H of solutions containing HCl and KCl at constant ionic strengths (Steinhardt and Harris, 1940a,b).

the curves changing shape and pivoting, as it were, around the isoionic point. Ionic strength has an important but a much smaller influence on the titration curves of soluble proteins (Cohn and Edsall, 1943; Steinhardt and Zaiser, 1955), but other fibrous proteins such as myosin show similar effects to those observed with wool. The titration experiments of Steinhardt and Harris (1940a,b), Steinhardt

262 w.

G. CREWTHER, R. D. B. FRASER, F. G . LENNOX, AND H. LINDLEY

et nl. (1940-1!)42b), Steirihardt (1!)42a,b), and Steinhardt and Zaiser (1950) have provided the following basic information: (u) Although with decreasing pII the acid uptake of wool falls off sharply at about 0.83 meq per gram there is evidence of further acid binding a t even lower pH values (Fig. 9). ACIDS

0 HYDROCHLORIC 0 NAPHTHALENE-B- SULFONIC 0 SULFURIC ( M E O ~ C D DIPHENYLSULF H O N I C 0 p-H YDROXYAZ OB ENZEN E-p?3JLFONK: 8 AN T H RAQU IN O N E-8- SU LFON I C @ p-DIPHENYLBENZENESULFONIC 8 ISOPROPYLNAPHTHALENESULFONIC

I

0

\

w

i 0.8 B I

B

v

DODECYL

SULFONIC

n

w 0.6

zrn I

0

-V

0.4

4

0.2

0 I

2

3

4

5

PH

FIG.9. Combination of various acids with wool as IL function of pH (Steinhardt e t al. 1942a).

( b ) I n the presence of high concentrations of salt the pH for half titration of wool by HC1 is displaced by approximately two pH units from the corresponding value in the absence of salt (Fig. 8). (c) The titration curves are displaced to higher pH values if the anions have a high affinity for the wool (Fig. 9). With acids such as dodecyl sulfuric and dodecyl sulfonic acids at 25OC the pH values for half-titration (ca. 4.0) approach pK, values for monovalent aliphatic carboxylic acids and for 0- arid y-carboxyl groups in soluble proteins. The affinities of various acids for wool are listed in Table XXIV. (d) Equivalent amounts of hydrogen ion and anion are absorbed by the fiber. (e) Salt effects and variations in cation affinity similar to those observed for absorption of H+ and anions are observed during the titration of wool with alkalies.

263

THE CHEMISTRY OF KERATINS

TABLEXXIV Anion Afinity Constuntsa

Acid

Molecular weight

p H of halfmaximum combination

98.0 97.1 36.5 126.1 80.9 63.0 152.2 158.2 172.2 174.2 186.2 163.4 203.2 178.0 237.6 98.1 227.1 248.2 208.2 245.1 229.1 314.2

2.155 2.22 2.32 2.33 2.47 2.58 2.58 2.63 2.66 2.66 2.71 2.73 2.86 2.94 3.07 3.08 3.13 3.17 3.24 3.64 3.86 4.24

-log KIA ~

Phosphoric Sulf amic Hydrochloric Ethylsulfuric Hydrobromic Nitric Isoamylsulfonic Benzenesulfonic p-Toluenesulf onic o-Phenolsulfonic o-Xylene-p-sulfonic Trichloroacetic o-Nitrobeneenesulfonic Pyrophosphoricb 4-Nitrochlorobenzene-2-sulfonic Sulf uricb 2,5-Dichlorobenzenesulfonic 2,4-Dinitrobenzenesulfonic Naphthalene-p-sulf onic 2,4,6-Trinitroresorcinol Picric Flavianicb$c

0.115 .20 .43 .44 .69 .89 .89 1.oo

1.035 1.035 1.12 1.16 1.39 1.53 1.76 1.78 1.87 1.94 2.06 2.73 3.08 3.67

KIA is as defined by Steinhardt and Harris (1940a,b) and values are calculated from the pH values of half-maximum combination a t 0°C. b The data given refer to combination with the divalent anion, but K’A is expressed, for purposes of comparison, as if the anion were monovalent. c The affinity given is minimal because equilibrium may not have been attained.

(f) Curves representing the relationship between pH and log A/ - A ) , where A is the amount of acid bound and A M is the capacity of wool for acid, are rectilinear a t constant ionic strength over a fairly wide pH range. At the pH of half-titration (pH+) they have a slope of -0.5 in the presence of salt concentrations greater than 0.01 M . In the absence of salt the slope is -1.0 (Fig. 10). (9) For titrations a t constant chloride concerltrations the relationship between pH, and log [Cl-] is rectilinear a t low ionic strength, but deviates at high ionic strength (Fig. 11). (h) The affinities of different anions and cations for the fiber are largely, but not solely, dependent on the size of the ion; for organic ions the length of the hydrocarbon chain is of particular importance. In general, acids (AM

2.0

-1.0

PH

FIG. 10. Relationship between log [A/(Av--A)I strengths (Steinhardt and Harris, 1940a,b).

2.0

-20

and p H for various ionic

0

-1.0

LOG

CHLORIDE

CONCENTRATION

FIG.11. Relationship between log CCI-I and pH+ for the reaction of wool with solutions containing HCl and KC1 at constant ionic strengths (Steinhardt and Harris, 1940a,b). 264

265

THE CHEMISTRY OF KERATINS

commonly used for precipitating proteins from solution are found to have a high affinity for wool (Table XXIV). (i)In mixtures of acids the amount of each anion bound is determined by the relative concentrations and affinities for wool as predicted by the law of mass action. Of relevance to the reaction of acids with wool is the demonstration of Larose (1953, 1955) that wool absorbs large amounts of dry HCl which he attributes to the binding of un-ionized molecules a t the peptide group. Larose and Donovan (1956) suggest that absorption of undissociated acid also contributes to acid uptake from aqueous solutions. Speakman and Stott (1934) made a similar proposal to account for the high capacity of wool for some weak acids. 6. Theories of the Combination of Ions with Wool

a. The Steinhardt-Harris Theory. Steinhardt and Harris (1940a,b) state the following as their basic assumptions in obtaining a theoretical relationship between pH and acid sorption: (a)Both H+ and Cl- combine with the wool proteins according to the law of mass action or the Langmuir adsorption law; ( b ) no recognition need be taken of differences in hydrogen ion concentration inside and outside the fiber; (c) variation in activity coefficients in the fiber phase may be disregarded. They consider the equilibria and mass action equations

+ H+$ WH+ (XV) W+ + A - e WA- (XVI) WH+ + A-= WHA (XVII) Wh

K A = [WH+l[A-I [WHAI

(3)

It follows that K H .KA

=

K'H KIA 3

In order to account for the near equivalence of anion and cation uptake it was assumed that K'A >> K A and K H >> KIH. If A is the amount of acid bound by the wool and A M is the amount bound when the wool is saturated with acid A _

A~ - [WHA]

[WHAI

+ [WH+l

+ [WH+l + [WA-I + [w+l

266

W. G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND 11. LINDLEY

Substituting from Eqs. (1) to (4)

A

1

where a H and aA are the activities of H+ and the anion A- in solution. i.e., at pH+, and when KIA>> a A >> K A , Equation When A / A M= (6) reduces t o

a,

pH+‘v PK‘H

+ log

UA

- log KIA

(7)

Equation (7) provides a satisfactory explanation for the experimentally determined relationship between pH+ and anion concentration (Fig. 11) for solutions of low ionic strength. From Eq. (6), however, when KIA>> UA >> KA,

A AM - A

-

(CII1+ a~

aA

aH ~

+ KA

1

~K’H

’ )

UA

. K‘A

and

With no added salt a H

=

ah, hence

On plotting log ( A / A M- A ) against p H these equations give slopes of -1.0 and -2.0, whereas the measured values given in Section IV,B,4(f) are -0.5 and - 1.0, respectively. By analogy with the titration of polyacrylic acid (Kern, 1939) Steinhardt and Harris (1940a,b) suggest that the twofold error in the slope of these curves is due t o the existence of a continuous range of pK‘H values resulting from electrostatic effects of ionized carboxyl groups. I n order to fit the experimental data Steinhardt (1942a) introduced the square roots of activities and dissociation constants as parameters in his equations, but this has no theoretical basis. b. Criticisms of the Steinhardt-Harris Theory. Peters and Speakman (1949) criticized the theory for its application of the mass action law t o the formation of ionic complexes which they consider are not real, and for its neglect of differences in ion concentration inside and outside the fiber. There is considerable evidence that the interaction of small ions with proteins follows the law of mass action (Scatchard, 1949; Klotz, 1953), and so

THE CHEMISTRY OF KERATINS

267

the objection is unlikely to be correct. The fault in the Steinhardt-Harris theory lies in the assumption that both anions and cations react at a single site, such as a salt linkage, the rupture of the salt linkage by reaction with an ion facilitating the reaction of an ion with opposite charge. The over-all equation H+ + A- + W * e WHA (XIXI leads inevitably to expressions giving an incorrect relationship between log (A/AM - A ) and pH. Regarding Peters and Speakman’s second criticism it follows from simple thermodynamic principles that the binding of ions in a solid phase in equilibrium with a liquid phase will be unaltered by the presence of a second liquid phase with which both are in equilibrium. It is not necessary therefore to take the internal liquid phase into account unless it is inseparable from the solid phase and of such dimensions that the analyses are affected by its presence. On the other hand, in experiments involving hydrolytic and other catalytic effects of H+, OH-, or other ions the “internal solution” cannot be ignored (Section IV,B,6). c. The Gilbert-Rideal Theory. Gilbert and Rideal (1944) assume that H+ and A- are bound unequally from very dilute solutions of HA until the resulting potential on the fiber is such that the over-all affinities of H+ and A- for the fiber are equal. As the excess of H+ which must be bound in order to produce such a potential is experimentally insignificant, essentially equal amounts of anion and cation are bound by the fiber. Gilbert and Rideal also assume that an anion is free to occupy any positive site that is not occupied already by an anion and that this freedom is not affected by its position relative to charged or uncharged carboxyl groups. Using the mass action terminology this could be written

[“W . COO-][H+] [“W . COOH]

-

[+W * NH,+][A-] =K [“W . NH,+A-]

“

where the superscript indicates the potential relative to the external solution. Fowler and Guggenheini (1939) have shown that the chemical potential or partial free energy 1.1 of an uncharged substance distributed a t random among a limited number of similar sites is given by

where e is the fraction of sitecoccupied and pais the value of .u at constant temperature and pressure when half of the sites are occupied. Taking account of the potential $ which was assumed to be constant over the whole fiber, the partial free energy of H+ within the fiber was given by

268 w.

G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

= PoH(T.P)fiber

MH

e

+

+ rC.F

In 1-8

(10)

and for the solution

+ RT 1nfH . [H+]

(11)

MH = M " H ( T , P ) ~ ~ I

where p o ~ ( ~ ~is, the ~ ~ )chemical s o ~ potential of H+ when the activity a t temperature l' and pressure P is unity and f H is the activity coefficient. If AM, = M'z(T,P)fibcr - M ~ ~ ( T , P ) ~on~ I equating , the chemical potentials a t equilibrium inside and outside the fiber.

RT In

OH

= -AMH

1-

~

+ RT In fH. [H+] - I,W

(12)

+ RT In

(13)

and similarly for a monovalent anion

RT ln

OA ~

1-

= OA

-A~A

f.4.

[A-]

+ @'

Adding Eqs. (12) and (13)

Assuming that the total number of acidic and basic sites in wool are equal and that equivalent amounts of anion and cation are absorbed a t any one time OH = @A Hence, for a pure acid Gilbert and Itideal obtain

or a t constant anion concentration

if effects of cations other than H+ are neglected and fE-I = j A = I. Equations (15) and (16) account satisfactorily for the slopes of the Curves relating experiniental values of log ( e H / ( 1 - O H ) ) aild pH. The relationship between pH, and log A-

~€14 log [A-I -

log e

is also satisfacbtory a t low ionic strength.

(APH 4-AMA)

(17)

269

THE CHEMISTRY OF KERATINS

From Eqs. (12) and (13) it follows that

For a pure acid since [H+] = [A-] this becomes -(APE - ApA)/2F, and if the affinities of H+ and A- for the fiber are equal the fiber is uncharged. In the presence of added salt a more complex expression holds, and the charge varies with salt concentration. d. Criticisms of the Gilbert-Rideal Theory. Peters and Speakman (1949) have criticized the Gilbert-Rideal theory because Eq. (17) suggests that there is no limit to the displacement of pH, with increasing salt concentration. Gilbert and Rideal clearly stated, however, that this equation applies only to dilute salt solutions, since it does not consider the effects of cations other than H+. At high salt concentrations the equation relating log [A-] and [H+]a t half-titration is

which reduces to Eq. (17) only at insignificant values of the term relating to “a+]. The deviation from rectilinearity of Fig. 11 is therefore predicted by the Gilbert-Rideal theory. Peters (1954) has further criticized the Gilbert-Rideal theory because the experimental values for ( A ~ H A&J/2.3RT and ( 2 A p ~ Ap804)/2.3RT calculated from data of Olofsson (1952) and Steinhardt and Harris (1940a,b) vary with pH. This means that the data cannot be accommodated by a single dissociation constant (Peters and Lister, 1954). Variations of 9 in different parts of the fiber could account for these small variations in affinity at different pH values. Such variations in the affinity of wool for acids may account in part for the slightly sigmoid character of the relationship between log [O/(l - O ) ] and pH for titration with HC1 that Peters and Speakman (1949) list as a criticism of the theory. This deviation from a rectilinear relationship between log [O/(l - O ) ] and pH is not apparent with all acids (Steinhardt et al., 1941). Horner (1954) constructed a theoretical curve for the titration of wool with alkali using amino acid analyses and the pK, values obtained for the side-chain groups of lysine, tyrosine, and arginine in the form of the free acids. He obtained a better fit using the Donnan expression of Peters and Speakman (1949) than he did with the Gilbert-Rideal (1944) expression. Obviously, much depends on the pK, values used; hydrogen bonding in a protein structure can shift these values by more than one pH unit. Also, the assumption was made that the affinity of sodium ions for the

+

+

270 w.

G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND Ir. LINDLEY

protein is zero. IIorner's conclusions are therefore open to some doubt arid the Gilbert-ltideal expression is probably satisfactory when used for monovalent anions and for the ionization of a single type of side-chain grouping in the fiber. In extending their theory to polyvalent ions, Gilbert and Rideal assumed that binding of each anion occurs a t a single positively charged site and that all the positively charged side-chain groups can function equally well as binding sites. This led to the equation for anions with charge z

In developing this equation the assumption that the afinities of monovalent anions and cations are equalized by the charge developed on the fiber is neglected. When all the carboxyl groups in the fiber are un-ionized there will be sites available for binding of AB- ions equal in number to l/zth the total number of charged basic groups, and the binding will take place with essentially the same free energy change for each site. Once these sites are filled, any further absorption of anions involves a decrease in the charge of the fiber and therefore of the affinity of the anions for the fiber. Because the Fowler-Guggenheim equation is based on the assumption that all sites have an equal affinity for the adsorbate, it is incorrect to use &/(i - e,) as the activity of a polyvalent ion i, where Bi is the fraction of all the basic sites occupied singly by anions. The activity should be expressed as &/[(l/z) - &I. As 0; = BH/z, this reduces to 8H/(1 - OH). Equation (20) then becomes

Olofsson (1951) also neglected this factor in obtaining a n equation for the amounts of sulfate and chloride bound by wool from a mixture of HC1 and H2S04. I n subsequent papers (Olofsson, 1952, 1954, 1956) he derived a n equation, identical with Eq. (21), based on the alternative assumption that each sulfate ion occupies two positively charged sites. The predictions of the two models were compared with experimental data, but no firm conclusion could be drawn. In part this may have been due to inaccuracies in the determination of the amounts of the various ions bound by the wool. Another criticism of the Gilbert-Rideal theory is its neglect of variations in the activity of water inside the fiber consequent upon the absorption of acids, bases, or salts. This criticism applies also to the Steinhardt-Harris

27 1

THE CHEMISTRY O F KERATINS

theory and t o the simplified form of the Peters-Speakman expression discussed below. A more rigorous expression derived by White (1955) takes into account all these factors, but insufficient accurate experimental data are available a t present for it to be applied. A further difficulty is the uncertainty as to activity coefficients of ions within the fiber. The experiments of Shore (1963) with cations in gelatin gels illustrate some of the difficulties likely to be encountered in investigating activity coefficients of ions in a protein gel. e. The Peters-Speakman Theory. This theory is often termed “the Donnaii Theory,” but it is only one possible application of the Donnan theory t o the titration of wool. Peters and Speakman (1949) assume that the external solution is in equilibrium with an internal solution and that as a result of preferential adsorption of H+ a t low acid concentrations the fiber is a t a potential $, assumed to be constant over the whole internal volume v. It is also assumed that the anions and cations entering the fiber are equivalent thereby maintaining J/ constant. I n mass action terminology this may be expressed

KH * [$W. COOH] = [+W * COO-][H+] [+H+]= [H+]/X [GA-] = X[A-] Where KH is the dissociation constant of the carboxyl groups, X is a constant [= exp (J/F/RT)],and the superscript $ indicates the potential of the solution or solid phase relative to the external solution. The system and terminology used by Peters and Speakman are shown in the following tabulation:

1

Internal phase concentrations

Total acid-binding capacity -COOH

A

-NHs+ H+ H,X(s-+O-

A

Ae

-coo-

A(1

External phase concentrations

- 8) H U X

h u

*-

X

Internal volume

V

1

3;xternal volume

1’

Applying the Donnan-Guggenheim (1932) expression for the electrochemical potential of hydrogen ions in the external solution pH =

+ RT 1 n H + RT InYH + I‘VE

pHo(T)

(22)

272 w.

G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

where yH is the activity coefficient and ~ T isH the partial molar volume of the hydrogen ion a t pressure equal to the mean pressure of the two phases. I n the internal phase ph

=

ph"(T)

+ RT In h 4- RT In + pvh + W Yh

(23)

A t equilibrium these are equal, and assuming that Mho(T)and pH"(T),and PHand v h are also equal we obtain by substituting ( R T / v w )In (w/W) for P - p , where W and w are the activities of water in the external and internal phases and mean of P and p

vwis its partial molar volume a t a pressure equal to the

+

YH.H R T . -1nRT In Yh

'

h

F H

vw

w W

=

F$

Hence

where r H is the ratio of the partial molar volume of the hydrogen ion and water. The corresponding expression for polyvalent x1- is

The assumption of electrical neutrality requires that

Ae

+ h = zx + 2 ( z - n)u

and

H

=

ZX

+ Z(Z - n)U

Furthermore, the amount of acid combined with the fiber, a', as determined by the difference in the amount originally present and that remaining in the external solution, is given by a'

=

v(A0

+ h + Znu) + V(H + 2 n U ) - (V + v)(H + ZnU)

In obtaining the last term it is assumed that the internal and external concentrations of H+ are equal, but the error is negligible because the value of v for wool immersed in aqueous solutions is very small. When these equations are combined and it is assumed that the acid is sufficiently strong to give insignificant values of u and U, the expression

THE CHEMISTRY OF KERATINS

273

is obtained, where

Peters and Speakman assumed that the activity constants of H+, X-, and the activity of water are identical inside and outside the fiber. The expression for a monovalent acid then becomes

H being small compared with a’/v.

In the presence of added salt

for low ionic strengths. Peters and Speakman have shown that the internal titration curves obtained with HC1 and with HC1-KC1 mixtures are almost identical and that a similar curve is obtained with HzSO, if appropriate corrections are made for the formation of HS04- at low pH values. It is apparent, too, that the relationship between pH, and anion concentration is given by pH+ = log X

+ constant

for low ionic strengths; the more exact expression for this relationship at high ionic strengths accounts for the deviation of Fig. 11 from a rectilinear relationship. f. Criticisms of the Peters-Speakman Theory. Olofsson (1952) criticized the theory for its inability to account for the different affinities of various anions for wool. He investigated experimentally the system wool-HClH,S04-water and calculated values of log K where

K

= a2cl,liq aso,,iiber/a2cl.riber.aso4,liq

The Speakman-Peters theory predicts a value of 0 for log K , whereas the Gilbert-Rideal theory could accommodate various values, depending on the relative affinities of the two ions for wool. Olofsson claimed his results showed that the value of logK is not 0. Peters (1954), however, considered that the accuracy of Olofsson’s data was insufficient to decide the point. The lack of precision is apparent from the fact that the amounts of C1- and SO4- bound by the wool differed markedly from the corresponding H+ uptake. There has been prolonged discussion on this point by Olofsson (1952, 1954, 1956) and Peters (1954, 1960) despite the fact that Steinhardt et al. (1941) had shown conclusively that different acids, and therefore pre-

274 w.

G.

CREWTHER, R. D.

B. FRASER,

F. G. LENNOX, AND €1. LINDLEY

suniably different anions, have different affinities for the fiber. Confirmation is provided by the careful experiments of Larose and Donovan (1962, 1963) who showed that the affinity of mono-, di-, and trichloroacetate ions for wool increases with increasing chlorine content. The binding of Rr- by horn has likewise been demonstrated by Wright (1950, 1953, 1954). Having first shown that the titration behavior of horn closely resembles that of wool (Wright, 1950) he studied self-diffusion rates of radioactive Br-, membrane conductivities, membrane potentials, and equilibrium sorption for the system, horn keratin-HBr (Wright, 1953, 1954). The measured membrane potentials were consistent with those calculated from self-diff usion/conductivity data. He was then able to show that as the amount of acid absorbed by horn was increased from 2 to 8 mmole per gram the ionic mobility of H+ in the horn increased some 600-fold, the change being most rapid near 0.8 mmole per gram, the saturation value for horn. The mobility of Br- increased fairly uniformly about threefold over the same range of acid absorption. This suggests that both H+ and Br- are bound by the solid phase, the fraction bound decreasing with increasing acid concentration. Wright also confirmed the equivalence of the number of cations and anions absorbed and showed that the ionic mobilities of Na+ and Br- were only about 1/1000 that expected from self-diffusion experiments. He concluded that diffusion was through a medium whose properties were not those of liquid water. This emphasizes our lack of knowledge concerning the activity of water inside the fiber. Hudson (1954), on the other hand, having obtained values of the same order for the diffusion of anions through wool as Wright obtained for diffusion of Br- through horn, concluded that the data is consistent with diffusion through a n aqueous phase. He considered that the diffusion rate is controlled by a small concentration of H+ in the aqueous phase. It is unfortunate that some of the primary data are not reported in these papers. Peters and Lister (1954) and Peters (1960) clearly recognized that anions differ in their affinities for wool, and in the latter paper Peters suggests that his revised model embraces both the Gilbert-Rideal and PetersSpeakman theories. In developing equations for K X and K M ,however, the dissociation constants for binding of anions and cations from the internal solution, Peters both fixes the value of the dissociation constant K x at zero and treats it as a variable. This follows from the assumption that d,the internal concentration of anions a t half-titration, is fixed at 1.5 if the internal pH of half-titration is fixed a t 4.6 which is true only if the wool has no affinity for the anions. As a result many conclusions reached in this paper require reassessment. For example, Peters concludes that the dissociation constants for cations and anions must be

THE CHEMISTRY OF KERATINS

275

approximately equal. It would be difficult t o accept such a conclusion for a cation such as Kf and an anion such as dodecyl sulfate. g. The Composite Theories of Vickerstaf and of Delmenico and Peters. Vickerstaff (1954) attempted to combine the Gilbert-Rideal and PetersSpeakman theories by considering the equilibrium between the internal solution and the solid phase assuming that the solid phase and the internal solution are a t the same potential J.. He obtained the equation

- (ApHO

+ ApxO) = R T In 1 -

OH

~

OH

*

Ox - EZTlnH1 - Ox

-~

The chief difficulty lies in assigning values to Ox. If it is assumed that OH/(1 - )0 , = &/(I - Ox), the expression becomes identical with the Gilbert-ltideal expression. If this assumption is not made the application of the Fowler-Guggenheim expression raises difficulties similar to those already discussed in connection with Gilbert and R.ideal's treatment of the uptake of polyvalent ions. Vickerstaff (1954) also derived an expression corresponding to the Peters-Speakman assumption that AFXO = 0. Since Ox/(l - Ox) approaches x as Apx0 approaches 0 we have

and as vx

=

vh

+ AOH and AOH >> vh APHO

2.3RT

A OH' log - = log ___ V 1 - OH

+ 2pH

The relationship between log [ e H z / ( l - OH)] and p H obtained from the data of Steinhardt and Harris (1940a,b) for reaction of HC1 and wool was slightly more nearly rectilinear than the corresponding relationship between log [O,/(l - OH)] and pH. The slope of the former curve, however, was 0.7 of the calculated value, whereas that of the latter was 0.9 of the calculated value. It should be recognized that Eq. (29) is based on the questionable assumptions that anions are as free to move in the internal solution as they are in the external solution and that the internal solution and solid phase are a t the same potential #. Delmenico and Peters (1962) have proposed a further variation of the Donnan equilibrium which they claim provides a means of evaluating J. and v. They assume that cations other than H+ have zero affinity for the fiber. Since it is impossible to determine the affinity of a cation for wool independent of the anion and since cations have variable affinities for wool (Steinhardt and Zaiser, 1950) there are insufficient data to decide whether

276 w.

G . CIIEWTIIER, K. D. B. FRASEI~, F. G. LENNOX, AND H. LINDLEY

the affinity of the particular cation used is sufficiently low for the theory to be approximately correct. h . Concluaions. The Gilbert-13ideal (1944) theory provides a relationship between the eqnilibriuni uptake of a wide range of acids on wool aiid the pH of the solution whicah is hi satisfactory agreement with experimental results. The theory is not conccriied with aiid is uiiaffected by any iiitcrmediate liquid phase if the internal volume is small. The Peters-Speakiiiaii theory on the other hand considers the equilibrium between the external solution aiid internal liquid phase of the fiber. Eecause the internal solution concentrations are determined by the interac%ionwith the solid phase and it is assumed in deteriiiiniiig these concentrations that the anioii has iio affinity for the fiber, the Peters-Speakinan expression is applicable only to acids in which the ariioii has negligible affinity for the fiber. If certain assumptions are made regarding the binding of anions within the solid phase, a niore comprehensive Doiinan expression can be derived. The Dorinan expression is particularly useful if information regarding the concentration of certain ions in the internal solution is needed. 6. The Internal Solution

Steinhardt arid lh g itt (1‘342) observed that for a particwlar acid coriwntration the amount of aiiinioiiia produced by hydrolysis of aniide side-chain groups in wool increased with increasing affinity of the anion. A similar increase in hydrolysis of amide arid peptide groups occurred with increasiiig salt concentration (Peters arid Speakman, 1949). This “catalyzed hydrolysis” was attributed by Steiiihardt and Fugitt (1942) to partial adsorption of anions a t the arnide-bearing side chains, thereby facilitating reaction of the arnide groups with hydrogen ions. Additions of dodecyl sodium sulfate to 0.05 M HC1 did not greatly affect the rate of hydrolysis until a concentration of 0.008 M was reached. The rate then increased greatly until a coilcentration of 0.02 M dodecyl sulfate was present. Further increase in roncentratioii did riot increase aiiiide hydrolysis, but the rate of hydrolysis of peptide bonds continued increasing. Steirihardt aiid Fugitt (1942) suggest that low concentrations of dodecyl sulfate favor ainide hydrolysis. The total amount of dodecyl sulfate present a t 0.008 M was equivalent to the ionized basic groups of the fiber and a t 0.02 M it was equivalent to basic groups plus amide. They assumed almost complete absorption a t both concentrations, but this is unlikely to be correct. Coniplete absorption of dodecyl sulfate would have involved an uptake of almost 2.0 meq per gram whereas Griffith (1961) found a maximum uptake of 0.99 meq per gram. The difference between rates of hydrolysis of peptide and sliiiide is probably related to the disaggregating and solubilizing effects of the dodecyl sodium sulfate a t the higher roncen-

THE CHEMISTRY OF KERATINS

277

tratioiis used. A more plausible explanation by Peters and Speaknian (1949) attributes the “catalysis” to an increase in the hydrogen ion concentration of the internal solution with increasing salt concentration or with increasing affinity of the anion for wool. Confirmation that acid daniage to wool is increased by increasing ionic strength was provided by Perynian (1954, 1956, 1957) who found a siniilar effect of salt a t low conceiitrations on alkali and acid daniage to wool. In particular the disulfide content of the fibers was decreased by the alkaline treatnieiit. Peryniaii attributes this to the greater uptake of alkali or acid by the wool in the presence of salt (Steinhardt and Harris, 1940a,b). Crewther and Dowliiig (1965) observed a similar major effect of ionic strength on the forniation of lanthioniiie in wool and other proteins a t pH 9.0. They showed that, irrespective of ionic strength, the points relating alkali uptake and larithionine formation fell on a smooth curve. Also lanthionine forniatioii was increased greatly in the presence of a cation with high affinity for the fiber. The results were attributed to an increase in the activity of hydroxyl ions in the internal solution or a t the liquid-solid interface with increased ionic strength or affinity of the cation. The concept of internal pH together with a suitably modified Doiiiian theory t o take into account ion affinity can be useful therefore in predicting catalytic effects of ions in solution. It docs not necessarily follow that the internal solution is a clearly defined phase. Alexander and Kitcheiier (1950) consider it in ternis of an electrical double layer, the positively charged solid being associated with a layer of solution containing more aiiioris and less cations than the bulk of the solution. The same general conclusions apply as in the case of the internal solution but quantitative treatnierit of the model is more difficult. 7 . Effects of Salts on Swelling and Chemical Reactiuity

In addition to the effects of increasing ionic strength comnion to all salts, and that of cations with high affinity on the forniatioii of lanthionine, specific ion effects are observed a t caoiiceiitrations (4 M ) too high to be explicable in ternis of “internal pH.” For example, ions such as Li+ and I-, which tend to swell and supercontract wool, give greater rates of lanthionine formation than ions such as K+ or C1- (Dowling and Crewther, 1965). This has been attributed to the greater reactivity of disulfide bonds when they are under stress as a result of swelling or ronforniational changes in the proteins (cf. Lindley and Human, 1957). The stabilization of wool a t pH 11 in the presence of cetyl triiiiethylariinioriiuIii bromide, as assessed by susceptibility to proteolysis (Crewther, 1956), has similarly been attributed to decreased nct-negative charge with a consequent decrease in swelling.

278 w.

G . CREWTHEK.,

n.

D . B. FHASER, F. G . LENNOX, AND H . LINDLEY

Shimizu and Oku (1957) studied the effects of salts on the solubility of wool in 0.1 M KOH. At low salt concentrations the effects of various ions followed the Hofiiieister series. Similarly, McPhee (1958b, 1959) has shown that whereas 56 % of wool was dissolved by 1.286 N NaOH a t 25°C in 2 hr, only 2 yo dissolved when the solution was first saturated with NaC1. There was a corresponding decrease in formation of primary amino groups and in loss of cystine. Not all salts were equally effective in protecting wool against alkali damage. The effectiveness of 2 M solutions of the sodium salts decreased in the order SZO3- > SO3= > citrate > COs’ > SO,= > acetate > C1- >_ Br- > NO3- > I- > CNS-. Cations followed the order Lit, Na+ > K+. Similar rates of alkali uptake were obtained with all salts at a concentration of 2 M . McPhee concludes that the effects of salts are not attributable to differences in rate of alkali upt,ake owing to differences in swelling. In saturated solutions of some salts, however, alkali uptake was retarded, and this may contribute to the protection of wool from alkali damage in very concentrated solutions. He showed that there was a direct relationship between the amount of alkali damage and the extent of swelling in the salt solution. The water absorbed preferentially by the fiber from each solution was determined by nieasuring the concentrations of solutions before and after equilibration with wool. In general, least preferential uptake of water occurred with solutions causing greatest swelling; only one of the solutions caused greater swelling than water. McPhee has interpreted these data in terms of Gibson’s (1934) concept of “effective pressure” exerted by salt solutions. This concept was used by McDevitt and Long (1952) to explain the “salting out” of nonpolar solutes from aqueous solution. Increases in the attractive forces between solvent molecules owing to polarizing effects of ions are considered equivalent to the imposition of an external pressure. Highly hydrated ions such as SO4=or Li+ increase this “pressure,” ions such as CNS- which tend to decrease solvent structure, decrease the “pressure.” McPhee (1959) suggests that the “solvent pressure” opposes swelling of the fiber and shows a direct relationship between the extent of swelling in 2 M salt solutions and the “solvent pressure” calculated from voluine changes during solution of the salt. This view i s open to a number of criticisms (Crewther, unpublished observations, 1964). ( a ) There are large differences between the dielectric constants of various salt solutions a t a concentration of 2 M (Robinson and Stokes, 1959). The values increase froin 34.3 to 58.3 over the series NaS04 < LiCl < NaCl < KCI. As swelling of an amphoteric polymer in neutral soIutions would be expected to be directly related to the dielectric constant of the

T H E C H E M I S T R Y O F KERATINS

279

medium, McPhee’s results could be explained adequately on this basis alone. I n fact any characteristic of the solution which is directly related to the extent of hydration of both ions in solution can provide a n apparent explanation for the effects of salts on the swelling of wool. ( b ) Absorbed salts have a major influence on the relationship between partial pressure of water vapor and regain of the wool-salt complex (Barnard et al., 1954). Insufficient data are available to predict relative effects of a large range of salts, but large differences have been demonstrated between the effects of LiBr and NaBr. ( c ) Differences in the water activities of the different solutions have been neglected. These alone cannot account for the observed swelling, but they would have an important effect (Barnard and White, 1954; Speakman and Whewell, 1936). ( d ) Wool itself is highly hydrated. Hence an “effective pressure” would already be established. No account has been taken of this fact. ( e ) The wool fiber is permeable to both solvent and ions (Barnard and White, 1954; Barnard et al., 1954). Hence there is no reason to assume that decreased bond lengths of secondary bonds in the solvent will decrease the over-all dimensions of the protein network constituting the fiber. McPhee (1960) has shown that high salt concentrations also retard the reaction of oxidizing agents with wool, arid Williams (1962) has extended this work to the reaction of permanganate with wool. Salt concentrations greater than 1 M decreased the rate of the reaction between wool and KMn04, but the subsequent clearing of the wool with bisulfite was accelerated by salt up to a t least 6 M . McPhee (1958b) found that NaCNS was the only salt tested a t 2 M concentration giving greater swelling than water (19% diametral swelling compared with 18 %). Barnard and White (1954), on the other hand, found that human hair swells more in concentrated solutions of LiC1, NaBr, or LiBr than it does in pure water. The swelling, defined as 100(V8 - V,)/V,, where V , is the fiber volume in water and V , is its volume in solution, increases with increasing salt concentration to values of 25 yoin 19.5 molal LiCl and 4.5 yoin 5.1 molal NaBr. Barnard et al. (1954) have determined directly the uptake of salts by human hair from aqueous solution and also the water content of the saltimpregnated hair after equilibration with the water vapor above the same solutions. The amounts of NaBr, KBr, and LiBr absorbed increased with increasing salt concentration and the relationship between the activity of the salt in the external solution and uptake was identical for each of the three salts. A different relationship held for NaC1, and Barnard et al. (1954) concluded that the anion is largely responsible for determining

280 w. G .

CREWTHER, R. D. B. FRASER,

F. G. LENNOX, AND 1-1. LINDLEY

the aniount of salt absorbcd a t a particular concentration. Iii studies 011 the sorption aiid desorption of radioartive ions by wool Underwood and White (1954, 1961) obtained evidence that SO4= is bound inore strongly than Na+. The uptake of water vapor by fibers contaitiiiig absorbed salt follows a differelit regain/relative huiiiidity isotherm for different salts and is greatly influenced by the cation (Rariiard et a / . , 1954). In the presence of absorbed salt the regain a t any value of’ relative humidity is greater than the corrcspoiiding regain when no salt is absorbed. The “interiial molality” of the salts, however, was greater than the external inolality of the solution in equilibrium with the fiber iiidicating that the salts were preferentially adsorbed. 8. Supereontractzon of Wool in Snll Solutions

The observation by Harrison (1937) that wool fibers contract and bcronie rubberlike when treated with solutions of ZnC1.L was followed by demonstrations that salts of silver and mercury have similar effects (Elod et al., 1942) and that fibers (.ontract in solutions of cupraninioniurii hydroxide, but return to their original length if iiiiniediately washed in dilute HCl (Whewell and Woods, 1944, 1946; Leveau, 1959b; Sotiriou-Provata aiid Vassiliadis, 1961). Nickel aiiinioiiiuin hydroxide (Be11 and Whewell, 1952, 1!)58) aiid other coordinate complexes of Cu++ (Vassiliadis, 1957, 1958; Whewell et al., 1959) have similar effects. Alexander (1951) has reported a reversible contraction in solutions of LiBr and he believed litliiuiii salts to be unique in this respect (Alexander aiid Hudson, 1954). He suggested that in solutions of LiBr sufficiently concentrated t o restrict the hydration of I,i ions, hydrogen donor groups in the proteiii such as NH or OH groups could be “draw11 within its coordination orbit.” This would involve breaking hydrogen bonds in the protein structure. A solution containing 50 giii I,iBr/100 giii water, the niininiuni cwnceiitration for supercontracting wool a t 85”C, (*ontains about ten water inoleculcs to each molecule of LiHr; Alexander suggests that this is a rcasoriable value for the riuinber of water molecdes iri the hydration shell of Li+. Crewther aiid Dowliiig (1956) showed that in fact a range of salt solutioiis caused wool fibers to supercontract. The effectiveness of aiiioris was in the order: acetate, formate < C1- < Br- < CNS-, I- and for cations K+ < Na+ < Li+ < Sr++ < Ca++. The results therefore resembled the effects of various ions on the shrinkage of collagen (Katz and Weidinger, 1933; Lennox, 1949), although much higher concentrations were required for supercontraction of wool. Crewther and Dowling pointed out that whereas the most highly hydrated rations are most effective in supercontracting wool, the least hydrated aiiioiis (Latimer, 1955) are most effective.

T H E CHEMISTRY O F KERATINS

28 1

Alexander’s (1951) explanation of the effects of Li salts is therefore inadequate for salts in general and cannot explain the large differences in concentration of LiI, arid LiC1 required for supercontraction of wool or the failure of lithium acetate and lithium forinate to superrontract wool a t concentrations up to 12 M . Crewther and Dowling (1956) suggested that the niore hydrophobic anions may be strongly absorbed on the protein, so increasing its net negative charge with consequent increased swelling and stress on the secondary bonds stabilizing the conformation. It was suggested that polarizing cations such as Lif would tend to increase the structure in the solvent and so favor adsorption of the niore hydrophobic anions. This hypothesis was tested using collagen as the contractile protein in order to avoid complications owing to the effects of pH on the stability of the disulfide bonds. At a pH value below the isoionic point increasing concentrations of salts such as Ca12, LiI, LiBr, NaI, and NaBr caused first ari increase in the stability of the collagen, then as the concentrations were further increased a decrease in stability (Crewther and Dowling, 1958). On the other hand at a p H value above the isoionic point the stability of the collagen decreased continuously with increasing salt concentration. The occurrence of a niaxinium in the stability curve a t the lower pH was predicted from the hypotheses of preferential anion adsorption. If the net charge on the protein were the only factor determining its therinostability, however, the same level of niaxinium stability should be observed with all salts. In fact there were large differences. Differences in the hydration of the cations absorbed as gegenious were considered an additional factor determining the stability of the collagen. Crewther and Dowling at present take the view that the stability of a fibrous protein in a salt solution is determined largely by the nuniber of sites available for hydration on ions bound by the fiber or absorbed as gegenions. The net vharge on the fiber is important in that it determines the nature and number of gegeriions absorbed. This hypothesis is in close agreement with the results and views presented by Barriard et al. (1954). It is also significant that salts of anions shown by Steinhardt et al. (1941, 1942a) to have a high affinity for wool are able to supercontract the fiber, whereas little or no contraction is obtained with ariioiis of low affinity. The sodium salts of trichloroacetic acid (Crewther and Dowling, 1956) and nitrated phenols (Crewther, 1959) are examples of the former; acetate is an example of the latter. Mandclkerri et al. (196%) like Alexander (1951) stress the importance of the lithium ion in the reaction of LiRr with proteins and consider possible binding of Li+ a t the peptide group. Like Be110 and Eello (1961, 1962) they consider the possible formation of an alternative crystalliiie structure

282 w.

G . CRMWTHER,

R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

in the protein involving Li+. The disappearance of the X-ray diffraction pattern for wool fibers in solutions of LiBr once the first stage of supercontraction is complete (Haly and Snaith, 1960) does not support this view. On the other hand Hambraeus and Steele (1951) claim that a t room temperature in LiBr solutions a t concentrations greater than 5 M , a new X-ray diffraction pattern is obtained. There can be no doubt that under certain conditions the cation reacts directly with the protein of the fiber. In reactions involving metal animonium and similar complexes (Whewell and Woods, 1946; Vassiliadis, 1957, 1958) the fiber is heavily stained with metal ions which can only be renioved by washing in acid. On the other hand, it is difficult to conceive the formation of similar strong interactions between potassium ions and the protein; yet solutions of KI readily supercontract wool. Even in the reaction of cuprammoniuni hydroxide with wool, the associated anion has a major influence on the amount of Cu++ bound and on the rate of contraction (Sotiriou-Provata and Vassiliadis, 1961). Although binding of Cu++ appears t o be essential for contraction of the fiber, there is no simple relationship between the aniount of copper bound by the fiber and the contra(.tion rate. Wool fibers do not contract a t all in solutions of ciipranimoniuni sulfate prepared by adding excess concentrated ammonia t o concentrated solutions of copper sulfate (Crewther, unpublished data, 1960) owing to the presence of sulfate ions. It has been demonstrated that LiBr complexes with urea (Geschwind, 1960) and also forms a crystalline complex with N-niethylacetamide in which each I,i+ is surrounded by four carbonyl oxygen atoms arid two oxygen atoms of water, while each Br- is surrounded by four NH groups and hydrogen atoms from two water molecules (Bello and Bello, 1961, 1962). Thus both ions are bound by groups similar to those found in peptide linkages or ainido side chains. These experinients, however, tell us nothing about the relative affinities of anions and (sations for the organic, niolecule. l~nrtherniore,infrared studies show that the hydrogen bonds between groups such as -OH or =NH and C1- are stronger than the corresponding bonds with I- (Waldron, 1957, Allerhand and Schleyer, 1963). Hence the much greater effect of I- than C1- on the conformation of proteins cannot be explained on the basis of hydrogen bonding with the N H of the peptide group. The uptake of ions niay be complicated by changes in the conforination of the protein such as may occur during swelling or supercontraction. For example, Hojo and Sugawara (1958) report a 60 % increase in uptake of Cu++ from CuS04 solution when wool fibers are stretched by 50 yo. The uptake of metal ions is awompanied by the release of hydrogen ions (Hojo and Hojo, 1958) from the fiber. The presence of natural pigment in animal

T H E CHEMISTRY O F KERATINS

283

fibers also facilitates binding of Cu++ or Fe++ from solutions of the corresponding sulfates a t pH 2.4 (Laxer and Whewell, 1953). It is apparent that no single hypothesis can account for all the effects of salts on the properties and reactions of a fibrous material such as wool. Salts have a general mass action effect on the uptake of acids and alkalies from solution, they can cause swelling or supercontraction of the fiber, an effect that decreases with increasing hydration of anion and increases with increasing hydration of the cation; a t high concentrations they may also decrease swelling. The latter effect may be due in part to removal of water from the fiber.

C. Photochemical Reactivity 1. Photochemical Changes in Wool

It has long been known that wool slowly decomposes on exposure to sunlight (Loebner, 1890). Much of the early research on the damage of wool by light has been reviewed by Hildebrand (1959). Sulfuric acid was identified early as a degradation product, and it was noted that during exposure the wool became progressively more swollen when immersed in 0.1 N NaOH (von Bergen, 1925). Exposure to a glass-enclosed carbon arc was found to increase the alkali solubility and decrease the concentration of cystine (Smith and Harris, 1936; Harris and Smith, 1936). NHOand Hi3 were evolved during irradiation in the absence of oxygen, but in the presence of air most of the H,S was oxidized to HzS04. Cysteic acid was also produced. The formation of H,S during irradiation in nitrogen was increased by the presence of moisture (Harris and Smith, 1938). Wool was shown to yellow approximately ten times as rapidly in the wet state as in the dry state (Milligan and Tucker, 1962). Modifications of the Allworden reaction (von Bergen, 1930), enhanced staining with Pauly’s reagent (Rimington, 1930), and increased dye uptake (Haly, 1958) following exposure to UV light have been reported. It is worth noting that whereas heat damage is retarded, UV irradiation damage to wool is acrelerated by the presence of acid (von Bergen, 1929). Yellowing has been ascribed to the degradation of tyrosine (Lundgren, 1956), tryptophan (Graham and Statham, 1956), proline (Hildebrand and Kersten, 1959), and cystine (Lennox, 1960), but complete amino acid analyses of wool yellowed by sunlight, by the 254 mp mercury vapor lamp, by a Sun Lamp, or by a Xenotest apparatus (Inglis and Lennox, 1963) showed no quantitative correlation between yellowing and destruction of any particular amino acid when the various methods were compared. Only tyrosine and tryptophan were degraded by all the sources. Free radic.als have been deterted in wool by their ability to initiate

284

W. G . CREWTHER, It. D. B. FRASEIl, F. G . LENNOX, AND H. LINDLEY

polynierization (Crawshaw and Speaknian, 1954) and by the signal obtained iisiiig the clcctron-spin r c s o ~ ~ a ~ technique icc (Rogle ct nl., 1962). The ESH, signal has bcen shown to increase in magnitude during UV irradiation. The cwbonyl content of the wool is also increased (Inglis, uiipublished observations, 1!)63). Wool exposed to sunlight in the presence of tritiated water uiidcrgoes irreversible tritium exchange a t CH sites in the prolyl, aspartyl, caystyl, and glycyl residues. These may be points of entry of energy into the wool proteins (Leach and Holt, unpublished observations, 1964). Possible exchange in tryptophyl residues was not investigated. Photodegradation of wool with the 254 mp mercury vapor lamp has been described as a n oxidative process involving partial destruction of cystine, tyrosine, tryptophan, and histidine (Maclaren, 1963). No relationship between the extent of cystine degradation and yellowing was found. Irradiation of wet wool fabric with 20 m p wavebands of UV light between 260 nip and 410 nip showed that yellowing increased as the wavelength decreased (Inglis and Lennox, unpublished observations, 1964). This was accompanied by a small decrease in the content of cystine, tryptophan, and tyrosine and an even smaller decrease of methionine. The eysteic acid content increased with increasing destruction of cystine. Spectroscopy has also been uEed to study the damaging effects of UV irradiation. Horsehair exhibits an absorption niaxiniuni a t 278-279 nip which is slightly higher than the value for tyrosine in aqueous solution (Bendit, 1960a). By niinirrsiiig wool in a liquid of equal refractive index, such as o-dichlorobenzene, arid obtaining a differential absorption curve it was possible t o drnionstrate the appearance of an absorption maxiniurn a t 310-330 nip during U V irradiation (I eiinox, 1959). Visible yellowing is presuniably due to the extension of this absorption band into the violet end of the visible spectrum. In addition to the 310-330 nip band, an additional band a t 630 mp (Lennox, 1960) has been detected in wool fabric inmediately after irradiation in the absence of moisture. This absorption band, which gives the fabric a greenish hue, fades on standing in moist air. These observations were confirmed by Laurier (1963a,b,c) who showed the absorption maxiniurn responsible for the green shade to he a t 600 nip using reflectance spectrophotometry. UV irradiation of tryptophan and tyrosine solutions also produces yellow pigments having absorption niaxiiiia a t 305 inp and 325 nip, respectively (Leaver and Lennox, unpublished observations, 1964), and the luminescence spectrum of wool following UV irradiation is similar to that of tryptophan (Haly and Stott, unpublished observations, 1963). The inforniation available so far on the photochemical degradation of wool presents a very complex picture. Tryptophan, tyrosine, and cystine

285

T H E CHEMISTRY OF KERATINS

appear to be the amino acid residues most susceptible to photodegradation, and the weight of the evidence suggests that tryptophan and tyrosine may be primarily responsible for the yellowing reaction. 2. Photochemical Changes in Amino Acids and Peptides

The photodegradation of cystine with UV radiation in acid and alkaline solution yielded cysteine, cysteic acid, serine, alanine, glycine, pyruvic acid, taurine, methane thiol, HzS, NH,, and COz (Mori, 1957). In more recent studies (Forbes and Savige, 1962a,b), cystine destruction was shown to proceed mainly by C-S bond fission at pH values above 5. Compounds containing S-S bonds, notably S-sulfocysteine CySS03H [Cy = NHzCH(COOH)CH2], were detected among the products together with pyruvic acid and NH,. The latter were probably derived from the unstable intermediary, aniinoacrylic acid. The peptide bis(glycy1)cystine appears to follow the same course of degradation as cystine judging from the products obtained after hydrolyzing the irradiated material with the proteolytic enzyme Pronase (Savige, unpublished observation, 1962). Yellowed wool, however, appears to contain no 8-sulfocysteine residues, since no SO2 was released when it was extracted with alkaline cyanide solution and the extract acidified (Milligan, unpublished observation, 1962). Nor was there any binding of radioactive thiol by wools yellowed under various conditions (Maclaren, unpublished observations, 1962). The formation of mixed disulfides would have been expected if 8-sulfo groups had been present (Swan, 1961). TABLEXXV Products Obtained by Irradiating Cystine Solutions with Sunlight or a Sun lamp. Products obtained a t pH

<5

Products obtained a t p H

>5

In nitrogen

In air.

In nitrogen

In air

CyHb (alanine) CySH cyssscy CyS0sHc CyOHd (serine) Glycine -

CySOaH cyssscy CyHd CyOHd Glycine -

CYH CySH cyssscy CySSOjH Cy SSSOaH CyOH cysssscy

Cy SSOjH CySOzH CySOjH CySOzSH cyssscy CySSSOjH Cy SSSSCyd

-

a Maximum 311 mr. Data from Forbes and Savige (1962a,b) and Savigc (personal communication, 1964). Cy represents NH2CH(COOH)CH2--. The formation of oxidized products may have been due to traces of O2 in the Nz. Formed only in traces. I n general, degradation proceeds much more rapidly in air than in NB.

286 w.

G. CREWTHER, 11. D. B. FRASER,

F. G . LENNOX, AND H. LINDLEY

When cystine is irradiated a t pH values below 5 in the presence of air and water, S-S fission predominates. Oxides such as CySOiSCy may be formed as intermediates, but the products isolated are mainly compounds such as cysteic acid, CyS03H. Alanine, serine, and glycine, produced in quantity in the absence of air, were formed only in minute amounts in its presence. Under all conditions of irradiation CySOzSHand CySSSCy were formed. When cystine was irradiated in the dry state cysteine, alanine, and lanthionine were formed. The results of irradiation experiments on cystine solutions are summarized in Table XXV. Savige (private communication, 1964) considers that products obtained by irradiation of cystine may arise by t>hefollowing mechanisms: 2CyS'

+cys + CySS'

Cy' Cy8'

+-

cysscy

+

cyscy

---t

cyssscy

---t

cyss + Cy'

+ c y s s c y * c y s s s c y + Cy' + c y s s c y s c y s + CySSSCy CySS' + CySS' cysssscy

Cy8'

CySS'

--t

Cy8'

---f

CySOEHand CyS03H

CySS

-+

CySSOIH and CyS02SH

CyHSS'

+

CySSSOaH

When mixed disulfides are exposed to sunlight or other fornis of UV radiation, disulfide interchange occurs readily. This reaction proceeds in solution even at acid p H values and in the solid state (Eager and Savige, 1963) and has been demonstrated for pairs of compounds including: cystine and cystamine, rystine and bis(2-carboxyethyl)disulfide, cystine and homocysthe, and bisglycylcystinylbisglyrine and bisglycylcystinylbisglutamic acid. Exchange reartions of this type may also proceed in the wool fiber. UV irradiation of tryptophan in acid, alkaline, or neutral solution in air yielded aspartic acid, serine, and alanine, in addition to several derivatives containing the indole ring system such as kynurenine (Matsuoa, 1953). More detailed studies revealed the presence of a wide variety of aromatic products including 2-hydroxytryptophan, formyl- and hydroxykynurenine, and various indole derivatives : indole acetaldehyde, indoleacetic wid, indole-3-aldehyde (Melchior, 1957). Structures have been suggested for urorosein (Harley-Mason and Bu'lock, 1952) and tryptochrome (Fearon and Boggust, 1950) which are derived from tryptophan on oxidation. They may be formed in wool also during photochemical yellowing (Graham and Stathani, 1956).

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287

Irradiation of tyrosine in solution with U V light introduces a second hydroxyl group into the benzene ring to form 3,4-dihydroxypheriylalariine. The solution also turns a reddish-brown color (Arnow, 1!137). Luse and MoLaren (1963) have reviewed published research 011 the photolysis products and yuantuni yields for the destrucbtion of aniiiio acids and have attributed the photochemical inactivation of the enzymes chyniotrypsin, lysozyme, ribonuclease, and trypsin by UV light at 254 mp primarily to destruction of the cystyl and tryptophyl residues. The destruction of these residues in proteins was suggested to be a function of the product of the number of residues present, the molecular extinction coefficient, and the yuantuni yield for destruction of each residue. Cysteine and tryptamine were identified among the irradiation products from cystine and tryptophan, respectively. Tyrosine, histidine, and phenylalanine were also shown t o be degraded by UV, histidine yielding histamine, urocanic acid, and other imidazole derivatives, and phenylalanine yielding tyrosine and dihydroxyphenylalanine. Destruction of these three amino acids was not considered to contribute appreciably to the enzyme inactivation. 3. Prevention of Photochemical Deyradatzon

The use of formaldehyde-thiourea resin has been patented for the prevention of yellowing of wool (Nakajo, 1951), but the “handle” of goods so treated is unsatisfactory. The use of formaldehyde alone gives a little protection (Milligan and Tucker, 1962), but much better protection is obtained using a solution containing both formaldehyde and thiourea (Milligan and Tucker, 1964) ; no resin is formed under these conditions. The photodegradation of tryptophan to yellow products can also be prevented by the addition of formaldehyde and thiourea (Rivett, unpublished observations, 1962). Several sulfonated hydroxybenzophenones applied to wool from hot aqueous solution a t about pH 1.5 also confer some protection against yellowing (Rose et al., 1961). It is well known that an improvement in color is obtained by exposing wool t o daylight under glass as in the solarium in some textile mills. By exposing sunlight-yellowed wool fabric to sunlight filtered through window glass, slight bleaching occurs (Milligan and Tucker, 1962). It would appear that whereas the UV irradiation in sunlight causes wool yellowing the visible radiation partly reverses this process.

V. MOLECULAR STRUCTURE OF KERATINS A . The Structure of a-Keratin The elucidation of the structure of a-keratin is a task of such enormous complexity that it has stimulated a great deal of research and led to a great

288 w.

G. CREWTHEIL,

n.

D. B. FRASER, F. G. LENNOX, AND H . LINDLEY

variety of proposals. In most instances attention has been concentrated on models which attempt to account for the discrete part of the X-ray diagram and hence refer only to the more highly organized regions of the keratin fiber. Models proposed to account for the chemical or mechanical properties, although useful in stimulating discussion, are in almost all instances too ill-defined to be capable of proof, or disproof, by physical methods arid will not be considered here. 1. Experimental F,vz d ence

a. Electron Microscopy. Our knowledge of the macromolecular structure of keratins is based largely on electron microscopy, and useful surveys of recent literature have been given by Lundgren and Ward (1962, 1963). I n all a-keratins so far examined the cortex appears, after staining with heavy metals arid a t moderate resolution, t o consist of rodlike niicrofibrils ca. 75 A in diameter embedded in a more densely stained matrix. The density and mode of packing of the microfibrils vary from cell to cell and keratin to keratin in a striking arid characteristic way. The contrast between the situations in the orthocortex and the paracortex of Merino wool is illustrated in Fig. 12 (Rogers and Filshie, 1963). The niicrofibrils are less readily visible in the orthocortex as less matrix is present and because the microfibrils in the outer layers of the whorls are inclined with respect to the whorl axes. The tendency for microfibrils to occur in orderly sheets (Jeffrey et al., 1956) is particularly evident in the orthocortex. In porcupine quill tip (Fig. 13) the packing is frequently near-crystalline (Rogers, 1959b). The most important discovery in recent years has been the clear evidence obtained by Icilshie and Rogers (1961) of an organized protofibrillar substructure within the microfibril. This substructure is illustrated in Fig. 13 together with an example of the less definite evidence for longitudinal substructure. As far as is known the size and substructure of the microfibril seem to be a constant feature of a-keratins from a variety of animals. Thus the microfibril niay be regarded as the fibrillar unit of structure, although it is to be anticipated that some species-to-species variation in detail will occur in view of the known variability in amino acid composition (Section III,B,1). The interpretation of the electron microscope images in terms of microfibrils and protofibrils has been criticized by Dobb and Sikorski (1961) and Johnson and Sikorski (1962) who consider that much of this detail is due to electron optical effects. Very little evidence of structural organization in the ground substance or matrix has been obtained. According to Sikorski and Woods (1960) the matrix contains “pseudoglobular” units ca. 50 A in diameter spaced

289

THE CHEMISTRY OF KERATINS

FIG.12. Electron inirrogrsphs of Mwino wool. Pmic.orlc?i (lowrr) (Rogers and Filshir, 1963).

(ii1)l)cr) ;

orthocortex

290 W. G .

CRE\STHER, R. D. B. FRASEK, F. G . LENNOX, AND H. IJNDLEY

FIG.13. Electron micrographs of porcupine quill tip. Cross section of cortex (upper) ; longitudinal section of cortex (lower) (Rogers and Filshie, unpublished observations, 1961).

THE CHEMISTRY OF KERATINS

29 1

at longitudinal intervals of 95 A. I n the electron micrographs of Rogers and Filshie the matrix contains densely stained particles, but these are about 20 A in diameter. More recently, Dobb (1964) has observed longitudinal periodicities of ca. 200 A and 40 A in negatively stained specimens, but these appear to be more closely related to microfibril substructure. b. X-Ray Difractaon. The X-ray diffraction pattern of a-keratin is of central importance in structural studies of this material because the agreement between calculated and observed intensities provides a searching test of the correctness of any proposed niodel. Early measurements by Astbury and co-workers were summarized by Astbury and Bell (1939), the eleven observed reflections being indexed on an orthorhonibic cell with a 10.3 A side parallel to the fiber axis. The cell sides perpendicular to the axis were given as 27 and 9.8 A in order to account for the two equatorial reflections observed a t the low instrumental resolution available. The 27 A equatorial reflection has always been regarded as an important feature of the pattern, although as early as 1936 Corey and Wyckoff stressed the importance of measuring the complete diffraction pattern and showed, by using highresolution X-ray cameras, that the 27 A reflection was only one member of a rich pattern of reflections a t low angles. MacArthur (1943) reported a very complete list of spacings out to 1.49A and suggested that the meridional reflections could be indexed as orders of a large unit of pattern along the fiber axis of length 658 A or alternatively 198 A. In the latter case it was noted that the strong 5.14 A reflection did not index well. Additional data on the spacings and intensities of the low-angle meridionals were reported by Bear (1944) and Bear and Rugo (1951). Lang (1956a,b) attempted a complete map of the diffracted intensity, but was handicapped by the low resolution inherent in the use of counter techniques with fibrous materials. Although quantitative data are highly desirable, they are very difficult to obtain from fibrous materials of large period. Measurements of nieridional intensities in various keratins reported by Onions et al. (1960) are of considerable interest in comparative studies, but cannot be used to check structures because the slit collimation used does not differentiate between ineridional and near-meridional reflections. The same difficulty applies to the intensities quoted by Bear (1944). The map of reflections given by MacArthur (1943) which covered reflections out to 3 A has recently been extended to 0.9 A by Fraser and MacRae (1961b). The spatial distribution of reflections is thus well catalogued, but there is a great need for precise intensity data. Nevertheless, the spatial distribution alone is sufficiently unique to provide a stern test of proposed structures. c. Infrared Spectra. Studies of infrared dichroisni and amide frequencies, which showed early promise of becoming a quantitative tool in

292 w.

G. CREWTHER,

R. D. B. FRAGER, F.

G . LENNOX, AND H . LINDLEY

thc elucidation of protein structure, lost much of their value because insufficient account was taken of the complex nature of the absorption processes. I n retrospect the most important contribution from infrared spectroscopy was the clear evidence that in the a-form of synthetic polypeptides and in a-keratin the CO and NH bands were preferentially oriented parallel to the molecular axis (Ambrose and Hanby, 1949; Ambrose and Elliott, 1951). Coniplications attending the quantitative determination of bond directions which were pointed out included the nonlocalized character of the aniide group vibrations and uncertainty in transition moment direction (Fraser and Price, 1952), the overlapping of side-chain absorptions (Lenorinant and Rlout, 1953; Ehrlich and Sutherland, 1953) and the presence of random coil forms (Elliott and Malcolm, 1956). The absorption spectra of proteins and polypeptides are now much better understood as a result of the vibrational analyses given by Miyazawa (1960, 1962, 1963), but it is still true that structures are more often used to test the interpretation of spectra rather than the reverse. Transition moment directions and coupling effects, however, are now sufficiently well understood for infrared dichroism measurements to provide a t least a semiquaiititative evaluation of some features of a model. 2. Structural Models of a-Keratin

Early attempts to devise chain coiifigurations consistent with the wideangle X-ray pattern have been summarized by Kendrew (1954), and the present account deals only with structures based on the a-helix. a. The Evolution of the a-Helix. Current ideas of the inolecular structure of a-keratin stem from a note published by Pauling and Corey (1950) in which they expressed the belief that a spiral configuration of the polypeptide chain, later termed the a-helix, was present in a-keratin. Subsequently, the application of an expression derived for the Fourier transform of a helical array of atoms by Cochran et al. (1952) established beyond reasonable doubt that the a-helix or a closely related structure was present in the specimens of the synthetic. polypeptide, poly-y-methyl-L-glutamate, prepared by Barnford et al. (1952). As originally described the a-helix embraces two distinct structures which for L-amino acids involve coiling either into a right-handed helix, corresponding to 0-carbon position 1 in the literature of that time, or into a left-handed helix, corresponding to P-carbon position 2. Early investigatioiis of synthetic polypeptides (Yakel et al., 1952) and proteins (Riley and Arndt, 1952) using X-ray diffraction favored position 2, although Huggins (1952) suggested that position 1 was more probable on stereochemical grounds. Studies of the X-ray pattern of poly-L-alanine (Brown and Trotter, 1956) again favored the left-handed helix, although neither

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possibility gave good agreement with the X-ray diffraction pattern. The question was finally resolved by Elliott and Malcolm (1959) who showed, by using the optical diffractometer, that the screw-sense of the a-helix in poly-L-alanine was undoubtedly right-handed, corresponding to position 1 of the /3-carbon atom. The important advance over earlier work was their recognition of the fact that the sense of the sequence -CONHalong the helix might be random as between neighboring molecules in a crystallite. The assignment of screw-sense in other helix-forming polypeptides is based on studies of the dispersion of optical rotation and in most rases it has been found to be right-handed for the L-enantiomorph (Urnes and Doty, 1961). Notable exceptions are poly-/3-benzyl-L-aspartate and poly-lbenzyl-L-histidine which form soniewhat unstable helices of opposite screw-sense. A direct confirmation of the occurrence of the a-helix in proteins was obtained in the 2 A Fourier synthesis of myoglobin by Kendrew et al. (1960). The screw-sense of the a-helix was found to be right-handed in all the helical sections of the molecule. b. Models of a-Keratin Based on the a-Helix. The earliest model of a-keratin structure based on the a-helix was that of Pauling and Corey (1951a), who suggested that a-keratin contained a-helices packed in pseudohexagonal array. No explaiiatioii of the low-angle equatorial X-ray pattern was offered, but the low-angle nieridional pattern was attributed to recurring sequences of residues a t axial intervals which were niultiples of the axial height per residue. The observation by MacArthur (1943) and Perutz (1951) of a nieridional reflection in a-keratin of spacing 1.5 A equal to the axial translation per residue in the a-helix provided considerable support for this iiiodel but, as noted by Pauling and Corey, the strong nieridioiial arc of spacing 5.15 A was riot accounted for without further assumptions. Crick (1952) pointed out that this difficulty could be overcome by supposing that the a-helices in a-keratin were distorted in a helical niariner to forni coiled coils as illustrated in Fig. 14. This distortion, which was claimed t o require only about 0.1 kcal per residue, enabled the side chains to pack more neatly. In subsequent papers Crick obtained an expression for the Fourier transform of a coiled coil (Crick, 1953a) and was able to show that this type of distortion could account in a general way for soiiie previously unexplained features of the X-ray pattern of a-keratin (Crick, 1953b) including the simultaneous appearance of 1.5 and 5.15 A meridional reflections. Detailed descriptions of two-strand and three-strand ropes of these coiled coils were given in which the pitch of the niajor helix was 186 A, and it was suggested that the three-strand niodel was appropriate

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0

FIG.14. (a) Distribution of residues in the three-strand coiled-coil rope. For clarity only one coiled coil is shown. ( b ) Distribution of residues in an undistorted a-helix.

to a-keratin. This choice was made on the basis of the position of the near-equatorial reflectioiis in the 1 0 A group. It was observed that the model did not explain the 27 A equatorial reflection nor the detailed meridional and near-meridional reflections. It was suggested that the latter might be due t o the presence of subunits joined end to end or to distortions in the structure. Pauling and Corey (19534 independently suggested that the a-helices in a-keratin were distorted into coiled coils, but argued that this was a result of the repetition of short sequences of residues. A detailed model

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was given in which seven-strand cables were packed in hexagonal array with single-coiled coils occupying the interstices between the cables. A somewhat more detailed version of Crick’s three-strand rope model was described by Lang (1956a’b) in which the major helix had a pitch of 197 A and radius 5.5 A giving a tilt of about 10’. The 0-carbon atom was supposed to be in position 2 which was favored at that time. Detailed calculations appeared to confirm Crick’s view that a model of this type could account for a strong nieridional reflection a t 5.15 A, but these are unlikely to be valid as it is now considered that position 1 is appropriate. Huggins (1957) called attention to the difficulty of transforming a-coiled coils into @-sheets during the a -+ p transforniation which takes place when keratin is stretched and proposed a model based on a three-chain unit to overcome this difficulty. The axes of the a-helices were supposed to be inclined to the triplet axis, but not to acquire any net twist around this axis. This was achieved by introducing periodic breaks in the a-helix to take out the twist acquired through tilting. Another important feature of this model was an attempt to explain the low-angle equatorial X-ray pattern by grouping the three-chain units into sets. Skertchly and Woods (1960) again stressed the difficulty of transforming Paulirig and Corey’s cabIe model t o a p-structure and pointed out that single coiled coils could be packed in such a way that the a-helix axes did not become entwined. In an attempt to reconcile the microfibrillar structure and the X-ray diffraction pattern with the coiled-coil hypothesis Swanbeck (1961) proposed a model in which a central three-strand coiled coil was surrounded further by four concentric layers of a-helices, the tilt increasing progressively up to a value of 50” in the outer layer of nineteen chains. In a later more detailed description (Swanbeck, 1963) it was proposed that the three inner layers consisted of “a-helices” of pitch 5.25, 5.65, and 6.40 A, that the fourth layer consisted of 3.010 helices (Donohue, 1953) and the outer layer was a p-structure. A possible modification of Crick’s coiled-coil models was suggested by Fraser and MacRae (1961a) in an attempt to improve agreement between the observed X-ray diffraction pattern and that predicted by a coiled coil. They argued that the distortion required to produce a rope structure might be concentrated a t particular residues rather than being continuous. This would lead to a “segmented rope” consisting of short straight sections of a-helix with axes tangential to the path of the major helix. It was suggested that the segment length coiild not exceed 20-30 A without significant departure from the “knob/hole” packing scheme envisaged by Crick. Following the suggestion by Filshie and Rogers (1961) that the microfibril contained a (9 2) arrangement of protofibrils Fraser et al. (1962) speculated that the microfibril might coiisist of a core

+

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W. G. CREWTHER, R. D. B. FRASEIZ, F. G. LENNOX, AND

H.

LINDLEY

of two three-strand ropes surrounded by a layer of nine three-strand ropes (Fig. 15). A variation of the segmented rope hypothesis in which the

b

-

0

50

A

FIG.15. Model of microfibril structure proposed by Fraser et al. (1962). (a) Protofibril containing tlirec similar but nonidentical sections of three-stand rope in a, 200A interval. (b) Microfibril formed from eleven protofibrils arranged in a (9 2) pattern. More recent work (Dobb, unpublished observations, 1964) supports the notion of a protofibrillar substructure, but casts doubt on the presence of the central pair, shown shaded in the diagram.

+

segment length was increased to 70 A was put forward by Lundgren and Ward (1962). This was incorporated in a very detailed model which encompassed both microfibril and matrix. 3. An Evaluation oj the Evidence a. Coiled Coils. In the two-strand rope niodel described by Crick (1953b) the axes of the a-helices are distorted so as to follow a helical path of pitch 186 A and radius 5.2 A. In the simplest case the two polypeptide chains have the same sense of chain direction (-CONH-) and are

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related by a twofold rotation axis coincident with the major helix axis. The screw-sense of the major helix is opposite to that of the a-helix and is thus likely t o be left-handed. In the three-strand rope, called the D3 rope by Pauling and Corey (1!)53a), the pitch and radius of the major helix would not be expected to be very different from the two-strand rope. Again in the simplest case the three polypeptide chains have the same sense of chain direction and are related by a threefold axis. An iniportant feature of the diffraction pattern predicted by these models is the occurrence of a series of meridional reflections which are orders of a 10.33 A periodicity. This periodicity is associated with the axial projection of the asyrnrnetric unit, which consists of seven residues. Astbury and Bell (1939) had noted such a periodicity in a-keratin and given spacings for the first four orders, whereas a spacing of 1.49 A, close to the seventh order, had been noted by MacArthur (1943). More recently, meridiorial scatter in the vicinity of the fifth, ninth, and eleventh orders has been reported (Fraser and MacRae, 1961b). In a field where structural models had been considered plausible if they predicted one reflection the suggestion of the coiled-coil models was thus a very significant advance. A second feature of the observed diagram successfully predicted by the coiled-coil model was the splitting of the 10 A equatorial reflection into equatorial and near-equatorial layers. The nature of the splitting is independent of the combination of chain-sense directions in a rope (Fraser et aZ.,1964a) and depends only on the ratio of the pitch of the major helix to the number of strands in the rope. Thus it should be easy to determine the number of a-helices in a rope, but unfortunately the splitting is not well resolved. According to Crick (l953b) the splitting is appropriate to a three-strand rope, but figures ranging from 65 to 90 A have been obtained for the ratio (pitch of major helix/number of chains) in recent measurements (Fraser et d.,1964b). When the effects of disorientation are taken into consideration the true value is likely to be a t the lower limit of the range. Although these measurements do not enable a clear choice to be made between the two- and three-strand rope, they would seem to eliminate the single coiled coil of Skertchley and Woods (1960). An equally cogent reason for doubting the latter model is that it does not explain why the a-helix should be distorted into a coiled coil. The first comprehensive test of Crick’s model was that reported by Fraser and MacItae (1961b) who showed that the optical transform of the coiled coil was in good agreement with certain broad features of the observed X-ray pattern. At very large angles of diffraction, however, it was found that the observed pattern agreed better with that of a tilted a-helix. This observation taken together with the considerable breadth of the wideangle meridionals and the near-equatorials suggests that the coiled coils

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G . CREWTHER, R. D. B. FHAHER, F. G . LENNOX, AND H. LINDLEY

do not persist with geometrical regularity over any great length of the molecule. The segmented rope model described in Section VA2b is only one of the many types of disorder which could produce the observed effects. The modification of the segment rope in which straight segments of a-helix 70 A long are envisaged (Lundgren and Ward, 1962) is not very plausible, as it is an extremely open structure witahabout 14 A between the helices toward the ends of the segments. The intensity transforms of Crick’s coiled-coil models have been calculated recently (Cohen and Holmes, 1963; Fraser et ul., 1964a), and there is a considerable measure of agreement with the observed pattern in the 10 A group of equatorial and near-equatorial reflections (Fraser et al., 1964b). The coiled-coil models do not of course predict the low-angle pattern or the fine lateral structure in the 5 A iiieridional or the 10 A equatorial groups of reflections. These features are clearly due to higher levels of organization and complexity. It is possible to calculate the infrared dichroism of the amide vibrations expected for a coiled coil. In the case of the amide A (NH stretching) vibration the dichroic ratio is calculated to be 10.1 compared with an observed value of 2.0 (Fraser and Suzuki, unpublished observations, 1964). This discrepancy is due in part to overlapping side-chain absorptions and to nonhelical proteins (Parker, quoted by Astbury, 1956) as is shown by the increased dichroic ratio observed after H -+ D exchange. Dichroic ratios as high as 5.5 have been observed after exchange which, allowing for natural imperfections in orientation, means that coiled coils could account for over 80 yoof the nonexchangeable hydrogen. 6. The Microfibril. Electron microscopy of ultrathin cross sections of cortex has provided striking evidence of a highly organized substructure within the microfibril (Filshie and Rogers, 1961). Although a complete resolution of this substructure has not been obtained the image (Fig. 13) suggests that the microfibril is bounded by a ring of about nine fibrils of the order of 20 A in diameter. These have been termed protofibrils, and it has been speculated that two more protofibrils are present within this ring (Fig. 15). Johnson and Sikorski (1962), however, regard the appearance of a ring of blobs as electron optical effects and consider that they cannot be regarded as end-on projections of protofibrils. While there is some evidence to cast doubt on the presence of a central pair of protofibrils, some tangible proof would seem to be desirable before rejecting the work of Filshie and R,ogers. The essential correctness of their notion of a protofibrillar substructure has recently been established by Dobb (unpublished observations, 1964) who found evidence for fibrils -20 A in diameter in negatively stained specimens of partially disintegrated wool. Although i t is too early to be dogmatic, the appearance of the sub-

299

T H E CHEMISTRY OF KERATINS

structure within the microfibril, with protofibrils only 20 A in diameter, would seem to be inconsistent with the models proposed by Pauling and Corey (1953a), Swanbeck (1961, 1963), Skertchley and Woods (1960), and Lundgren and Ward (1962). Clearly, the confirmation of the electron microscope evidence with its sweeping implications is a topic of the greatest importance. Independent but less definite evidence for a protofibrillar structure of the type suggested by electron microscopy has been obtained recently from the X-ray diffraction pattern. In the region of the 10 A equatorial group of reflections and along layer lines near the 5.15 A meridional reflection there is clear evidence of an oscillation in the transform with a lateral periodicity of about 20 A. The interference function for a “9 2” structure has been calculated (Wilson, 1963; Fraser et al., 1964b), and while showing similarities to the observed pattern the detailed agreement is poor. This could be due to the omission of scattering material attached to the protofibrils and projecting into the matrix. While it has not been proved that this structure is correct in detail, the evidence nevertheless supports the idea of a subdivision into protofibrils -20 A in diameter. The omission of the two central protofibrils has little effect on the calculated pattern and their presence or absence is unlikely to be established by X-ray studies. Although a considerable correlation of low-angle X-ray diffraction with electron microscopy has been obtained for equatorial reflections, there is little manifestation of the elaborate longitudinal regularity suggested by the low-angle meridional pattern. This pattern indicates a very regular distribution of chemical units parallel to the fiber axis, and it is of considerable interest that the intensities vary from keratin to keratin (Onions et al., 1960). When a-keratins are “stained” with heavy atoms (Fraser and MacRae, 1957, 1958, 1961b; Sikorski and Woods, 1960; Sinipson and Woods, 1960) this causes considerable changes in the distribution of intensities, but no new periodicities are revealed. It s e e m niost likely therefore that the 197 A period is due to a repeating pattern of residues in the axial projection of the microfibril. The true period must be many tinies greater than 197 A, as the 5.15 A reflection and numerous other reflections do not index on this period. It has been generally assumed that the 197 A periodicity is due to the pitch of a coiled coil, but there is no evidence to support this view (Fraser and MacRae, 1961b). It is more likely that the meridionals are due to zero-order Bessel terms in the transform associated with a large scale helical geometry in the microfibril. Studies of these reflections give information about the distribution of residues within the microfibril and the mapping of this distribution is potentially possible if resolution of the longitudinal structure can be attained in the electron microscope. Although the electron niicroscope is a powerful tool in the investigation

+

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G . CREWTHER, R. D. B. FRASER, F. G . LENNOX, AND H. IJNDLEY

of macroniolec~ularstructure, it is important to realize that large deposits of heavy atoms are required within the structure to produce suitable contrast in the image. It is thus important that findings from electron microscopy be cross-checked as far as possible by methods such as X-ray diffraction and itifrared spectrometry which can be used with the native material. Examples of such checks are the demonstration that the lowangle equatorial X-ray pattern is consistent with a microfibril/matrix structure when the microfibril diameter is about 75 A (Fraser and MacRae, 1958) and the evidence from the wide-angle pattern supporting a protofibrillar structure (Fraser et al., 1062). I n conclusion it should be stressed that a ring of about nine protofibriis, each containing two or three chaiiis, only accounts for a fraction of the protein in the cortex. The nature and organization of the material in the venter of the microfibril is unknown as is the organization of the matrix protein.

R. The Structure of &Keratin I t is now generally accepted that the pleated-sheet models suggested by Paiiling and Corey (195lb, l953b) are the basis of the extended configuration of the polypeptide chain which yields the characteristic 0-pattern by X-ray diffraction (Astbury arid Street, 1931; Astbury and Woods, 1933). Paulirig and Corey (1953b) describe two pleated-sheet models; in the first the axial translation per residue is 3.5 A and the CONH sequence in alternatc chains is oppositely directed, in the second the translation is 3.25 A per residue and the chain senses are all parallel. Because the translation in the latter case was closer to the observed meridional spacing of 3.34 A, it was put forward as a model of p-keratin structure. Later Miyazawa arid Blout (1961) claimed that the absence of a weak band at 1690 cm-' iu the infrared spectrum of p-krratin confirmed Pauling and Corey's conclusion, but it was pointed out (Fraser and MacRae, 1962b) that this component is in fact present, thus suggesting that aiitiparallel (ahairis arc present. Rradbury and Elliott (1962) independently called attention to this discrepancy and remeasured the spectrum of stretched horsehair in order to confirm the presence of the 1690 cn-l band. Although the original work of Astbury and Woods (1933) showed clear evidence of the presenm of antiparallel rhains, the climate of opinion during the last decade has been influencard very murh by Paulirig and Corey's proposals. A factor which they did not consider and which is now believed to be important in deteriiiining the structurr is the nature of the substituciits on the 0-carbon atom. I n a recent study l'rascr and MacHae (l962b) reported five reflections in the X-ray diagram which require two chains in the unit of structure, as in the aiitiparallel-c,hain pleated-sheet. Thr intensity distribution, how-

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301

ever, calculated for this configuration did not agree well with the observed pattern. Better agreeiiient was obtained by taking a mixture of parallelchain sheets and antiparallel-chain sheets. It was suggested that this may reflect restrictions on the coinbinations of chain direction which can occur, owing to the predetermined arrangement within the microfibrils in the native material. Very little is known about the fate of the microfibril during the course of the a -+ 0 transition. MacArthur (1943) reported that the axial long spacings related to the niacromolecular structure are lost beyond 2 % extension, and Kratky (1951) gives a value of 67 A for the innermost lowangle equatorial reflection at 60 yo extension compared with a value of 86 A in the untreated material. This suggests that the microfibril preserves its identity during the a 0 transformation, although this has not been confirmed so far by electron microscopy. --f

C. The Structure of Feather Keratin The X-ray diffraction pattern obtained froin feather keratin is in some respects siniilar to that obtained from 0-keratin although of very muc*h greater coniplexity. Attempts to devise model structures which would account for the elaborate pattern are of two types. In the first it is assunied that the structural units are distributed on a two-dirriensional net (Bear and Iiugo, 1951; Fraser and MacRae, 1959; Astbury and Reighton, l96l), and in the second it is assumed that rodlike fibrils with helical features are present (Hamachandran and Dweltz, 1962; Schor and Kriniiii, 1961b; Fraser and Macltae, 1963). The discovery by Filshie and Rogers (1962) that feather keratin contains rodlike fibrils about 30 A in diameter (Fig. 16) greatly favors the latter type of model, although it does not exclude detailed considerations about chain configuration and symmetry in the earlier publications The first fibrillar model was described by Krinini and Schor (1956), and in later publications (Enonioto and Krinini, 1961; Krinini, 1961, 1962; Schor and Kriinm, 1961a,b; Westover et al., 1962) it was claimed that this model accounted for a large body of experimental evidence. Nevertheless, the /%helix model, as it was called, is so much at variance with the observed X-ray pattern that it is unlikely to be correct in its present form (Fraser and MacRae, 1963). Despite this, the recognition by Krimm and his colleagues that the X-ray diagram was consistent with a helical structure was an important step forward. Fraser and MacHae (1963) pointed out that the data presented by Bear and Rugo (1951) in terms of a two-dimensional net could be interpreted equally well in terms of helical diffraction. Although this reinterpretation does not enable a detailed model to be deduced, it leads to a description of

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G . CREWTHER, R. D. B. FIZASER, F. G. LENNOX, AND H. LINDLEY

--

FIG.16. Electron micrographs (upper) of a cross section of feather rachis (Filshie and Rogers, 1962) ; (lower) of fibrils obtained by reconstituting extracted feather proteins (Filshie et al., 1964).

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the geometry of the helix which is consistent with the evidence from electron microscopy. It has been speculated that the structural units of the helix are protein particles which may be regarded as very small 0-crystallites (Fraser and Macltae, 1962a, 1963). This contrasts with the @-helixmodel of Krimm et al. in which it was supposed that the 0-chains were distorted to follow a helical path. It is perhaps naive to think that the structure of feather keratin can be deduced purely from stereochemical considerations as was the case, for example, with the a-helix. Future progress will most likely depend on further correlation between evidence from electron microscopy, X-ray diffraction, sequence analysis, and solution studies. A promising line of investigation is the study of the fibrous material regenerated from solutions of feather proteins (Woodin, 1956; Fraser arid MacRae, 1959, 1963; Filshie et al., 1964). An example of fibrils obtained in this way is shown in Fig. 16. VI. RELATIOXSHIP BETWEEX THE PHYSICAL PROPERTIES AEU‘DCHEMICAL STRUCTURE OF KERATIN

A . Introduction This section is concerned primarily with the effects of chemical niodificasetting, tions of keratins on their physical properties-supcrcontraction, swelling, load-extension characteristics, and other mechanical properties. Much of this work could be described by the term “mechanochemical” coined by Speakman (1947). The complexity of the cellular and subcellular structure of keratins necessitates the use of simplifying assumptions in the interpretation of mechanochemical experiments. One of the chief difficulties in attempting to relate the physical properties of the fiber to its chemical structure is the nonspecific nature of many chemical treatments applied to the fiber. Frequently, effects ascribed to the modification of one residue are explained equally well in terms of side reactions with other amino acid residues. The cystine residues, because they may act as cross-linkages between protein chains, have been studied more closely than other residues in keratin. Burley’s (1956a) use of the concept of thiol-disulfide interchange to explain the effects of chemical modification on the physical properties of wool fibers has stimulated further work on cysteine and cystine residues.

R. Physical Properties o j Animal Fibers 1. Stress-Strain Relationships

The stress-strain curve for wool or hair in water a t room temperature (Harrison, 1918; Shorter, 1924) suggests an obvious division into three parts

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G . CREWTHER,

R . D. B. FRASEH., F. G. LENNOX, AND H. LINDLEY

(Fig. 17). (a) A Hookean Region from 0 to 2 70strain in which strain is proportional to stress. ( b ) A yield region ranging from 2 to 30 70strain in which a small increase in stress results in a large increase in strain. ( c ) A post-yield region in which the increase of stress required to produce a given increase in strain is greater than in the yield region. The extension at which the fiber breaks is determined by the conditions and rate of stretching. A dry fiber breaks before reaching the post-yield region, whereas in water the fiber may be stretched about 50 % a t 0°C. If Stretching is very slow or if the temperature is raised to between 90 and 100°C the fiber may be stretched by 65 to 90 70(Fig. 17) depending on the type of wool used eo 70

u z

I

60

W

A

50

-I

4

-3I-

40

0 LOAD

(GM./cM.~ INITIAL

AREA)

FIG.17. Stress-strwn curves for Cotswold wool In water at 0", 32.9", and 75.2"C (Sprakman, 1927). The Hookean, yield, and post-yield regions are Indicvitcd.

(Speakman, 1927, 1928; Astbury and Street, 1931; Ripa and Speakman, 1951). Speaknian (1947) showed that if a wool fiber is stretched rapidly by <30 % in water and inimediately released it recovers its original length and elastic properties upon standing in water. He has used the work required to extend by 30 yo as a measure of changes in the fiber indured by chemical treatment. The work done in stretching the fiber 30 yo in water is measured, the fiber is immediately released and held 24 hr in water; it is then modified chemically aiid the work done in stretching 30 yo again determined. A complete knowledge of the chemical or physical changes

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accoiiipariyiiig thc treatnient is iiecessary to permit a correvt interpretation of the results. The extensive investigatioiis by Astbury and Woods (1933) of the X-ray diffraction patterns of aninial fibers before and after stretching, setting, or superrontraction (Section VI,B,2) have had a major iiifiueiic~eor1 the inodels proposed to account for the load-extension properties. Astbury and Woods showed that when aniinal fibers are stretched, the characteristic a-pattern decreases in intensity with the siniultaneous appearance of a /?-pattern siiiiilar t o that obtained from silk. The sharpness and intensity of the @-patterii increases with strain, with teniperature, aiid u ith t h e under strain. In the Hookeaii region the 5.1 il nieridional spaciiig increases by up t o 2 % (Astbury and Haggith, 1953). Astbury and Woods (1933) stated that virtually no @-patternappears imtil the fiber has been stretcbhed by a t least 20 yo,and for 25 years this observatioii doiiiiiiated the inodels proposed to explain the elastic properties of wool (Alexander and Hudson, 1954). A quantitative investigation of the X-ray diffraction pattern of a-keratin by Bendit (1957, 1960c) showed that the a-pattern begins to disappear and the @-spacingsbegin to appear when the fiber has been extended by as little as 5 yo. There is then a smooth continuous decrease in the intensity of the a-pattern and an increase in the intensity of the P-pattern until the fiber breaks. This observation and the coiifiriiiatory studies by Skertchly and Woods (1960) impose an iniportant new reqiiiremeiit on niecahanical iiiodels for the wool fiber. 2. Supercontractton and Set

The ability of keratin fibers to assume a more or less permanent change in shape or length when steamed under stress has been exploited in the clothing and hairdressing trades for centuries. The term “setting” is used to describe this property. Astbury and Woods (1933) found that if woo1 fibers were stretched in cold water, steamed in the stretched state, and then released in steam, the final length could be either greater or less than the original (Fig. 18). Short periods of steaniirig in the extended state resulted in a decrease in length, and to distinguish this contraction froni that occurring when a stretched fiber is allowed to revert to its original length they coined the term “supercontraction.” The percentage increase in length of fibers treated in the stretched condition for long periods is ternied the “set” acquired by the fiber. Set has been further classified according to its permanency to aqueous treatmeiits. Wool fibers remain extended when stretched in water arid dried, but return to their original length when again iiiiinersed in water.

306 m. G.

C R E W T H E I ~ , R. D. B. FRASER, F. G . TXNNOX, AND H . LINDLEY

This is generally ternied “cohesive set.” When steamed or boiled in water and released in cold water stretched fibers retain much of the extension, but 011 ininiersion in boiling water this decreases rapidly a t first and then more slowly over a prolonged period. The set which is lost in 1 hr is arbitrarily designated “temporary set” and the residual set “permanent set.” It is probable that several reactions occur simultaneously and the so-called permanent set continues to decrease with continued boiling of the slack fibers for several hours (Blackburn and Lindley, 1948). 40 30

-

5 40 YI

FIG.18 Relationship between final length of wool fihers and time and temperature of trentmmt with water while extended 50% The fibers were released and steamed for 1 hr before measurement (Astbury and Woods, 1933).

With short setting times an additional section appears in the loadextension curves (Feughelnian, 1960) between the Hookean and yield regions (Fig. 19). As the time of setting increases, the extent of the new section increases until the Hookean region disappears (Speakman, 1929; Feughelman, 1960). Feughelman attributes this to the transforniatioii of certain regions of the fiber to an elastomer which, because of its small elastic modulus, extends more readily than the material that normally gives rise to the yield region. The load-extension curves suggest that the newly formed structures are in series with unmodified material. The presence of modified keratin in set fibers is confirmed by the niarked increase in longitudinal swelling of wool fibers in water when they undergo setting treatments (Astbury and Woods 1933; Menkart and Sakol, 1962; Ihghelnian et nl., 1962; Haly and Snaith 1963). It has also been shown that setting increases the number of hydrogen atoms capable of rapid exchange with deuterium (Bendit, private communication, 1964). Chemical treatments also may cause supercontraction. Thus Speakmari

T H E CHEMISTRY OF KERATINS

307

(1931) showed that set fibers when treated with solutions of NaOH or NazS contract t o lengths less than the original, and Astbury and Woods (1933) observed contractions of up to 10 % in fibers stretched 80-90 % and then released in cold 1 NaOH. These fibers retained their a-diffraction pat-

LOAD

FIG.19. Typical load-extension curve of

-

Corriedale fiber in water at about 20°C after boiling for less than 20 min at, n constant strain of 1070 (Feughelman, 1960). R

tern but on steaming contracted further by a total of about 50 %; a partially disoriented @-patternwas then obtained. Table XXVI lists other chemical reagents or treatments known to cause supercontraction of keratin fibers. Layers from the epiderniis of the cow’s lip and from the wall of the cow’s hoof contract in hot water (Rudall, 1946), and feather keratin (Mandelkern et al., 1962b) and porcupine quill (Crewther, unpublished observations, 1964) contract in solutions of LiBr. 3. Plasticity, Creep, and Stress Relaxation

Under a constant large load, instantaneously applied, animal fibers elongate rapidly by about 30 % in cold water and then continue to creep for a prolonged period (Speakman, 1926). In the post-yield region, after

TABLEXXVI Reagents and Treatments Producing Supereontractwn i n Keratins

No.

Reagent

Concentration

Supercontraction

Temperature ("C)

Time

(% 1

Fiber used

X-ray diffraction pattern

Reference

A . Reagents primarily breaking disulfide bond8 1 NaOH 2 As 1 followed by steaming 3 NaB 4 NaHSOs

1%

Room temp.

Various

Cotswold wool

ca. 10

Disoriented a

-

100

-

Cotswold wool

45

Disoriented 0

0.0325 N 5%

Room temp. 100

-

1 hr

Cotswold wool Wool

24

Wool Wool Lincoln wool

13 28 40

24

5 KCN 0.65% Saturated 6 AgzSOd 7 N-chlorosulfamic acid 0.5 gm available (or N-monochlorourea) C1 per gram wool 8 Chlorine dioxide l.25N Water 9 As 8 followed by 10 As 8 followed by 0.1 N HCl 11 Sodium sulfoxylate 2 yo 12 Sodium hydrosullite 0.2 % 0.5 M followed by 13 Sodium thioglycolate 0.01 N HC1 14 Peracetic acid 2% 15 A s 14 treated in water 16 Tetrakis (hydroxy1% methy1)phosphonium chloride 17 Brz in 4 M HC1 4 x 10-2 M

-

6

Disoriented 0

-

Speakman (19311, Astbury and Woods (1933) Astbury and Woods (1933) Speakman (1931) Speakman (1933a), Jeffrey t-t al. (1955) Speakman (1936a) Speakman (1933a) .4lexander et al. (1951b)

100 100 40

30 rnin 5 hr

22 100 100 100 100 100

120 hr 1 hr 1 hr 30 rnin 30 rnin 20 hr

Lincoln wool Lincoln wool Lincoln wool Wool Wool

-

62 28 26 28

20 95 100

24 hr ? 15 rnin

Lincoln wool Lincoln wool Wool

20 41 38

Disoriented a Random @

-

Alexander (1951) Alexander (1951) Bajpai el al. (1960)

98.5

90 rnin

Corriedale wool

45

-

Crewther and Dowling (1961b)

?

50

-

-

Das and Speakman (1950) Das and Speakman (1950) Speakman (1941) Brown and Harris (1948) Brown et al. (1950) Elijd and Zahn (1949b)

B. Reagents primarily heaking hydrogen bonds 18 Formamide

so%

19 Phenol

50%

so%

100 118 100

3 hr 3 hr 20 min

Horsehair Horsehair Wool

25 45 43

-

-

Elijd and Zahn (1944) E M and Zahn (1944) Zabn (1947, 1949a,b). Elod and Zahn (1949b)

20 Substituted phenols

-

-

Wool, horsehair

-

-

21 Sodium phenylpropionate 22 Sodium salts of other aromatic acids

100

6 . 5 hr

Corriedale wool

20

-

-

-

Wool

-

-

23 Formic acid

Boiling

3 hr

Lincoln wool

15

24 Urea 25 Dimethylsulfoxide

120

105

lhr 1 hr

Wool Wool

33 28

26 HCI

98.5

35 min

Corriedale wool

27

30

27 Cuprammonium hydroxide

9 g m Cu per liter

Room temp.

75 min

Lincoln wool

28 Copper ammines

Various

Room temp.

-

Lincoln wool

32 (max)

29 Nickelammonium hydroxide 30 LiBr

10 g m Ni per liter

20

16 hr

Lincoln wool

40

100 gm/100 ml HzO

100

2hr

Lincoln wool

14

31 LiBr (bromine-free)

100 gm/100 ml He0

100

3hr

Crossbred

50

32 Other salts 33 Stretching and release in steam

Various

100

1 hr

Corriedale wool Cotswold wool

-

100

-

34

E16d and Zahn (1949b) Crewther (1959) Crewther (1959)

Alexander and Hudson (1954). Crewther and Dowling (1956), Crewther (1959) Stoves (1946). Jeffrey et al. Disoriented p after washing (1955) Schmidt (1932) Disoriented 0 Koenig and O’Connell (1960) after washing Crewther and Dowling (1959), see also Elod and Zahn (1949b) Pattern Whewell and Woods (1946) eliminated, restored by dilute acid treatment Whewell el al. (1959)

-

Bell and Whewell (1952. 1953)

Disoriented 0 Alexander (1951) after washing Pattern Griffith and Alexander (1957), eliminated in Haly and Snaith (1960, LiBr solution 1961) Crewther and Dowling (1956) Disoriented (3 Astbury and Woods (1933)

W 0

co

310 w.

G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

+

the initial extension a t constant load, log (Eli, - E ) = a kt, where t is the time of extension, E is the extension, and Elilnis the limiting extension-usually 75 to 80 yo for Lincoln wool-and a and k are constants (Ripa and Speakman, 1950, 1951). The constant k is used as a measure of the plasticity of the fibers and values ranging from -3 X sec-1 have been recorded. Feughelman (1954,1958) has shown that with smaller loads creep occurs in both the Hookean and yield regions of the loadextension curve; in the yield region the limiting extension is about 30 %. The associated phenomenon of stress relaxation (continuous decrease in stress a t constant strain) has been demonstrated in animal fibers a t all levels of elongation (Rigby, 1955, 1959; Feughelman and Mitchell, 1959).

4. Torsional Properties Feughelman (1959) and Feughelman and Mitchell (1981) have drawn conclusions concerning the properties of the matrix from measurements of the torsional elastic niodulus of wet and dry wool. They concluded that in the dry state the Young’s moduli of microfibrils and matrix are approximately equal, whereas in water, although both structures are softened, the modulus of the microfibrils is many times that of the matrix. Like longitudinal stresses, torsional stresses undergo relaxation a t constant twist (Feughelman and Mitchell, 1961). Similar effects have been observed in experiments with horn (Warburton, 1958). 5. Nature of Retractive Forces in Stretched Fibers Retractive forces in animal fibers stretched into the Hookean region of the stress-strain curve are generally attributed to the stretching of chemical bonds and hence to an increase in the internal energy of the fiber (Astbury and Haggith, 1953; Peters, 1956). On the other hand the nature of the retractive forces in the yield and post-yield regions has been the subject of much controversy. Bull (1945), Woods (1946a,b), Astbury (1947), Elod and Zahn (1949a), arid Breuer (1982) have concluded from the effects of temperature on the retractive forces that entropy contributes very little to retractive forces a t strains up to 30 %. Meyer and Haselbach (1949) and Meyer et al. (1952), however, consider that the fibers must reach an equilibrium condition before measurements are made and conclude that the forces are entirely entropic. There can be no doubt that after stress relaxation a t high temperatures the residual force is largely entropic (Feughelman and Mitchell, 1959), but this force is only a fraction of the initial force. On the other hand there is clear evidence that the forces acting in wool fibers which have been supercontracted and then stretched to a limited

THE CHEMISTRY O F KERATINS

311

extent are largely entropic (Woods, 1946a,b; Elod and Zahn, 1949a; Haly and Feughelman, 1957). Hence the changes which take place during supercontraction of keratin fibers are similar to those occurring when collagen or aligned crystalline polymers are converted to their elastomeric form (Mandelkern, 1959).

C. Efects of Chemical Modi$cation on Physical Properties of Keratin Fibers 1. The Chemistry of Creep and Stress Relaxation

Early studies of the plasticity of wool fibers revealed an apparent relationship between sulfur content and plasticity (R.ipa and Speakman, 1950, 1951), but this was later shown to be fortuitous (Dry et al., 1952). Differences in plasticity of individual fibers were found to be related to their origin (Dry et al., 1952; Burley and Speakman, 1953), primary follicles producing less plastic fibers than secondary follicles. LeRoux and Speakman (1957) demonstrated a direct relationship between the diametral swelling of fibers in water or formic acid and their rate of creep. They also suggested (Leltoux and Speakman, 1955, 1957) that there is a relationship between the plasticities and the tyrosine contents of individual fibers as determined radiometrically by iodination. It was thought that tyrosine residues contribute to disorder and hence to greater plasticity in the fiber. It is not known, however, whether tyrosine residues in different wool fibers are equally reactive with iodine; also there is evidence that tyrosine is not confined to noncrystalline regions (Fraser and MacRae, 1957) or lacking from proteins considered to arise from the microfibrils (Section 11). Burley (1956a) has shown that the rate of creep is greatly increased by increasing the thiol content of the fibers and decreased equally dramatically by blocking existing thiol groups with reagents such as N-ethylmaleimide or l-fluoro-2,4-dinitrophenol. He attributed these results to a n interchange reaction between thiols and disulfide cross-linkages facilitating conformational changes in the fiber. Burley (1960) showed further that wool from sheep on a copper-deficient diet has both a high thiol content and a high rate of creep. In view of the wide range of thiol contents reported in the literature for various wool samples (Zahn et al., 1962) it is probable that this is the niajor factor determining the plasticity of individual wool fibers. Changes in thiol content may also have a bearing on the observation (Whiteley and Speakman, 1959) that the plasticity of wool is increased by lowering the level of nutrition of the sheep. Stress-relaxation measurements confirm Burley’s interchange hypothesis. Fibers pretreated with iodine or nitrous acid, both of which oxidize disulfide groups and probably also thiol groups (Sookne and Harris, 1937; Cockburri et al., 1948; Crewther and Dowling, 1961a), were characterized by

312 w.

G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLET

very slow stress relaxation (Rigby, 1959; Feughelmari and Mitchell, 1959), whereas pretreatment with thioglycolate accelerated stress relaxation. Hence there can be little doubt that the rate of stress relaxation is determined largely by the thiol content of the fiber. These conclusions apply to stress relaxation for strains between 20 and 40 % and to creep for even greater strains. They have an obvious application in the setting of fibers at such strains. 2. Supercontraction

a. Bonds Limiting Contraction. The supercontraction of animal fibers in chemical reagents has been used as a tool for demonstrating the presence of new cross-linkages or for investigating the nature arid location of existing cross-linkages. Alexander (1951) observed a maximum contraction in aqueous LiBr for wool fibers of about 14 % which was completely reversed by washing immediately in water. Griffith and Alexander (1957) obtained variable results with this reagent and showed this to be due mainly to the presence of free Br2 or Bra- in the solutions. When this was removed the fibers contracted by almost 50 in boiling LiBr solutions. Fully contracted fibers, however, were no longer capable of even partial length recovery in water. Haly and Feughelman (1957) observed that the contraction of animal fibers in bromine-free LiBr solutions takes place in two stages which be-

z

0 I-

< c

TIME

(MINUTES)

Fro. 20. Relationship between supercontraction of keratin fibers and time of treatment in 8.3 M LiBr at 89°C (Haly and Feughelman, 1957).

313

T H E CHEMISTRY O F KERATINS

come well defined as the temperature decreases (Fig. 20). The extent of both stages is characteristic of particular fiber types. Haly and Feughelman suggested that the total contraction was determined by the number of disulfide cross-linkages in the fiber, whereas the first stage of contraction was thought t o be limited by strong secondary bonds which break during the second stage of contraction. Solutions of salts such as LiBr or N a l containing 1 N HCI, however, give contractions a t 100°C of about 75 % (Crewther and Dowling, 1959). It was concluded that acid-labile bonds or ionic interactions are responsible for limiting the extent of supercontraction in neutral solutions of LiBr. Crewther and Dowling (1960a) showed that conversion of 94 % of the disulfide bonds of wool to S-methyl groups does not affect the total supercontraction of Lincoln fibers. Hence disulfide bonds place little if any restriction on the extent of total supercontraction of these fibers.

I 100 IS-S-]

I

200 OR [-S-CH,-C%-S-]

I 300

I

400

(@OLE/CM)

FIG.21. Relationships between ( x ) extent of first stage of supercontraction and residual disulfide content of reduced and methylated wool fibers and ( 0 )becross-linkages. tween total supercontraction and content of (--S-CH,-CH2--S-) The theoretical relationship is shown by the unbroken line (Crewther, 196413). On the other hand the extent of the first stage of contraction increases with decreasing disulfide content (Crewther and Dowling, 1961c; Crewther, 1964b) approaching the value for total supercontraction at zero disulfide content (Fig. 21). The conversion of disulfide bonds into -S-CH2-CH2-Scross-linkages does riot appreciably affect the extent of the first stage of contraction, but decreases the total contraction.

314 w.

G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

The relationship between -S-CHz-CHz-S-content and total contraction in neutral solution is similar to the relationship between residual disulfide content of S-methylated wool and the first stage of contraction (Fig. 21). I n this study -S-CHz--CHZ-Scontent was equated to the loss of disulfide during the treatment. Analysis of these wool samples for S,S'-di-3-alanylethanedithiol and S-(2-hydroxymethyl)cysteiiie (Crewther et al., 1964a) showed the presence of appreciable amounts of the latter compound when the cystine loss was large. The true relationship between total contraction and -S-CH2-CH2-Scontent therefore falls a little below that in Fig. 21. A similar relationship holds between lanthionine content of alkali-treated or cyanide-treated fibers and the extent of total supercontraction (Crewther et al., 1964a). Since the thiol and disulfide contents of wool fibers do not change appreciably during supercontraction in 6 M LiBr a t pH 6, it was concluded (Crewther and Dowling, 1 9 6 1 ~ Crewther, ; l964b) that the first stage of contraction is limited by both disulfide and acid-labile cross-linkages and that the restrictions owing to the former are eliminated by a process of thiol-disulfide interchange during the second stage of contraction. Acidlabile bonds then limit the extent of total contraction. b. Thiol-Disulfide Interchange. There is a great deal of evidence suggesting that thiol-disulfide interchange takes place during the second stage of supercontraction. The reaction of wool with N-ethylmaleimide decreases both the thiol content and the rate of supercontraction in phenol (Burley, 1956a), and a similar effect is observed in aqueous LiRr (Crewther and Dowling, 196lb). In 6 M LiBr a t p H 6 free bromine accelerates the first stage of contraction, but almost completely represses the second stage; in 4 M LiBr a t pH 8 both stages are inhibited. I n the presence of sufficient Brz to eliminate thiol groups froni the fiber the rates of contraction of untreated fibers and those treated with N-ethylmaleimide become identical. Similarly, conversion of a large proportion of the disulfide groups of wool to Smethyl or -S-CHz--CH2--Sgroups almost eliminates the inhibitory effects of free bromine. Iodination in various solvents (Haly et al., 1957; Crewther and Dowling, 1962), nitration (Crewther and Dowling, l960b), oxidation with peracetic acid (Crewther and Dowling, 1962), and treatment with methyl iodide (Crewther, 1964b), all of which decrease the thiol content of the fiber, retard the second stage of supercontraction in 6 or 8 M LiBr near neutrality. Treatment with thioglycolate accelerates supercontraction over the whole range of contraction (Haly and Feughelman, 1960). Zahn et al. (1960) have suggested that lanthionine may be an inevitable side product of thiol-disulfide interchange reactions involving cysteine and

THE CHEMISTRY OF KERATINS

315

cystine. Since the disulfide content of wool does iiot decrease during the complete supercontraction of wool in 6 M LiBr a t p H 6, this suggestion is probably incorrect. In drawing this conclusion it is assumed that most of the disulfide bonds in the fiber are involved in interchange reactions during supercontraction. c. Acid-Labile Bonds and Contraction in L i B r Solutions Containing HCI. S-Methylated fibers reach maximum contraction very rapidly in neutral 8 M LiBr at relatively low temperatures niaking it possible to study the effect of acid concentration on the additional contraction caused by the acid (Crewther, 1964~). The rate of this additional contraction is directly proportional t o the acid concentration over a wide range of concentrations. Hence hydrogen ions have a catalytic action such as would be expected in hydrolysis of covalent bonds. Since alkaline solutions also give increased total contraction of S-methylated fibers the restriction of contraction in neutral solution is attributed to the presence of covalent cross-linkages other than disulfide bonds. It has not been possible to test the suggestion (Crewther, 1964c) that these cross-linkages may be ester linkages, as the methods currently used to demonstrate the presence of esters in proteins do not differentiate between esters and side-chain amide groups (Crewther, unpublished observations 1963). Since the extent of the first stage of contraction in 4 M LiBr/l N HCl is identical with that in neutral LiBr solutions (Crewther, 1964c) and the elimination of disulfide bonds does not cause appreciable changes in the total contraction, a reaction occurs which eliminates the effects of the disulfide bonds during contraction in acid solutions. Crewther (1964c) suggests that when under stresses imposed by entropic forces the disulfide bonds undergo interchange reactions with Sf groups a t lower acid concentrations than those known to catalyze disulfide interchange in mode1 systems. No explanation has been provided for the failure of alkali treatments producing lanthionine in the fibers t o depress the level of total supercontraction in 4 M LiBr/l N HCl a t 98.5"C (Crewther et al., 1964a). d. The Contribution of Microfibrils and Matrix to Supercontraction. The rate of the first stage of supercontraction in 4 M LiBr a t pH 8-8.5 is decreased by reacting the wool fibers with thiol-blocking reagents (Crewther and Dowling, 1960a, 1961b; Crewther, 196413; Crewther et al., 1964a). It is suggested (Crewther and Dowling 1960a, Crewther 196413) that the contraction of aligned molecules in the microfibrils deforms a parallel noncontractile matrix which is plastic rather than elastic in its properties. The matrix proteins are thought of as globular niolecules stabilized by many intrachain disulfide bonds and containing a few intermolecular disulfide bonds which, together with hydrogen bonds between side-chain

316 w.

G . CHEWTHER, R. D. B. FRASER, F. G. LENNOX, AND H . LINDLKY

groups, maintain the shape of the matrix. X-Ray diffraction data also suggest the preseiicc of a globular matrix (Praser, 1961). The viscosity of such a structure is determined by the rate of thiol-disulfide interchange when under stress and by solvation. Gluten is believed to have such a structure (Frater et al., 1960, 1961). Sikorski (1960) and Woods (1960) have criticized the concept of a noncontractile matrix on the basis of the studies of Jeffrey el al. (1955). Electron micrographs of preparations froin the interiors of cortical cells derived from supercontracted fibers showed no apparent twisting of the fibrillar material, yet X-ray diffraction patterns showed marked disorientation of the a- or /%structures in the fibers. Jeffrey et al. (1955) attributed this result t o contraction of the matrix. A disoriented a-pattern, however, could arise from the collapse of a super helix when restraints imposed by disulfide bonds are removed (as in peracetic acid or caustic soda solutions). Disoriented patterns, either a! or ,8, could equally well arise from partial solution of proteins within the fibers and deposition within the cells upon washing out the solubilizing agent and drying (Happey et al., 1953; Harrap, 1963). Using a n expression for the mean length of a random polypeptide chain (Crewther, 1964a) and assuming that the helices are contiriuous in the niicrofibrils and that the matrix does not affect the extent of supercontraction, the number of residues between acid-labile bonds can be calculated from the extent of total supercontraction (Crewther, 1964b). This value together with the contraction a t first stage make possible an estimate of the cystine content of the aligned proteins. Although the calculated values are in satisfactory agreement with the over-all amino acid analyses for five types of keratin fiber (Crewther, 1964b; Crewther et al., 1964b), recent studies on partially digested low-sulfur proteins (Crewther and Harrap, unpublished data, 1964) suggest that some of the cystine residues of the low-sulfur proteins do not form part of the aligned helices in the fiber. Hence this relationship may be indirect. In general, the experimental values relating disulfide and first stage of contraction for a number of keratin fibers (Fig. 21) fall below the theoretical curve based on the assumption that disulfide bonds are equally spaced along the chains (Crewther, 1964b)c). This indicates that the distribution of disulfide bonds along the chains is uneven. Calculations of cross-link density from stress-strain and volume-swelling measurements on supercontracted wool fibers immersed in solutions of LiBr (Haly, 1963a) gave values differing by factors of 3 to 4 from those calculated from the supercontraction data of Crewther (1964b). When extrapolated to zero disulfide content, the stress-strain data for S-methylated wool fibers (Haly, 1963a) suggested that the cross-link density of the

T H E CHEMISTRY O F KERATINS

317

wool would be decreased by a factor of 15 during complete reduction and alkylation of the disulfide bonds. Similar calculations from supercontraction data (Crewther, 196413) gave a factor of only 2.5. The approximations and assumptions made in obtaining these values probably contribute to these discrepancies, but differences in the chemical modifications undergone by the fibers appear to be partly responsible for this lack of agreement. Both Crewther (unpublished observations, 1961) and Haly (196313) have observed changes in the extent of total supercontraction of animal fibers after repeated reduction and alkylation, but Crewther found these changes to be small or insignificant. On the other hand, Haly observed a large increase in the extent of total supercontraction with the same X-methylated fibers used in assessing cross-link density. It is possible that new stable cross-linkages were formed during the reduction and alkylation of Crewther’s samples. Alternatively, acid-labile cross-linkages may have been ruptured during the reduction and alkylation of Haly’s samples. Either of these possibilities could contribute to the observed discrepancies. 3. Set

a. Covalent Linkage Formation. There is general agreement that setting of wool fibers involves the rupture of linkages during extension of the fibers followed by the formation of similar or new linkages in the extended position. Speakman (1936a) put forward the view that in order to set a keratin fiber it is necessary to break disulfide bonds and, after rearranging the protein chains, to form new disulfide bonds or other covalent bonds. He suggested that the reactions RI-S-S-Rz Rz-SOH

+ HzO

+ HZN-R,

RI-SH

+ Rt-SOH

S Rz-S-NH-Ra

(XXU

take place. Later, Speakman (1945) accepting a suggestion by Phillips (1936) assumed that the reactions Rz-CHZ-SOH Hz-CHO

+ HgN-Ra

Rz-CHO

+ HzS

S Rz-CH=N-Rx

(XXII)

also take place. The isolation and identification of lanthionine from hydrolyzates of alkali-treated wool (Horn et al., 1941, 1942a,b) introduced an additional type of covalent linkage which could contribute to maintaining set produced under alkaline conditions. J. €3. Speakman (1936a) attributes the optimum pH of 9-10 for setting in water t o the need for hydrolytic rupture of disulfide bonds. Treatment of wool with nitrous acid (Speakman, 1936a), adsorption of anions (Elliott and Speakman, 1943), the sulfation of the fiber (Speakman and Elliott,

318 w.

G. CREWTHER,

R. D.

B. FRASER, F. G. LENNOX, AND H. LINDLEY

1946), and reaction with 1-fluoro-2,4-dinitrophenol (Asquith et al., 1957) prevent or retard setting. Set fibers also contain fewer reactive lysine side chains (Asquith and Speakman, 1952, 1956; Asquith et al., 1957). I n each case these effects have heen interpreted in terms of the reactions of amino groups (reactions X X I and X X I I ) . Neither of the linkages formed by reactions (=I) or (XXII) has been demonstrated in alkali-treated wool, but Patchornik and Sokolovsky (1964) and Bohak (1964) have demonstrated recently the presence of c-N-(~,~-2-amino-2 carboxymethy1)-L-lysine in hydrolyzates of proteins treated with alkali. This they attribute to the reaction of the r-NH2 group of lysine residues with a-aminoacrylic acid residues formed from cystine residues by the p-elimination reaction (Section V,A,5). Ziegler (1964) has shown that this amino acid residue is formed during the alkali treatment of wool in both the stretched and unstretched states. No assessment of the importance of these linkages in retaining set has yet been reported. P. T . Speakman (1961) obtained a linear relationship between set in borate solutions and [loss of ~ y s t i n e ] ~Using . Flory’s (1956) expression for the isotropic length of a cross-linked polymer he concluded that lanthionine cross-linkages determined the set length of the fibers. Two main assumptions were made: (1) that cystine loss equals the lanthionine formed, and (2) that the fiber is an elastomer in hot water. Other attempts to investigate the formation of covalent bonds other than disulfides are equivocal (P. T. Speakrnan 1957, 1958), as insufficient information is known concerning the chemical and physical effects of boiling bisulfite solutions on wool. On the other hand there can be no doubt that reduction of disulfide bonds followed by stretching and reforniation of disulfides by oxidation (Patterson et al., 1940) gives a high degree of permanent set. Cuthbertson and Phillips (1945) could not detect any significant loss of amino nitrogen, increase in thiol content, or formation of aldehyde groups on treating wool with alkali. About one-half of the total cystine residues were converted to lanthionine residues and the remainder converted to two residues of a-aminoacrylic ac4d per residue of cystine destroyed. Blackburn and Lindley (1948) point out that the formation of lanthionine and a-aminoacrylic arid is very slow and unlikely to account for the speed of the setting reaction as observed by Astbury and Woods (1933). Asquith et al. (1957) suggest that thiol groups formed during setting are eliminated during hydrolysis for cystrine analysis, but this is not confirmed by thiol determinations on unhydrolyzed set fibers (Caldwell et al., 196413). An alternative explanation for the retardation of setting by deamination, dinitrophenylation, anion adsorption, and sulfation is provided by the

319

THE CHEMISTRY OF KERATINS

decrease in hydroxyl ion activity within the fiber that these treatments entail (Crewther, 1965a). Improved setting following esterification (Blackburn and Lindley, 1948) is likewise explained on this basis. b. Hydrogen Bond Formation and Crystallization. The view that hydrogen bonding contributes largely to setting arose from the observed change in diffraction pattern of fibers set by prolonged steaming while extended (Astbury and Woods, 1933). This has been strengthened by the observed reversion of fibers to their original length and X-ray diffraction pattern when set for short periods, immersed in saturated urea solutions, and washed (Itudall, 1946). Similar reversions to the a-pattern have been obtained on treating set fibers with cold solutions of NaOH, LiBr, or cupraminoniuin hydroxide (Sikorski, 1952). The cuprammoniuni reagent can release set even after fairly prolonged setting treatments. With this reagent, however, a dilute acid treatment is necessary to remove bound copper from the fiber (Whewell and Woods, 1946). Farnworth (1957, 1960) showed that chemical treatments which rupture disulfide bonds, such as reduction followed by niethylation or treatment with sodium bisulfite solutions, increase the rate of setting in boiling water and decrease the temperature required to give a predetermined set in a fixed time. Set fibers contract in boiling formic acid or 7 M LiBr to lengths less than their original length (Table XXVII). Farnworth conTABLE XXVII Release of Wool and S-Methyluted Wool Fibers i n Formic Acid or LiBr Solutions Ajter Setting Change in length (% of original)

Treatmenta Boiling H20 Boiling HzO followed by formic acid Boiling HzO followed by lithium bromide

Untreated +22

-t16 -31

Reduced and methylated +25 -20 -20

4 Fibers set a t 40 % extension in boiling water for 1 hr, released in boiling water for 1 hr, released in boiling water and then in formic acid (98-100 %, 30 min, 20OC) or lithium bromide solution (7 M , 80"C, 1 hr).

cluded that setting is accelerated by rupturing disulfide bonds and that this facilitates rearrangements and the formation of new hydrogen bonds which are largely responsible for maintaining the set form of the fiber. This view is confirmed by the more intense /I-pattern given by set S-methylated fibers as compared with normal fibers set under identical conditions (Jenkins and Wolfram, 1964). The studies of Diorio et al.

320 w.

G. CREWTHER, R. D. B. FRASER, F. G . LENNOX, AND H . LINDLEY

(1962) on the crystallization of the 0-phase in stretched keratin fibers on transferring from solutions of LiBr to boiling water have similar iniplioations. The fact, however, that set fibers contract less, in ternis of the original length, than unset fibers when boiled in 7 M LiBr (see Table XXVII) suggests that bonds other than hydrogen bonds have been foriucd or rearranged in the fibers. A variety of compounds capable of rupturing disulfide bonds have been shown to accelerate setting. Sulfite mixed with ninhydrin (Speakman and Speakman, 1956), tetrakis(hydroxymethy1)phosphonium chloride (Bajpai et al., 1960, 1961; Zahn and Vassiliadis, 1962; Jenkins and Wolfram, 1963)) thiourea dioxide (Bajpai and Whewell, 1961), arid peracetic acid (Jenkins arid Wolfram, 1964) are examples. c. Mechanisms i n Setting. The change in leiigth of the fiber after setting is a summation of the effects of (i) formation of p-keratin, (ii) covalent cross-linking of partially aligned chains, (iii) restoration of a-keratin structure, (iv) randomization of peptide chains, and (v) disorientation of CY- or p-keratin; (i) and (ii) tend to increase the net length, (iii) to maintain the original length, and (iv) and (v) to decrease the length of the fiber. There is evidence that all effects contribute to the length of a set fiber. One aspect of setting that has received little attention, although it follows logically from the work of Burley (1956a) and the data on creep and stress relaxation (Section VI,C,l), is the role of thiol-disulfide interchange in setting. The existing evidence has been considered elsewhere (Crewther, 1965~). It may be sumniarized as follows: (i) Creep, stress relaxation (Section VI,C,l), and setting of wool fibers are accelerated by reductive treatments (Farnworth, 1957; Feughelman and Mitchell, 1959; Bajpai and Whewell, 1961; Bajpai et al., 1960, 1961; Wolfram and Speakman, 1960) and retarded by oxidative treatments (Mitchell and Feughelman, 1958; Haly and Feughelman, 1960; Wolfram and Speakman, 1960; Caldwell et al., 1964a). (ii) Oxidative inhibition is reversed by reduction (Wolfram and Speakman, 1960; Bajpai et al., 1961). (iii) Stress relaxation (Feughelman and Mitchell, 1959) and setting (Speakman, 1936a; Whiteley, 1962) are prevented or retarded by treatments which convert cystine residues t o lanthionine or to S1S’-di-3-alanylethanedithiol residues. (iv) Reaction of wool with reagents that block thiol groups such as l-fluoro-2,4-dinitrobenzene (Asquith and Speakman, 1956; Asquith et al., 1957) or N-ethylmaleimide (Zahn et al., 1961a) retards setting and this effect is reversed in the presence of small concentrations of NaHS03 (Crewther, 1965~). (v) Setting occurs a t pH values a t which thiol-disulfide interchange is catalyzed by ionization of thiol groups (Speakman, 1936a).

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(vi) There is also evidence that thiol-disulfide interchange facilitates recovery of the fiber after release (Burley, 1956a; P. T. Speakman, 1959; Caldwell et al., 1964a). The following summarizes the views of Crewther (1964~)concerning the mechanism of setting a t 40 yoextension in boiling water. (i) When stretched in cold water by 40 yothe a-helices are partly opened out, including some portions closely associated with disulfide bonds which are under stress, and the matrix has undergone plastic deformation. (ii)At 100°C the remaining a-helices tend toward the random coil form, and some of the matrix proteins may undergo denaturation. Thioldisulfide interchange is greatly accelerated by both changes due to greater accessibility of reacting groups. (iii) Each half-cystine residue takes part in several interchanges before the chains are suitably arranged to form p-sheets which are thus stabilized by disulfide bonds. (iv) On release in boiling water the noncrystalline structures assunie their most probable length in their existing cross-linked condition. This imposes entropic forces on disulfide bonds that undergo thiol-disulfide interchange reactions and permit parts of the fiber to assume the random state. The smaller p-crystallites may also revert to the random-coil state. (v) On cooling, some a-helices or P-crystallites tend to form where the disulfide bonds permit these conformations. Other sections retain a random form. This view (Crewther, 1960) is directly opposed to the suggestion by Skertchly and Woods (1960) that polypeptide chains in portions of the fiber cross-linked by disulfide bonds will extend in phase and assume a p-sheet structure more readily than long sections of a-helix containing no disulfide bonds. It has much in common with the view expressed by Bendit (1960b), but is not in accord with the suggestion by Bendit that matrix proteins contribute to the formation of p-keratin. It is difficult to visualize the formation of p-sheets in the fiber from the high-sulfur proteins in which one residue in four to six residues is half-cystine and one residue in seven is proline. I n the form of the soluble S-carboxymethyl derivatives, however, the high-sulfur proteins can be induced to form structures giving a p-pattern (Bendit, private communication, 1964). d. The Use of Protein Denaturants in Setting. Farnworth (1957) has shown that wool can be set a t 30°C by treating with 50 yo urea in 5 % sodium bisulfite solution followed by rinsing in the extended position and the usual release in boiling water for 1 hr. He attributes this to the simultaneous rupture of hydrogen bonds and disulfide bonds followed by their reformation in the extended fiber. On the other hand Jenkins and Wolfram (1964) were unable to obtain a similar result with S-niethylated wool containing 170 residual cystine

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after stretching in cold urea solutions containing no bisulfite. They suggest that molecular slippage is responsible for the setting of S-methylated fibers in hot water and that unfolding of peptide chains in phase can only occur in a highly cross-linked system. Jenkins and Wolfram claim that these views are supported by their observations that (i) a linear relationship exists between the set of S-methylated fibers in hot water and the extension during setting. An extension of 20 yo was required to give zero set. (ii) Maximum setting was obtained in neutral solutions decreasing very greatly with increasing pH. (ii2) S-methylated wool gave a sharp oriented P-pattern which was largely disoriented in 97 % formic acid and then showed some a-pattern on removing the acid. Similar treatment of wool containing 4.5 yo residual cystine gave a less well-defined p-pattern on setting and a little more a-pattern on treatment with formic acid and washing. The first result would bc expected on most hypotheses; the second, together with the absence of set in cold urea solutions, suggests that the S-methylated fibers cannot set in the highly swollen or solvated state; the third indicates that better alignment of the polypeptide chains is achieved when the disulfide content is decreased. These results are in agreement with the view presented in the preceding subsection, but they do not denioristrate that molecular slippage occurs during setting of S-methylated wool. On the contrary under conditions that favor molecular slippagepresence of urca or high pH-fibers do not set readily. The important difference between the cold setting experiments of Farnworth and those of Jenkins and Wolfram is the presence of a considerable number of thiol groups and disulfide bonds in Iparnworth’s fibers under conditions where disulfide rearrangement is facilitated; a t the end of the setting process the disulfide bonds are optimally arranged to preserve a highly ordered and therefore stable B-sheet structure.

4. Stress-Strain Relationships a. Rupture of Disulfide Bonds. The stress required to produce a fixed extension in animal fibers immersed in water is progressively decreased when the disulfide bonds are ruptured (Patterson et al., 1941; Harris et al., 1942; Sobue, 1956; Lindley, 1957; Feughelman and Haly, 1961; Feughelman, 1963; Crewther, 1965b). This is largely due to the decrease in the stress a t which the Hookean region ends. The relationship between the work t o stretch fibers by 30 Yo and their residual disulfide content after oxidation with peracetic acid (Alexander et al., 1951a) or reduction followed by methylation (Patterson et al., 1941) is approxiniately linear. As the disulfide content of Sniethylated fibers approaches zero, the stress a t which the stress-strain curve first deviates from a linear relation-

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ship also approaches zero (Crewther, 1965b). The stresses a t the end of the yield region and a t the break similarly decrease with decreasing disulfide content, and there is an increase in the strain both a t the end of the yield region and a t the break. A breaking strain of about 80 % is reached at a disulfide content of about 20 pmole per gram. The curves relating residual disulfide of S-methylated fibers and strain a t the end of the yield region are identical within experimental error for a number of different keratin fibers. This suggests that disulfide bonds in both high-sulfur and low-sulfur proteins contribute to the properties of the fiber in the post-yield region. The slopes of the stress-strain curves in both yield and post-yield regions decrease with decreasing disulfide content and approach zero at zero disulfide content. With fibers stressed a t a rate of about lo9 dyne cm-2 min-' the ratio of these slopes remains constant a t about 10: 1. The conversion of disulfide bonds to thiol groups has a much greater effect on the physical properties of wool than conversion t o S-methyl groups (Crewther, 1965b). Reduction of relatively few disulfides and the consequent small increase in thiol content give maximum extensions > 100 % and high strains a t the end of the yield region. The magnitude of the changes in longitudinal and torsional stiffness of wool caused by rupturing disulfide bonds is very dependent on the relative humidity used for stressing the fibers. In dry fibers the effects of disulfide rupture are small or not apparent (Harris and Brown, 1946; Feughelman, 1963; Feughelman and Mitchell, 1964). The stress-strain behavior of iodinated fibers in water illustrates further the contribution of disulfide bonds to the stiffness of the fiber (Hoare et al., 1962). When strained into the post-yield region wool fibers containing loosely combined iodine are drastically modified and on removal of iodine with NazSz03have the physical properties of wool containing few disulfide bonds. It has been suggested that the disulfide bonds become more reactive when stressed (cf. Speakman, 1936a; Lindley and Human, 1957). b. Extensions in Solvents Other than Water. The stress a t which the Hookean region ends is greatly decreased by immersing the fibers in aqueous phenol (Alexander 1951), aqueous LiBr (Alexander, 1951; Crewther, 1 9 6 5 ~ aqueous )~ urea (Crewther, 1 9 6 5 ~ or ) ~98-100 % formic acid (Speakman, 1932; Szucht, 1962) instead of water. In formic acid the Hookean region disappears altogether. Mixtures of mpropanol and water also decrease the stiffness of the fiber more than water does (Atkinson et al., 1959; Zahn and Blankenburg, 1964). Since the Hookean region of the stress-strain curves can be eliminated by rupturing either disulfide or secondary bonds, these bonds probably cooperate in stabilizing a single structure.

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G. CHEWTHER, R. D . B. FIZASER, F. G . LENNOX, AND 13. LINDLEY

13yuilibratioii of wool with aqueous urea or LiRr, treatments which would be expected to facilitate chain movement and hence also thioldisulfide interchange, increases the strain at the transition point between the yield and post-yield regions of the stress-strain curves (Crewther, 1965~). c. Blocking of Thzol Groups and Related Modifications. The reduction of wool with thioglycolate and alkylation with triniethylene dibromide (Harris et al., 1942) or ethylene dibroniide (Lindley 1957; Crewther, 1965b) does not cause a major change in the elastic properties of the fibers, such as might be expected if thiol-disulfide interchange took place during stretching. The efferts are equivalent to the conversion of a small proportion of the disulfide bonds to S-methyl groups and are attributable to the formation of S-(2-hydroxyethyl)cysteine residues (Crewther et al., 1964a). The consequent rupture of disulfide bonds may mask reverse effects owing to the conversion of disulfide bonds to the less reactive -S-CH2-CH2-Slinkages. The stress-strain characteristics of alkali-treated wools are variable (Satlow, 1959). Probably two main reactions affect the stiffness of the fibers; conversion of disulfide bonds to thioether linkages and disruption of cystine residues to foriii a-aininoacrylic acid residues (Cuthbertson and Phillips, 1945). The reaction of wool with iV-ethylmaleimide or l-fluoro-2,4-dinitrobenzene sharpens the transitions between the Hookean and yield regions and between the yield arid post-yield regions of the stress-strain curve (Crewther, 1965~). Both reactions decrease the straiii at the yield/postyield transition and this is particularly evident with extensions in hot water. Treatment with iiitrous acid (l+ughelman et al., 1959) has a similar effcct. These changes may be due to inhibition of thiol-disulfide interchange during stretching. d. Rate of Loading and “Transition Temperature.” The shape of the load-extension curve for untreated wool is iiiflueiiced by the rate of loading (Speakman, 1927). Under “instantaneous” loading the transitions between Hookean, yield, and post-yield regions are sharper and their corresponding stresses are greater than with slower loading. There is also a decrease in strain at the transition between the yield and post-yield regioiis. These observations have been confirmed for various fiber types and under various conditions of loading (Schiefer et al., 1956; Feughelman et al., 1959; Szucht, 1962; Crewther, 196513). S-Methylated fibers containing few disulfide bonds do not show the latter effect (Crewther, 1965b). Similarly, Sikorski and Woods (1950) have shown that the Youiig’s Modulus of wool and hair fibers increases with increased rate of loading. Thus creep or stress relaxation is sufficiently rapid a t room temperature to influence the

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shape of the stress-strain curve; at high temperatures when stress relaxation is rapid compared with loading the effects are more pronounced. Feughelnian et al. (1959) have shown that the strain a t the transition point between the yield and post-yield regions remains almost constant at constant rate of loading up to a certain temperature, but then increases with temperature. The temperature a t which this change becomes apparent is raised by increasing the rate of loading or by chemical treatments such as iodination or reaction with nitrous acid. It is decreased by decreasing the rate of loading or by treatment with thioglycolate. Feughelnian et al. (1959) have called this the “transition temperature” of the fiber. The data are consistent with relaxation of stress (Section VI,C,l) by thioldisulfide interphange reactions which are not sufficiently rapid a t high rates of loading and low temperature to affect the load-extension curves. With increased temperature or thiol content, however, or with decreased rate of loading, this reaction releases the stress by rearranging sufficient disulfide bonds for the fiber to behave as though it contained fewer such bonds. This suggests that the transition temperature is a rate phenomenon and has no thermodynamic significance. It should be clearly distinguished from the “melting” of structures in the wool fiber which may be disclosed by other methods (Mason, 1964a,b). No transition temperature has been observed in torsional properties of the wool fiber (Feughelman and Mitchell, 196l), although the torsional rigidity and stress relaxation change rapidly over the temperature range studied (0’44°C). Nevertheless, the rigidity modulus decreases and the torque relaxation increases in rate on treating the fibers with thioglycolate solutions, which suggests that thiol-disulfide interchange occurs during deformation of the matrix (Feughelman and Mitchell, 1964). e. Hydrolysis of Peptide Linkages. The hydrolysis of relatively few peptide bonds causes a marked decrease in the breaking load of single wool fibers (Elod et al., 1940). Treatment of wool with limited amounts of dilute HzS04produces chiefly aspartic acid N-terminal residues (Crewther and Dowling, 1960a). As the low-sulfur proteins have three times the aspartic acid content of the high-sulfur proteins (Section 1I1C,4),this suggests a preferential attack on the microfibrils. Treatment with restricted amounts of concentrated H&04 produces chiefly serine and threonine N-terminal residues. The greater content of these amino acids in the high-sulfur proteins suggests a preferential attack on the matrix. The former treatment decreases the breaking load in water more than that a t 50 % relative humidity (R.H.), the latter treatment has about equal effects a t both R.H. values (Crewther and Dowling, 1960a). This suggests that in wet fibers the microfibrils bear most of the breaking stress, whereas a t lower R.H. values the matrix bears a significant part of the breaking stress.

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W. G. CREWTHER, R. D. B. FRASER, F. G. LENNOX, AND H. LINDLEY

Feugheliiian (1959) has drawn similar conclusions for small stresses from a study of torsional properties of the fibers. 5. Sorption and Swelling

a. Sorption of Water. The equilibrium regain of wool is determined by the I1.H. of the environment (Fig. 22), but measured regains differ during sorption and desorption (Speakman, 1936b) and are also dependent on the magnitude of the change in R.H. (Watt, 1960) and on the previous history of the fiber (Watt and Kennett, 1960). Nuclear magnetic resonance studies (Shaw et al., 1960, Haly et al., 1962) have coiifirined earlier

PERCENTAGE

RELATIVE

HUMIDITY

FIG. 22. The adsorption isotherms relating regain and relative humidity for wool (----) and for S-methylated wool (-) containing 10% of the original disulfide bonds (Watt, 1963).

conclusions (Alexander and Hudson, 1954) that a t low R.H. values water is firmly bound by the wool, but as the R,.H. increases from 50 to 100 % the water in the fiber becomes more mobile. X-Ray diffraction studies indicate that most of the water is located in the matrix (Fraser et al., 1959). The conversion of disulfide groups to thiol groups does not greatly affect the water sorption isotherm (Mellon et al., 1949; Watt, 1963), the maximum regain increasing by only 1 yo. Extensive reduction in n-propanol, however, followed by methylation in the same solvent (Maclaren, 1962) causes

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a decrease in regain a t low R.H. vdues and a large increase in water uptake

at high R.H. values (Fig. 22). These properties resemble those of supercontracted or set fibers (Haly and Snaith, 1963). Attempts to determine the extent of cross-linking in wool fibers, whether untreated or reduced and alkylated, by measurement of swelling in aqueous LiBr or similar solvents (Haly, 1963a; Atkinson and Speakman, 1960; Speaknian and Stone, 1962, 1963) give results of doubtful significance because of the inhoniogeneity of the wool fiber. b. Eflects of Adsorbates on Regain and Mechanical Properties. The uptake of water by wool is decreased by the adsorption of anions particularly those with high affinity for the fiber (Speakman and Elliott, 1946; Nicholls and Speakman, 1954; Larose, 19.54) such as anionic dyes. The increase in volume of fibers a t 65 % R.H. owing to the absorption of dyes is approximately equal to the volume of dye adsorbed (Larose, 1956), and there is evidence from swelling measurements that the dye may exclude water from the fiber (Speakman and Elliott, 1946; Brown, 1959). Similarly, benzylation of wool giving about 10 yoincrease in weight almost halves the regain at 65 % R.H. (Oku and Ishibashi, 1960), and deposition of ninhydrin and related compounds in the fiber decreases swelling (Cockburn and Speakman, 1956a,b). The ninhydrin treatment caused a 25 % increase in work needed to extend by 30 yo (Cockburn eta,?., 1953, Cockburn and Speakman, 1956a,b), and Feughelman and Watt (1964) conclude that ninhydrin-treated fibers behave in water like untreated fibers a t 90 % R..H. Likewise, fibers containing adsorbed dye are stiffened (Speakman and Elliott, 1946) and alkylation of reduced fibers with benzyl chloride gives stiffer fibers (in water) than alkylation with metliyl iodide (Harris et al., 1942). There is a t present insufficient data to determine whether these effects of large adsorbed molecules are due to (1) increasing cohesion between adjacent peptide chains, i.e., a direct physical effect of the adsorbed material (Speakman and Elliott, 1946), (2) exclusion of water (Haly, 1963a; Feughelman and Watt, 1964), (3) cross-linking (Haly, 1963a; Cockburn and Speakman, 1956a), (4) other chemical or physical effects such as changes in electrostriction (Kauzmann et al., 1962; Kasarda, (1961), or (5) inhibition of thiol-disulfide interchange, either by changing the “internal pH” or by direct oxidative or blocking effects. Probably more than one effect is operative. c. The Pore Size of Keratin. ICarly studies by Speaknian (1930) showed that wool fibers stretched in water, methanol, ethanol, or n-propanol showed increasing work to stretch 30 % in that order; with n-butanol or n-amyl alcohol the fibers behaved like anhydrous fibers. As there was a progressive decrease in the amounts of the various alcohols sorbed by the

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fibers to a rnininiuni of 0.3 % with n-amyl alcohol, Speakniari suggested that the larger molecules could not penetrate the fine structure of the fiber. The high degree of iodination of tyrosine residues of wool in ethanol (Richards and Speakman, 1955, 1956), the incomplete iodination in n-propanol, and the absence of appreciable iodination in n-butanol (Harrison and Speakman, 1958) have been used to support this view. Bradbury and Leeder (1963a,b,c), however, have shown that with prolonged equilibration in anhydrous alcohols wool fibers absorb essentially equal volurnes of water, methanol, ethanol, n-propanol, isopropanol, and n-butanol amounting to 34.1 m1/100 gin. Furthermore, considerable amounts of higher alcohols are also sorbed. They suggest that the “pores” iri wool vary in size owing to thermal fluctuations and swelling. They also demonstrated a considerable volume contraction when water was adsorbed by wool and an even greater effect with ethanol. The partial iodination of tyrosine in wool by propanolic iodine and its lack of reaction in butanol are explained by the observation that N-acetyltyrosine ethyl ester is more extensively iodinated in ethanol than in propanol (Crewther and Dowling, unpublished observations, 1962). Solutions of I2 in n-butanol do not react with the tyrosine derivative. The results of iodination in different solvents are therefore attributable to differences in chemical equilibria. 6 . Models of the Wool Fiber Based o n Physical Properties

Many models have been proposed to describe or explain the physical properties of wool fibers. Some simple mechanical models, such as those of Shorter (1924) and Burte and Halsey (1947) describe the behavior of fibers in terms of idealized mechanical components which can be treated mathematically (Feughelman, 1954; Feughelman and Rigby, 1956; Ruoff and Eyring, 1956; Warburton, 1956). Others relate the physical properties of the fiber to observed or hypothetical cellular or subcellular components within the fiber. None of the proposed models accounts for all the physical and chemical data available. Some models (Astbury arid Woods, 1933; Feughelman and Haly, 1959, 1960; Skertchly, 1964; Munakata, 1964) consist of a t least three phases of which two or more are in series along the fiber axis. As an example of a model comprising different phases the most rerent version of the serieszone model proposed by Feughelman and Haly (1959, 1960) will be considered. It consists of microfibrils, containing aligned a-helices, surrounded by a disorganized matrix in which disulfide cross-linkages show a periodic rise and fall in density along the fiber giving rise to zones in series (Feughelman, 1963). Upon stretching the fiber in the yield region the helices are

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opened out adjacent to the lightly cross-linked zones in the matrix with a progressive decrease in a-structure arid the appearance of 0-crystallites. When the lightly bonded sections of the matrix are fully extended, additional stress is needed to extend the heavily cross-linked zones in the matrix, giving rise to the post-yield region. The model provides a simple explanation for Bendit’s (1957, 1960b) intensity measurements on the conversion of a-keratin to 0-keratin. When the model was first proposed it was suggested (Feughelman and Haly, 1959, 1960) that the ratios, extent of first stage of supercaontraction/evtent of second stage of supercontraction, extension in the yield region/extension in the post-yield region, and length of zone X/length of zone Y are equal. Although the model describes the behavior of wool fibers in general terms, its acceptance as a true repi esentation of the subcellular structure of wool encounters several difficulties : (1) The interrelationship between supercontraction data and stress-strain data summarized above is not identical for all keratin fibers (Crcwther, unpublished observations, 1962). (2) The effects of acid treatment on the wet and dry breaking strengths of wool fibers indicate that in watw most of the breaking stress is borne by the microfibrils. In the model the matrix bears a large proportion of the stress. (3) The proteins of the corlkal cells are remarkably resistant to protease attack. Disorganized protckins capable of absorbing large amounts of water would not be expected to show this characteristic. (4) The breaking load of wool decreases contini tously with decreasing disulfide content, whereas Gee (1947) has shown that the breaking load of rubbers reaches a maximum a t a low level of cross-linking and then steadily decreases. This suggests that the main stress is not borne by a heavily cross-linked rubberlike structure at the time of rupture. (6) Other difficulties have been indicated elsewhere (Mandelkern et al., 1962a; Crewther, 196413). Other models seek to explain the properties of the fiber in terms of known subcellular structures and cross-liakages. In the model of Crewther and Dowling (1961~;Crewther, 19641) the microfibrils consist mainly of a-helices linked by disulfide and acid-labile covalent bonds to each other and to the matrix. The matrix is considered to be plastic rather than elastic and to consist of globular protein molecules with an ordered structure stabilized by intrachain disulfide bonds and linked to similar molecules or to the microfibrils by a few disulfide bonds and hydrogen bonds. The viscosity of the matrix is believed to be determined by the rate of thioldisulfide interchange when streswd and by the number of intramolecular hydrogen bonds (cf. gluten, Frator et al., 1960, 1961). The resistance of wool to proteolysis (Geiger et al., 1941; Geiger arid Harris, 1942; Lennox, 1952; Crewther, 1956) despite th(8 large amount of water absorbed by the

330 w.

G. CIZEWTHER, 11. D. B. FIIASER,

F. G. LENNOX, AND H. LINDLEY

matrix may be explained on this basis. The explanation for two-stage supercontractiori has already been outlined in terms of this model (Section VI,C,2). The stress-strain properties of keratin fibers are attributed to stretching of a variety of bonds in the Hookean region and opening out of the a-helices in the yield region together with plastic flow of the matrix. It is suggested that a t a certain strain the rigidity of individual disulfide bonds hinders the further opening out of the a-helices and other hydrogen-bonded parts of the fiber. This gives rise to the post-yield region. Thus the post-yield region is considered to reflect the viscoelastic properties of many different units in the fiber. Stress relaxation is attributed to the rearrangement of strained disulfide cross-linkages by a disulfide-interchange mechanism (see Sections VI,C,I and 3). Experimental facts which have not been explained in terms of the model are: (1) The acid-labile bonds have little if any influence on the strain at the end of the yield region (Crewther, 1964b). ( 2 ) The yield region of the stress-strain curve, in which the matrix is visualized as undergoing chiefly plastic flow, is not markedly changed in slope by treating the fiber with thiol-blocking agents (Crewther, 1 9 6 4 ~ ) . Although the structural models described in Section V,A,2 have not yet been integrated with those arising from the study of physical and mechanochemical properties of the fiber, recent observations and current research using many different techniques make such a synthesis imminent. ACKNOWLEDGMENTS The authors are grateful to their colleagues in the Division of Protein Chemistry for assistance in assembling and interpreting the data presented and for making available for inclusion in this review much of thcir unpublished work.

REFERENCES Alexander, P. (1951). Ann. N . Y . Acad. Sci. 53, 653. Alexander, P., and Earland, C. F. (1950) Nature 166, 396. Alexander, P., and Hudson, R. F. (1954). “Wool, Its Chemistry and Physics.” Chapman & Hall, London. Alexander, P., and Kitchener, J. A. (1950). Textile Res. J. 20, 203. Alexander, P., and Smith, L. F. (1956). P ~ o c Intern. . Wool Textile Res. Con!., Australia, 1956 B, 56. Alexander, P., Fox, M., and Hudson, R. F. (1951a). Biochem. J . 49, 129. Alexander, P., Carter, D., and Earland, C. (1951b). J. SOC.Dyers Colourists 67, 17. Allen, A. K., Lindley, H., and Rogers, G . E. (1964). Proc. 6th Intern. Congr. Biochem., New York, 5B, 20. Allerhand, A., and Schleyer, P. von R. (1963). J . Am. Chem. SOC.85, 1233. Ambrose, E. J., and Elliott, A. (1951). Proc. Roll. SOC.Ser. AZ&, 206. Ambrose, E, J., and Hanby, W. E. (1949). Nature 163, 483. Amow, L. E. (1937). J . Biol. Chem. 120, 151.

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Author Index Kumbers in italic show the page on wliic.11 the complete reference is listed.

A Abraham, E. P., 56, 106, 123, 137, 141, 187, 189

Akabori, S., 97, 107, 132, 160, 177, 178, 181, 189

Akcroyd, J. H., 2, 35 Alexander, A. E., 309, 312, 386 Alexander, P., 146, 181, 206, 214, 216, 220, 242, 247, 277, 280, 281, 305, 308, 312, 322, 323, 326, 330 Allen, A. K., 219, 330 Allende, J. E., 96, 99 Allerhand, A,, 282, 330 Allison, H., 52, 106 Altenschopfer, T., 151, 152, 153, 156, 185 Altgelt, K., 117, 118, 119, 181 Altman, K. I., 147, 164, 170, 186 Ambrose, E. J., 292, 330 Ames, W. M., 111, 114, 141, 181 Annn, K., 96, 99 Anderson, N., 259, 341 Andersen, S. O., 110, 181 Anderer, F. A., 69, 75, 99 Anderson, B., 93, 99, 177, 181 Anderson-Cedcrgren, E., 24, 34 Andrejeva, A,, 178, 182 Anesey, J., 114, 118, 119, 189 Anfinsen, C. B., 39, 55, 63, 67, 87, 94, 96, 98, 99, 100, 101, 105 Anson, M. L., 95, 99 Antonini, E., 97, 99 Armstrong, M. D., 123, 186 Arndt, U. W., 292, 342 Arnow, L. E., 287, 330 Astrup, H. N., 181 Asquith, R. S., 318, 320, 881 Ashworth, J., 280, 309, 346 Astbury, W. T., 259, 291, 297, 298, 300, 301, 304, 305, 306, 307, 308, 309, 310, 319, 328, 331 Atkinson, J. C., 254, 323, 327, 331 Azzone, G. F., 24, 34

B Baddiley, J., 145, 181 Bailey, J. L., 208, 249, $31

Bailey, K., 96, 100 Bajpai, L. S., 308, 320, 331 Baker, J. R., 238, 331 Bakerman, S., 118, 181 Balch, D. A,, 238, 331 Bnldwin, R. L., 97, 98, 105 Ballerini, G., 9, 32 Balls, A. K., 156, 181 Balog, I., 244, 343 Balog, J., 53, 106 Balsamo, C. A,, 241, 340 Bamford, C. H., 292, 331 Baptist, V. H., 132, 181 BBrAny, K., 19, 32 Bkbny, M., 18, 19, 24, 32 Barker, S. A., 181 Barkin, S. M., 115, 181 Barnard, W. S., 279, 280, 281, 331 Bnrnett, L. M., 67, 103 Barritt, J., 230, 234, 331 Bnrtsch, R. G., 53, 93, 101 Bartulovich, J. J., 203, 233, 234, 331, 3 9 , 345

Bass, L. W., 131, 187 Battaglia, F. C., 61, 106 Battersby, A. R., 134, 181 Baum, W. E., 96, 100 Bauters, L. L., 229, 331 Bear, R. S., 291, 301, 331 Beck, S., 96, 105 Bedford, G. R., 110, 181 Beeley, J. G., 181 Behrens, 0. K., 65, 81, 100 Beighton, E., 301, 331 Bell, F. O., 291, 297, 331 Bell, J. W., 280, 309, 331 Bello, H. R., 150, 151, 152, 181, 281, 282, 331 Bello, J., 150, 151, 152, 181, 281, 282, 331 Bencze, W. L., 58, 99 Bender, M. L., 65, 99 Bendit, E. G., 284, 305, 321, 329, 331 Benesch, R., 248, 331 Benesch, R. E., 248, 331 Benitez, R., 2, 35

348

AUTHOR INDEX

Bensusan, H. B., 140, 182 Bentley, J. P., 119, 186, ID0 Berger, A,, 67, 104 Bergmann, F., 155, 152 Bergmann, M., 40, 51, 55, 64, 90, 99, 106 Bernard, W., 3, 32 Bernhard, S. A., 56, 99 Bertsch, I,., 67, 103 Bessis, M., 3, 32 Betheil, J. J., 140, 182 Bettelheim-Jevons, F. R., 178, 182 Bettex-Gulland, M., 5 , 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 25, 26, 32, 34 Bidnicatl, D. S., 180, 182 Bierman, H. R., 3, 34 Bigwood, E. J., 105, 229, 230, 343 Uinkley, C. H., 228, 237, 346 Biserte, G., 52, 99, 203, 208, 331 Bizzozero, J., 2, 32 Blackburn, S., 52, 99, 206, 208, 210, 216, 225, 243, 245, 253, 306, 318, 319, 332 Blackwell, P. M., 10, 33 Blankenburg, G., 242, 323, 346 Blau, K., 63, 100 Blook, R. J., 332 Bloom, G., 10, 22, 32 Blout, E. R., 241, 292, 300, 339, 3 4 Blumenthal, D., 230, 332 Blumenfeld, 0. O., 146, 147, 148, 151, 152, 155, 158, 159, 161, 162, 168, 173, 174, 177, 179, 182 Bodanszky, A., 327, 338 Boedtker, H., 111, 182 Boydanov, V. P., 178, 182, 186 Boggust, W. A,, 286, 334 Bogle, G. C., 284, 332 Bohak, Z., 318, 332 Bonjour, G., 50, 100 Bonsma, F. W., 235, 332 Borchgrevink, C. F., 8, 9, 22, 32, 34 Barman, A,, 97, 106 Born, G. V. R., 7, 16, 32 Borrelli, J., 3, 5, 8, 9, 36 Bow, S. M., 112, 124, 132, 135, 136, 138, 139, 140, 151, 153, 164, 179, 182, 186 Botvinik, M. M., 182 Bounameaux, Y., 6, 8, 9, 17, 21, 33, 36 Bourrillon, R., 177, 178, 182 Bovarnick, M., 123, 131, fS2

Bovey, F. .4.,74, 100 Bowes, J. H., 113, 137, 138, 139, 140, 141, 179, 182 Boyer, P. D., 97, 101, 248, 331 Braconnot, H., 37, 100 Bradbury, E. M., 300, 332 Bradbury, J. H., 160, 182, 214, 215, 233, 234, 243, 328, 332 Brand, E., 51, 0.9 Braunitzer, G., 156, 157, 182 Braunsteiner, H., 3, 33 Bresler, S. E., 96, 100 Breuer, M. M., 310, 332 Bricas, E., 121, 122, 182 Briggs, F. N., 24, 33 Bromcr, W. W., 65, 81, 100 Brown, A. E., 308, 323, 332, 337 Brown, D. M., 59, 90, 91, 102, 105, 10G Brown, J. C., 327, 332 Brown, L., 292, 331 Brown, P. C., 112, 182 Brown, R. K., 10T Bruckner, V., 124, 126, 182, 186 Buchanan, D. L., 122, 123, 182 Buehanan, J. M., 87, 92, 93, 100, 102 Buckingham, D. A,, 62, 100 Buddecke, E., 182 Budte-Olsen, 0 . E., 9, 10, 33 Bull, H. B., 40, 100, 132, 181, 310, 332 Bullock, E., 144, 182 Bu’lock, J. D., 286, 337 Burgess, V. R., 284, 332 Burkhardt, H.-J., 182 Burley, R. W., 218, 224, 236, 243, 303, 311, 314, 320, 321, 338, 334 Bursa, F., 123, 126, 134, 135, 144, 186 Burt, N., 53, 103 Burte, H., 328, 332 Butler, J. A. V., 96, 100

C Cabrera, G., 140, 187 Caesar, R., 29, 33 Calam, D. H., 122, 182 Caldwell, B., 318, 320, 321, 332 Canfield, R. E., 39, 64, 69, 72, 75, 81, 87, 88, 100 Caputo, A., 97, 99 Carpenter, F. H., 96, 97, 100, 106 Carter, D., 206, 308, 330

349

AUTHOR INDEX

C a d , A., 50, 51, 100 Castaldi, P. A., 10, 33 Castaiieda-Agull6, M., 65, 100 Cater, C. W., 113, 114, 182 Cawkwell, J. M., 24, 25, 35 Cecil, R., 247, 332 Chaikoff, L. L., 90, 107 Chapman, G. V., 233, 234, 332 Chen, T. L., 8, 33 Chernikov, M. P., 100 Chervenka, C. H., 61, 96, 100 Chibnall, A. C., 41, 51, 58, 90, 100, 125, 132, 144, 146, 148, 182, 183 Chinard, F. P., 225, 332 Christensen, H. N., 41, 45, 100 Chun, E. H. L., 118, 119, 182 Chung, D., 64, 69, 72, 103 Clarke, H. T., 140, 185, 230, 248, 332 Clayton, D. W., 134, 183 Clifford, K. J., 10, 33 Cochran, W., 292, 332 Cockburn, R., 311, 327, 332, 333 Cohen, C., 298, 333 Cohen, E., 59, 60,66, 103, 107 Cohen, J., 114, 116, 118, 119, 1S9 Cohn, E. J., 220, 259, 261, 333 Cohnheim, O., 90, 100 Cole, R. D., 47, 50, 66, 68, 96, 100, 102, 103, 105, 199, 249, 331, 342 Cole, S. N. J., 90, 103 Collman, J. P., 62, 100 Condliffe, P. G., 96, 102 Conley, C. L., 6, 9, 33, 36 Consden, R., 112, 182, 244, 246, 333 Corey, R. R., 291, 292, 293, 294, 297, 299, 300, 333, 341, 346 Corfield, M. C., 206, 207, 215, 216, 227, 228, 229, 230, 333 Cormick, J., 54, 64, 75, 88, 89, 106 Corn, M., 33 Courts, A., 113, 114, 115, 116, 138, 139, 143, 183, 214, 333 Craig, L. C., 123, 137, 185 Crawhall, J. C., 148, 183 Crawshaw, G. H., 284, 333 Creeth, J. M., 96, 100 Crestfield, A. M., 92, 100, 204, 223, 333 Cretius, K., 24, 25, 33 Crewther, W. G., 194, 213, 216, 223, 225, 226, 244, 251, 253, 277, 280, 281, 308,

309, 311, 313, 314, 315, 316, 317, 319, 320, 321, 322, 323, 324, 325, 329, 330, 333, 334 Crick, F. H. C., 110, 188, 292, 293, 296, 297, 332, 333 Crosby, N. T., 115, 117, 183 Crounse, R. G., 241, 333 Csapo, A., 29, 31, 33 Cunningham, L. W., 93, 100, 177, 183, 187 Cunningham, R. S., 18s Cuthbertson, W. R., 229, 252, 318, 324, 333 Czaky, T. Z., 155, 183 Czerkawski, J. W., 127, 137, 183

D Dahlquist, C. A,, 145, 169 Dakin, H. D., 123, 131, 183 Damodaran, M., 90, 100 Danilczenko, A,, 169, 183 Das, D. B., 211, 308, 333, 334 D a s h , W., 179, 183 Davie, E. W., 97, 100 Davies, D. R., 293, 338 Davis, H. F., 51, 105 Davis, N. C., 39, 91, 100 Davis, P., 183 Davison, P. T., 115, 119, 175, 188 Dawid, I. B., 87, 92, 93, 100, 101 Dawson, J. A. T., 259, 331 Deasy, C., 132, 180, 183 Deatherage, F. E., 47, 107 Decroix, G., 225, 230, 253, 334 De Deunvaerder, R., 203, 210, 211, 334 Dekker, C. A,, 144, 183 de Kock, W. T., 243, 332 de la Burde, R., 160, 161, 180, 183 Delaney, R., 107 del Castillo, L. M., 65, 100 Delmenico, J., 275, 334 De Marsh, G. B., 2, 33 de Milstein, C. P., 96, 100 Desnuelle, P., 38, 50, 51, 60, 61, 68, 100, 144, 183 Deutch, H. F., 97, 100 DBvBnyi, T., 67, 101 Dickerson, R. E., 293, 338 Dillon, R. T., 39, 40, 107 Diorio, A. F., 281, 307, 319, 329, 334, 340 Dische, Z., 169, 183

350

AUTHOR INDEX

Discombe, G., 10, 33 Dixon, C . H., 81, 88, 101 Dixon, J., 96, 103 Dixon, M., 124, 183 Dobb, M. G., 203, 210, 288, 291, 334 Donnan, F . G., 271, 334 Donne, A,, 2, 33 Donohue, J., 295, 334 Donovan, R., 265, 274, 338 Dorfman, A,, 118, 187 Doty, P., 111, 118, 119, 172, 183, 293, 345 Dowling, L. M., 216, 223, 225, 226, 251, 253, 277, 280, 281, 308, 309, 313, 314, 315, 324, 325, 329, 333, Dowmont, Y. P., 127, 183 Downes, A. M., 219, 334 Drake, M. P., 183 Drechsler, E. R., 97, 101 Dreyer, W. J., 67, 104 Dreee, A,, 58, 62, 101 Drucker, B., 311, 333 Dry, F. W., 311, 334 Dumanskii, A. V., 257, 334 Dumanskii, 0. A., 257, 334 Dumsha, B., 163, 185 Dunne, C. J., 2, 35 Dunnill, P. M., 122, 183 Durante, M., 119, 179, 187 Dus, K., 53, 93, 101 Dusenbury, J. H., 233, 334 du Vignexutl, V., 96, 97, 101 Dweltz, N. E., 301, 342

Elliott, I). I?., 51, 101, 148, 183 Elliott, G. H., 226, 258, 259, 317, 318, 327, 334, 344 Elliott, R. G., 113, 137, 138, 140, 141, 182 Ellis, W. J., 204, 334 Elod, E., 257, 280, 308, 309, 310, 311, 325, 33/,

185,

244, 311, 334

Elsden, D. F., 110, 176, 188, 189 Elsken, R. H., 326, 343 Elswortli, F. F., 199, 254, 339 Endres, H., 132, 146, 147, 148, 150, 162, 163, 164, 166, 167, 170, 184, 165 Engel, J., 111, 116, 117, 118, 183, 185 Engle, R. I,., 227, 346 Enmoto, S., 301, 334 Eriksson-Quenscl, I. B., 94, 107 Eveland, W. C., 2, 35 Eylxr, E. €I., 177, 178, 183 Eyring, H., 328, 342

F

Fabre, C., 60, 100 Farmworth, A . J., 319, 320, 321, 334 Fearon, W. R., 286, 334 Feissly, R., 3, 33 Fell, M., 245, 334 Fessler, J. H., 183 Feughelman, M., 306, 310, 311, 312, 314, 320, 322, 323, 324, 325, 326, 327, 328, 329, 334, 335, 336, 337, 340 Fietzek, P., 115, 119, 175, 186 Filshie, B. K., 209, 210, 223, 233, 288, 289, 295, 298, 301, 302, 303, 335, 339, 342 Finck, H., 32, 33 E Finn, F., 64, 102 Firkin, B. G., 10, 33 Eager, J. E., 252, 286, 334 Earlnnd, C. F., 206, 220, 255, 308, 330, Fischer, E., 144, 183 Fischer, E. H., 93, 101 33.6 Fishman, L., 140, 187 Easley, C. W., 105 Fleischman, J. B., 95, 96, 98, 101, 161, Eastoe, B., 169, 163 183 Eastoe, J. E., 110, 141, 169, 183 Flcscli, P., 239, 240, 335, 345 Etmtli, J. C., 2, 3, 33 Fletcher, J. C., 224, 230, 231, 253, 355 Edmnn, P., 51, 103 Fletchrr, A. P., 178, i84 E:tlmundson, A. R., 64, 101 Flory, P. J., 112, 115, 116, 184, 318, 335 Etisall, J. T., 20, 36, 259, 261, 3% Folk, J. E., 68, 10Y, 225, %$.$ Edw:irds, Q . A , , 29, 33 Pokers, I<., 136, 188 I<&enberger, D. N., 189 Fonio, A,, 3, 21, 33 Ehrlich, G., 292, a34 Forbes, W. F., 284, 285, 332, 335 Eigner, E. A,, 117, 118, 188 Fourt, L., 322, 324, 327, 337 Elliot.t, A,, 292, 293, 330, 331, 332, 334

AUTHOR INDEX

Fowden, L., 122, 183 Fowler, R. H., 267, 335 Fox, M., 206, 322, 330 Fox, S. W., 120, 18,$ Fraenkel-Conrat, H., 58, 60, 62, 81, 95, 97, 101, l O Y , 137, 184 Frankel, E. M., 90, 101 Franzblau, C., 121, 129, 131, 135, 136, 143, 158, 160, 164, 172, 173, 176, 184, 189 Fraser, R. D. B., 238, 239, 291, 292, 295, 296, 297, 298, 299, 300, 301, 302, 303, 311, 326, 335 Frater, R., 256, 316, 329, 335 French, T. C., 87, 92, 93, 100, 101 Frenkel, S . Y., 96, 100 Friend, J. A., 211, 335 Fries, G., 167, 168, 172, 174, 186 Fritz, H., 1SB Fromageot, C., 121, 122, 124, 182, 184 Fruton, J. S., 61, 106, 127, 144, 183, 184 Fuchs, F., 24, 33 Fugii, S., 45, 48, 55, 10% Fugitt, C. H., 259, 261, 262, 269, 273, 276, 281, 344 Funatsu, M., 96, 107 Fysh, D., 112, 113, 184

G Gaarder, A., 7, 33 Gallop, P. M., 128, 129, 131, 135, 136, 140, 146, 147, 148, 149, 150, 151, 152, 154, 155, 156, 157, 158, 159, 161, 162, 164, 168, 169, 172, 173, 174, 177, 179, 182, 184, 1SB Garrett, R. R., 115, 116, lS.4 Gautier, A,, 3, 33, 34 Gee, G., 329, 335 Geiger, W. B., 318, 322, 329, 335, 336, S 4 l Gergely, J., 24, 36 Gerthsen, T., 224, 225, 311, 346 Geschwind, I. I., 96, 103, 282, 336 Gibson, R. E., 278, 330 Gilbert, J. B., 41, 101 Gilbert, G. A., 267, 269, 276, 336 Gillespie, J. M., 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 206, 209, 211, 213, 215, 216, 217, 218, 220, 223, 236, 243, 248, 256, 316, 333, 336, 33Y, 344 Ginsberg, A,, 94, 96, 101

35 1

Giroud, A., 240, 336 Gish, D. T., 81, 1U7 Gladner, J. A., 67, 96, 101, 107 Glanzmann, E., 10, 33 Glasstone, S., 41, 102 Glazer, A. N., 55, 101 Glazer, N., 163, 186 Glick, J. H., Jr., 97, 101 Glikina, M. V., 96, 100 Glynn, L. E., 112, 182 Goddard, D. R., 195, 336 Godfrey, J. E., 95, 96, 98, 104 Godin, C., 243, 338 Goldberger, R. F., 67, 101 Golden, R. L., 233, 234, 336 Goldenberg, H., 68, 101 Goldenberg, V., 68, 101 Goldhaber, D., 119, 187 Goldstein, J., 76, 77, 78, 79, 83, 84, 85, 88, 101, 103 Golsch, E., 254, 346 Golubow, J., 96, 97, 101 Gonon, W., 310, 340 Goodall, F. L., 227, 259, 336 Gordillo, G., 97, 101 Gordon, A. H., 40, 45, 50, 53, 101, 244, 246, 333 Gorshkova, T. A., 182 Got, R., 177, 178, 182 Gottschalk, A., 150, 177, 184, 187 Grabar, P., 184 Graham, C . C., 228, 238, 239, 336 Graham, D. R., 226, 283, 286, 336 Graham, E. R. B., 150, 177, 184 Gralen, N., 211, 341 Grannis, G. F., 52, 101, 106 Grassmann, W., 116, 117, 118, 131, 132, 145, 146, 147, 148, 150, 151, 153, 162, 163, 164, 166, 167, 168, 170, 183, 184, 185, 186

Green, F. C., 53, 69, 106 Green, N. M., 68, 74, 101 Greenstein, J. P., 37, 38, 39, 41, 55, 101 Grette, K., 6, 11, 12, 13, 15, 17, 20, 25, 33 Grice, M., 52, 106 Griffith, J. C., 276, 309, 312, 314, 336, 337 Gross, E., 225, 336 Gross, J., 113, 117, 118, 119, 120, 163, 185, 187, 188

Gross, R., 6, 9, 33, 34

352

AUTHOR INDEX

Harrington, W. F., 94, 95, 96, 97, 98, 102, Grossowicz, N., 155, 156, 190 104, 107, 113, 116, 117, 185 Groves, M. L., 61, 102 Harris, J. I., 47, 50, 53, 62, 64, 67, 75, Growitz, F., 140, 190 81, 95, 96, 97, 101, 102, 103 Grutzmacher, H. I?., 49, 102 Harris, M., 226, 256, 257, 259, 260, 261, Guba, F., 19, 32 262, 263, 264, 265, 266, 269, 273, 275, Giintelberg, A. V., 80, 101 277, 281, 283, 308, 311, 318, 322, 323, Guggenheim, E. A., 267, 271, 334, 336 324, 327, 329, 332, 336, 336, 337, 341, Gugler, E., 6, 33 Guidotti, G., 66, 88, 101 343, 344 Harrison, D., 328, 337 Gundladi, H . G., 61, 101 Harrison, W., 280, 303, 337 Gurevitch, J., 2, S4 Harrold, S. P., 260, 337 Gurin, S., 140, 185 Gustavson, K. H., 112, 113, 114, 116, 140, Hart, R. G., 293, 338 Hartley, B. S., 56, 68, 102, lo4 141, 146, 150, 179, 185 Hartley, F. K., 177, 186 Gutnikov, G., 155, 185 Haruna, I., 132, 160, 181 H Haselbach, C., 41, 58, 100, 132, 148, 182, 310, 340 Haas, W., 102 Hass, W., 64, 102 Hahnel, R., 243, 336 Hasselbach, W., 23, 24, 25, 26, 28, 33 Hafter, R., 115, 119, 170, 184, 186 Haugaard, E. S., 81, 102 Haggith, J . W., 305, 310, 331 Haugaard, N., 81, 103 Hagihara, B., 80, 101, 102 Haurowitz, F., 123, 126, 127, 134, 135, Hahn, J. W., 40, 100 Haley, E. E., 122, 123, 182 144, 186 Halpern, Y. S., 155, 156, 190 Haussmann, W., 123, 137, 185 Halpin, J. C., 281, 307, 329, 340 Hayashida, T., 96, 103 Halsey, G., 328, 332 Hayem, G., 2, 33 Haly, A. R., 233, 282, 283, 306, 309, 311, Haylett, T., 207, 211, 337 312, 314, 316, 317, 320, 322, 324, 325, Heikkinen, E., 113, 187 Heilbrunn, L. V., 9, 33 326, 327, 328, 329, 335, 3% Hambraeus, E., 282, 337 Hellem, A. J., 7, 33 Hamilton, P., 39, 40, 107 Herhst, F. S. M., 239, 340 Hammel, E. F., Jr., 41, 102 Hersh, R. T., 118, 181 Hess, G. P., 96, 102 Hanafusa, H., 132, 160, 181 Hmby, WT.E., 292, 330, 331 Heyns, K., 49, 102, 115, 132, 138, 139, Hmes, C. W., 60, 102 143, 185 Hannig, K., 111, 115, 116, 117, 118, 167, Hiepler, E., 7, 35 Higgs, D. G., 116, 186 168, 172, 174, 183, 184, 185, 186 High, L. M., 221, 346 Hnnson, J., 28, 29, 53, 34 Hanson, L. A , , 96, 1/22 Highberger, J . H., 115, 118, 186 Happey, F., 316, 537 Highton, T. C., 112, 189 Hardin, R. I,., 126, 134, 135, 185 Hildebrand, D., 254, 283, 314, 337, 346 Harfenist, E., 45, 60, 106 Hill, J., 337 Harington, C. R., 121, 122, 185 Hill, R. J., 66, 69, 72, 73, 75, 76, 77, 78, Harkness, R. D., 112, 185 79, 83, 84, 85, 88, 101, 102, 103, lo4 Harley-Mason, J., 286, 337 Hill, It. L., 57, 58, 61, 65, 67, 87, 88, 89, Harnish, D. P., 254, 345 90, 91, 92, 95, 97, 99, 102, 106, 106, Harrap, B. S., 199, 202, 211, 213, 215, 217, 220, 337 218, 221, 222, 223, 236, 237, 244, 302, Hill, U. T., 155, 186 303, 316, 333, 334, 335, 336, 337

353

AUTHOR INDEX

Hille, E., 214, 223, 224, 226, 230, 242, 337, 344 Hilson, H., 323, 331 Hipp, N. J., 61, 102 Hir, S. W., 228, 238, 239, 336 Hird, F. J. R., 60,102, 256, 316, 329, 336 Hirohata, R., 45, 48, 49, 55, 102, 104 Hirs, C. H. W., 57, 59, 64, 69, 72, 75, 88, 92, 102 Hirst, M. C., 257, 258, 259, 344 Hoare, J . L., 323, 337 Hobday, C., 227, 259, 336 Hoch, H., 96, 105 Hodge, A. J., 117, 118, 119, 181 Hormann, H., 115, 116, 117, 119, 131, 138, 139, 147, 148, 150, 151, 152, 153, 156, 158, 163, 164, 165, 166, 167, 168, 170, 171, 172, 173, 174, 175, 176, 180, 184, 185, 186 Hoffman, P., 93, 99, 177, 181 Hoffmann-Berling, H., 2, 10, 15, 25, 26, 27, 30, 33 Hofmann, K., 64, 89, 92, 102 Hofmann, U., 163, 164, 166, 167, 168, 184,

Humes, J. L., 64, 89, 92, 102 Hutter, R. V. P., 3, 34 Huxley, A. F., 29, 34 Huxley, H. E., 28, 29, 34

I Ibrom, H., 3, 34 Ikawa, M., 59, 103 Ikenaka, T., 132, 160, 181 Inagami, T., 65, 68, 103 Inglis, A. S., 203, 215, 227, 233, 243, 283, 314, 315, 316, 324, 333, 336, 938 Inglis, I., 248, 256, 344 Ingram, V. M., 53, 66, 103 Irion, W., 252, 538 Irving, J. T., 138, 139, 189 Isherwood, F. A,, 60, 102 Ishibashi, H., 327, 341 Izak, G., 2, 34 Izumi, K., 177, 186 Izumiya, N., 45, 65, 102, 103

J

Jaaback, G., 90, 100 186 Jackson, D. D., 6, 9, 33, 36 Hofschneider, H . P., 23, 33 Jackson, D. S., 117, 166, 186 Hojo, H., 282, 337 Jacobs, S., 177, 186 Hojo, N., 282, 337 Jacobsen, C. F., 39, 103, 259, 338 Holeysousky, V., 66, 107 Jagger, L. G., 327, 333 Holmes, K. C., 298, 333 Jaisle, F., 18, 24, 25, 38, 33, 34 Holtzer, H., 32, 33 Janus, J. W., 140, 186 Honne, I,., 53, 69, 106 Jean, G., 3, 34 Hoover, S. R., 95, 104, 326, 340 Jeanloz, R. W., 178, 186 Hopkins, F. G., 90, 103, 135, 186 Jpffrey, G. M., 288, 308, 316, 338 Hopton, J . W., lS1 Jrnkins, A. D., 319, 320, 321, 338 Horden, I?. W. A,, 218, 236, 332 Jcvons, F. R., 93, 103, 177, 181, 185, 186 Horn, M. J., 252, 317, 337 Jorgensen, L., 8, 9, 22, 34 Horner, J . L., 260, 269, 337 Johansen, P. G., 93, 103, 177, 186 Howitt, F. O., 257, 337 Jolrmsson, B. G., 96, 102 Horowitz, J., 126, 127, 134, 135, 185 Johnson, A. W., 144, 186 Horro, T., 97, 105 Johnson, D. J., 288, 298, 338 Horvath, E., 67, 101 Jones, C. B., 202, 208, 538 Hovig, T., 6, 8, 34 Jones, D. B., 252, 317, 337 Hsiao, S., 96, 103 Hudson, R. F., 206, 247, 274, 280, 305, Jones, R. T., 54, 64, 75, 88, 89, 106 Jonsen, J., 7, 33 309, 322, 326, 35'0, 337 Josefsson, 51, 103 Huggins, M. L., 292, 295, 337 Joseph, K. T., 112, 124, 132, 135, 136, Hugues, J., 6, 8, 17, 21, 34, 36 138, 139, 140, 151, 153, 164, 179, 182, Human, J. P. E., 211, 235, 248, 277, 323, 186 338, 339

354

AUTHOR INDEX

Kiiiiincl, H., 52, 106 Kimmel, J. R., 57, 59, 66, 67, 83, 87, 91, 92, 102, 103, 104, 106 Kincaid, J. M., Jr., 66, 100 K King, A. T., 234, 331 King, N . L., 234, 333 Kaser-Glanzmann, R., 6, 7, 22, 33, 34 Kinosita, R., 3, 34 Kaiser, E. T., 65, 99 Kirk, P. L., 38, 103 Kakimoto, Y., 123, 186 Icitagawa, W., 65, 103 Kamen, M. D., 53, 93,101 Iiitai, R., 52, 55, 57, 66, 105, 244, 342 Kaminski, M., 94, 96, 103 Kitchener, J. A,, 277, 330 Kamiyama, S., 177, 178, 186 Kiaer, A,, 220, 343 K m d a , Y., 45, 48, 49, 55, 102, 104 Iijaerhcim, -q., 8, 34 Kandel, I., 126, 186 Klee, W. A,, 61, 103 Kandel, M., 126, 166 Iilenk, M., 151, 152, 156, 186 Kancko, T., 135, 189 I
Joubert, F. J., 207, 211, 337 Jutisz, M., 124, 184

355

AUTHOR INDEX

Kunina, 0. V. (see Kounina, 0. V.) Kunitz, F. W., 254, 314, 320, 346

1 Lablond, C. P., 240, 336 La France, N. H., 230, 245, 334, 338 Laland, S., 7, 33 Landucci, J. M., 119, 179, 187 Lang, A. R., 291, 295, 338 Langhof, H., 240, 241, 341 Lapiere, Ch. M., 8, 34 Lapresle, C., 96, 103 Larose, P., 265, 274, 327, 338 Latimer, W. M., 280, 338 Launer, H. F., 284, 338 Lawrence, L., 41, 47, 62, 103 Laxer, G., 283, 339 Layne, E., 38, 39, 103 Leach, A. A,, 140, 169, 186 Leach, S. J., 38, 39, 61, 103, 208, 209, 210, 223, 224, 225, 229, 230, 237, 249, 250, 251, 260, 318, 320, 321, 332, 337, 339, 340

Lerhler, E., 9, 34 Ledermair, O., 24, 25, 26, 28, 33, 34 Lee, G. R., 52, 99, 225, 243, 245, 253, 332 Lee, Y. C., 177, 187 Leeder, J. D., 328, 332 Lees, K., 199, 254, 339 LeGette, J., 53, 106 Legler, G., 115, 132, 143, 185 Leikhim, E., 107 Lemin, D. P. L., 257, 339 Len, J., 96, 103 Lennox, F. G., 195, 196, 199, 280, 283, 284, 329, 336, 338, 339 Lenormant, H., 292, 339 Ldonis, J., 39, 64, 69, 72, 103 Leplus, R., 3, 33 Le Roux, P. I,., 311, 339 Lc Sourd, L., 9, 34 Lcttr6, H., 34 Levcau, M., 233, 280, S39 Levene, C. I., 119, 179, 187 Levene, P. A,, 131, 187 Lever, W. F., 241, 242, 339 Levcnson, S. M., 118, 186, 188 Levinc, L., 107 Levy, A. L., 60, 62, 95, 101, 103 Levy, M., 39, 103, 137, 140, 143, 144, 187

Lewis, B., 223, 230, 236, 237, 238, 339, 343 Lewis, H. B., 234, 339 I,ewis, J. H., 2, 35 Lewis, M. S., 117, 118, 120, 187, 188 Ley, F. J., 180, 182 Li, C. H., 64, 67, 69, 72, 96, 97, 102, 103 Lieflander, M., 150, 153, 156, 157, 187 Liener, I. E., 66, 103 Lietze, A,, 126, 134, 135, 185 Light, A., 38, 69, 72, 103, 104 Lightbody, H. D., 234, 339 Lillycrop, J. E., 41, 44, 47, 48, 104 Linderstr@m-Lang,K. U., 94, 95, 10.4, 259, 338 Lindley, H., 67, 104, 209, 219, 229, 253, 255, 256, 277, 306, 311, 318, 319, 322, 323, 324, 330, 332, 333, 339 Lineweaver, H., 95, 104 Lipmann, F., 155, 187 Lippincott, E. R., 319, 334 Lisie, S.G., 126, 134, 135, 185 Liss, M., 241, 242, 339 Lister, G. H., 269, 274, 342 Little, K., 116, 183 Litwin, J., 124, 149, 156, 189 Liu, T. Y., 64, 89, 92, 102 LO,T., 103 Lockhart, I. M., 123, 137, 141, 187 Loebner, C. H., 283, 339 Loewy, A. G., 22, 34 Long, D. A., 41, 43, 44, 47, 48, lo4 Long, F. A., 278, 340 Lorimer, J. W., 118, 190 Louw, D. F., 207, 211, 225, 235, 236, 337, 339

Lowther, A. G., 206, 332 I,owy, J., 29, 33 Luscher, E. F., 5, 6, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 22, 25, 26, 32, 33, 34 T,ukin, M., 128, 129, 135, 154, 184 Ixndgren, H. P., 191, 192, 209, 221, 282, 288, 296, 298, 299, 339, 345 Lundin, R. E., 326, 343 T,use, R. A., 287, 339 Lustig, B., 201, 339, 340 Lute, R., 23, 33 Lynn, L. T., 105

M Mnc~,.Zrtliur,I., 291, 293, 297, 301, 340 McConnell, W. 13., 51, 105, 107

356

AUTHOR INDEX

Matikknla, E. J., 122, 187 McCrackin, F. I,., 324, 342 Matoltsy, A. G., 239, 240, 241, 340 McDevitt, N. B., 10, 35 Matsubara, H., 66, 72, 80, 88, 102, 104 McDevitt, W. F., 278, 340 Matsuoa, G., 286, 340 MacFayden, D. A,, 39, 40, 107 Matsushima, T., 132, 160, 181 McKenzie, B. F., 135, 186 Maupin, B., 6, 10, 32, 34 McLaren, A. D., 68, 101, 287, 339 Maclaren, J. A., 203, 223, 225, 226, 248, Maeingue, G., 225, 229, 230, 253, 333, 346 251, 252, 256, 260, 284, 314, 315, 324, Maeur, J., 259, 340 Mead, T. H., 121, 122, 185 326, 333, 334, 340,342, 344 Mecham, D. K., 202, 208, 338 McMeekin, T. I,., 61, 102 McPhee, J. R., 247, 261, 278, 279, 332, 340 Mechanic, G. L., 137, 143, 144, 187, 189 MacRae, T. P., 238, 239, 291, 295, 296, Meichelbeck, H., 224, 225, 311, 320, 346 297, 298, 299, 300, 301, 302, 303, 311, Meienhofer, J., 194, 346 Meilman, E., 128, 129, 135, 146, 147, 149, 316, 326, 335, 337 150, 152, 154, 155, 156, 157, 158, 159, Magnua, I. A., 240, 340 173, 174, 184, I S 9 Mahowald, T. A,, 59, 104 Meinenhofer, J., 103 Makar, J. A,, 236, $40 Mclchior, G. H., 286, 340 Makino, M., 177, 178, 186, 190 Mellon, E. F., 326, 340 Malcolm, B. R., 292, 293, 334 Menkart, J., 233, 306, 334, 340 Malmstrom, G. B., 96, 97, 104 Mandelkern, I,,, 281, 307, 311, 319, 329, Mercer, E. H., 208, 209, 211, 213, 233, 234, 310, 336, 340 334, 340 Merignn, T. C., 67, 104 Mangan, J. L., 41, 58, 100, 132, 148, 182 Meriwether, B. P., 67, 102 Mann, G., 37, 104 Meriwether, I,., 62, l U 4 MarIcek, V., 238, 239, 340 Meschers, A,, 224, 225, 320, 321, 332, 339 Mnrcovici, J., 3, 33, 34 Meschia, G., 61, 106 Mardashev, S. R., 340 Margolinsh, E., 53, 67, 69, 72, 84, 87, 88, Meunier, L., 257, 340 Meyer, A. E., 188 91, 104 Meper, D., 177, 182 Markiw, R. T., 122, 123, 182 Meycr, K., 93, 99, 177, 181, 184 Marko, A. M., 181, 185 Meycr, K. H., 310, 3.$0 Marks, G. S., 178, 184, 187 Micliaelis, L., 195, 336 Mitrsh, B. B., 20, 24, 34 Michaels, S., 155, 156, 159, 189 Marshall, J. M., 32, 53 Marshall, R. D., 93, 103, 177, 178, 184, Middlebrook, W. R., 242, 243, 248, 255, 331, 340 1S6, 1x7 Mihalyi, E., 94, 95, 96, 98, 102, lo4 Martin, A,, 279, 280, 281, 331 Martin, A. J. P., 40, 45, 50, 53, 101, 104, Mijal, C. F., 225, 344 Mikes, O., 66, 107 244, 246, 353 Martin, G. R., 117, 118, 119, 120, 187, 188 Mikkonm, I,., 113, 186 Milch, R. A,, 179, 187 Mart,in, N., 53, lOG, 244, 343 Miller, A,, 297, 298, 299, 335 Martin, R. B., 44, 104 Milligan, B., 249, 250, 283, 287, 318, 320, Mxrston, H., 257, 340 321, 332, 340 Martonosi, A,, 34 Milliser, R. V., 179, 183 Marx, R., 3, 34 Miro, P., 226, 340 Masamune, H., 177, 187 Mirsky, A . E., 95, 9.9 Mason, H. L., 135, 186 Mitchell, H. K., 182 Mason, P., 325, 340 Mitchell, T. W., 310, 312, 320, 323, 325, Mzisurov, V. I., 117, 118, 187, 188 335, 340 Mathews, M. B., 118, 187

AUTHOR INDEX

357

Nelken, D., 2, 34 Miyazawa, T., 292, 300, 340, 341 Mizell, L. R., 318, 322, 324, 327, 329, 336, Nemoto, Y., 224, 341 Neuberger, A,, 90, 93, 103, 104, 177, 178, 337, 341 184, 185, 186, 187 Moffitt, W., 213, 341 Neumann, N. P., 61, 104 Montagna, W., 240, 341 Neurath, H., 51, 65, 66, 68, 74, 81, 88, 96, Montgomery, R., 177, 178, 187 97, 100, 103, 104, 106, 107 Moore, D. H., 201, 340 Nicholls, C. H., 327, 341 Moore, J. E., 287, 342 Moore, S., 39, 40, 41, 44, 55, 57, 59, 61, Nicol, D. S. H. W., 96, lo4 64, 69, 72, 75, 88, 89, 92, 100, 101, Niedergerke, R., 29, 34 102, 104, 105, 106, 204, 223, 227, 229, Niemann, C., 110, 144, 188 Nikkari, T., 117, 118, 119, 120, 138, 151, 230, 237, 333, 241, 343, 344 152, 187 Moore, W. J., 41, 47, 62, 103 Nischwitz, E., 113, 140, 190, 225, 346 Morgan, F. R., 187 Nishihara, T., 115, 118, 119, 172, 183, 187 Mori, S., 285, 341 Noboru, Y., 64, 89, 92, 102 Morris, C. J., 122, 187 Nolan, C., 92, 93, 104, 177, 178, 187 Morton, J. I., 97, 100 Noltmann, E. A,, 59, 104 Morton, T., 252, 334 Nomoto, M., 80, 82, 87, 104, 106 Moschetto, Y., 203, 331 Moss, J. A,, 113, 137, 138, 139, 140, 141, Northrop, J. H., 96, 105 Nowotny, H., 280, 325, 334 162, 163, 182, 187 Nozaki, M., 97, 105 Moss, H. J., 256, 316, 329, 336 Nuenke, B. J., 93, 100, 177, 183 Motulsky, A. G., 2, 33 Xuenke, R. H., 93, 100, 177, 183, 1W Moyer, D. L., 110, 145, 189 Muting, D., 240, 241, 341 0 Muir, H. M., 93, 104, 162, 177, 186, 186, O’Callaghan, F., 211, 341 187 O’Connell, R. A,, 309, 338 Mumaw, V. R., 140, 181 O’Donnell, I. J., 97, 98, 105, 196, 197, 198, Munakata, H., 328, 341 201, 203, 204, 205, 206, 207, 211, 215, Munger, N., 53, 103, 106, 236, 237, 238, 216, 217, 220, 223, 227, 228, 243, 248, 244, 343 336, 336, 340, S41, 345 Murakami, M., 80, 82, 87, 104, 105 Ohno, K., 133, 160, 181, 188 Muramatu, M., 45, 48, 49, 55, 104 Ohno, S., 3, 34 Murphy, W. H., 150, 177, 184, 187 Okada, Y., 132, 160, 181 Muscatello, U., 24, 34 Oku, M., 278, 327, 341, 343 N Okunuki, K., 80, 97, 102, 106 Olcott, H. S., 58, 105 Nakajo, N., 287, 341 Olofsson, B., 208, 211, 259, 269, 270, 273, Nagamatsu, A., 45, 48, 102 340, 341 Nagy, H., 124, 182 Oneson, I., 115, 163, 164, 170, 172, 181, Nakajima, H., 23, 34 188 Nakamura, M., 45, 102 Onions, W. J., 291, 299, 341 Nakamura, Y., 224, 341 Ono, T., 45, 48, 49, 55, 102 Nakai, M., 80, 102 Namagatsu, A,, 45, 48, 49, 55, 61, 104, 106 Ooi, T., 96, 99, 105 Orekhovitch, V. N., 117, 118, 187, 188 Narahashi, Y., 80, 82, 87, 104, 106 Osborne, T. B., 37, 107 Narita, K., 156, 157, 160, 188 Oster, G., 254, 342 Naughton, M. A., 56, 102, 104 Ottesen, M., 95, 97, 101, 105 Needham, D. M., 24, 25, 35 Owren, P. A., 7, 33 Neidle, A., 57, 103

358

AUTHOR INDEX

P l’agnicaz, l’., 9, 84 I’aintw, J. C., 10, 35 Paiva, A. C. M., 66, 92, 103 I’akesch, F., 3, 33 Papenhcimcr, A. M., Jr., 96, 105 Papkoff, M., 96, 103 Papkoff, H., 177, 178, 187 Pardoe, G. I., 181 Park, J . H., 67, 109 Parkcr, A. J., 255, 341 Parker, C. J., Jr., 24, 35 Parkhill, E. M. J., 229, 260, 339 Parmcggiani, A., 5, 8, 22, 35 Partridge, S. M., 110, 176, 188, 189 Patchornik, A., 318, 341 Patridac. S. M.. 51, 105 Patterson, W. I., 318, 322, 329, 336, 341 Pauling, I,,, 60, 105, 110, 144, 188, 292, 293, 294, 297, 299, 300, 341, 546 Paz, M. A,, 168, 173, 174, 182 Peacock, N., 211, 341 Peck, R. L., 136, 188 Pcckliam, L., 160, 161, 180, 183 Pelet-Jolivet, I,., 259, 2341 Pcnasse, L., 184 Pendergrass, J. H., 308, 336 Perkins, H. R., 127, 137, 185 Perkoff, G. T., 91, 106 Perlmann, C:. E., 60, 96, 105, 145, 188 Perry, S. V., 22, 23, 24, 55 Perutz, M. F., 293, 341 Peryman, R. V., 254, 277, 339, S41 Petcrman, M. L., 96, 105 Pet,ers, E. L., 58, 90, 91, 92, 107 Peters, L., 259, 266, 269, 271, 273, 274, 276, 277, 310, 341, 349 Peters, R. H., 275, 554 Pethira, €3. A,, 260, 337 Pfahl, D., 115, 119, 175, 188 Pfannmiiller, M., 230, 337 Phillips, D. C., 293, 358 Phillips, D. M. P., 96, 100 Phillips, H., 229, 252, 253, 255, 317, 318, 324, 333, 930, 340, 349 Pickard, J. W., 234, 331 Piez, K. A,, 113, 117, 118, 120, 187, 188 Pigache, P., 52, XI, 208, 331 Pirie, A,, 164, I S 8

Pon, N. G., 47, 50, 96, 102 Poole, N. F., 188 Pope, C. G., 95, 96, 105 Porter, R. R., 60, 95, 96, 98, 101, 105, 139, 161, 163, 18s Portzehl, H., 11, 15, 18, 19, 20, 23, 24, 32, 35 Posner, A. S., 281, 329, $40 Pouradier, J., 119, 179, 187 Pratt, A. N., 140, 187 Press, E. M., 95, 96, 98, 101, 161, 183 Price, V. E., 41, 101 Price, W. C., 292, 335 Primosigh, J., 105 Pucher, G. W., 41, 107 Pusztai, A,, 186 l’utnam, F.W., 96, 103, I06

Q Quick, A. J., 10, 35 Qiireshi, M., 180

R Raacke, I., 96, 103 Raftery, M. A,, 68, 105, 199, 342 Ramachandran, G. N., 301, 342 Rainnchantlran, J., 64, 103 Ramachandran, L. K., 51, 105, 156, 157, 160, 188 Raspcr, J., 327, 338 Raven, D. J., 334 Reardon, G. V., 112, 113, 144, 173, 189 Redfield, R. R., 39, 63, 67, 94, 99 Reed, R., 116, 185, 188 Rees, E. D., 160, 188 Rees, M. W., 41, 51, 58, 100, 106, 125, 132, 146, 148, 186, 183 Reichenctler, E., 121, 127, 189 Reis, P. J., 206, 209, 218, 236, 336, 342 Rey, G., 257, 340 Rhinesmitli, H. S., GO, 105 Rice, R. V., 116, 188 Rich, A,, 110, 188 Richard, A. J., 96, 105 Richards, F. M., 61, 96, 97, 98, 99, 101, 103, 105, 125, 148, 185 Richards, H. R., 235, 236, 328, 342 Rideal, E. K., 267, 269, 276, 336

AUTHOR INDEX

Riedel, A,, 151, 152, 153, 156, 184, 185, 186

Rigby, B. J., 310, 312, 324, 325, 328, 336, 342

Riley, D. P., 292, 342 Rimington, C., 234, 283, 331, 342 Ringel, S. J., 252, 317, 337 Ripa, O., 304, 310, 311, 323, 337, 348 Rivett, D. E., 252, 334 Robinson, J. C., 134, 181 Robinson, R. A,, 278, 342 Robson, A., 206, 215, 216, 223, 224, 227, 228, 229, 230, 231, 253, 333, 335, 339 Rodman, N. F., Jr., 10, 35 Roe, D. A., 239, 342 Rogers, G. E., 204, 209, 210, 218, 219, 223, 233, 234, 235, 288, 289, 295, 296, 298, 300, 301, 302, 326, 330, $34, 356, 339, 342 Rogers, H. J., 127, 137, 183 Roos, P., 53, 64, 75, 81, 102 Rose, W. G., 287, 342 Rosen, H., 118, 186, 188 Rosenthal, N. A,, 254, 342 Rosenthal, R. I,., 5, 35 Rosevear, J. W., 93, 105, 177, 178, 188 Roskam, J., 8, 21, 36 Ross, D. A,, 236, 342 Rossi-Fanelli, il., 97, 09 Rothen, A., 96, 105 Rothfus, J. A., 178, 188 Rougvie, M. A,, 221, 342 Rovery, M., 60, 100 Roxburgh, C. M., 252, 334 Rubin, A. L., 115, 119, 175, 188 Rudall, K. M., 240, 307, 319, 342 Ruegg, J. C., 26, 35 Rugo, H. J., 291, 301, 331 Ruoff, A. L., 328, 342 Rupley, J. A,, 96, 98, 99, 105 Ruskn, H., 29, 33 Rybicki, P., 140, 186 Ryle, A. P., 54, 55, 105, 244, 348

S Sakol, E., 306, 340 Sailer, S., 3, 33 Sajgo, M., 67, 101 Salmenpera, A,, 187 Salo, T. P., 140, 188

359

Sanger, F., 38, 51, 52, 54, 55, 56, 57, 59, 60, 61, 63, 66, 69, 72, 75, 102, 104, 105, 143, 180, 188, 214, 244, 342 Satlow, G., 324, 342 Savige, W. E., 204, 225, 251, 252, 253, 284, 285, 286, 332, 334, 336, 342 Scanu, A. W., 140, 182 Scatchard, G., 266, 342 Schachman, H. K., 94, 96, 101 Schenk, G. H., 155, 185 Scheraga, H. A,, 96, 98, 99, 105 Schiefer, H. F., 324, 5'42 Schimmelbusch, C., 2, 3, 33 Schinkel, P. G., 206, 209, 218, 236, 336, 348

Schlack, P., 126, 131, 132, 188 Schleich, H., 162, 184 Schleyer, M., 116, 117, 118, 119, 185, 189 Schleyer, P. von R., 282, 330 Schluetter, R . J., 180, 189 Schmid, K., 58, 99, 177, 178, 186 Schmid, H. J., 6, 36 Schmidt, W. J., 309, 343 Schmidt, W. R., 58, 67, 87, 88, 90, 91, 92, 102, 104

Schmitt, F. O., 115, 117, 118, 119, 175, 181, 188, 189

Schnabel, E., 103 Schneider, F., 145, 162, 166, 167, 170, 189 Schneider, G., 25, 33 Schneider, R. B., 110, 145, 189 Schoberl, A., 248, 252, 253, 254, 343 Schoenberg, C. F., 29, 35 Schoniger, W., 230, 343 Schor, R., 301, 338, 343 Schram, E., 229, 230, 343 Schramm, G., 18, 23, 3.5, 69, 75, 99, 105 Rchricker, K. T., 3, 8, 35 Schroedcr, W. A,, 53, 54, 60, 64, 69, 75, 88, 89, 95, 97, 103, 105, 106, 236, 237, 238, 244, 343 Schrohenloher, R. E., 96, 106 Srhultz, J., 52, 106 Schulz, H., 7, 35 Schwarts, D. R,., 248, 331 Schwarts, H. C., 87, 89, 102 Srhwendener, J., 3, 21, 33 Schwert, G. W., 65, 106 Schwimmer, S., 220, 343 Seegers, W. H., 9, 32

360

AUTHOR INDEX

Segnl, R., 155, 182 Seifter, S., 128, 129, 131, 135, 136, 146, 147, 149, 150, 151, 152, 154, 155, 156, 157, 158, 159, 164, 168, 172, 173, 174, 182, 184, 189

Sela, M., 63, 67, 99, 103 Setna, S. S., 5, 35 Sharry, L. F., 219, 334 Shaw, D. C., 56, 104 Shaw, T. M., 326, 343 Shay, H., 52, 106 Shelton, J. B., 54, 64, 75, 88, 89, 106 Shelton, J. R., 54, 64, 75, 88, 89, 106 Sheppard, R. C., 134, 183 Shiba, T., 135, 189 Shibuya, S., 45, 48, 49, 55, 104 Shields, G. S., 61, 65, 106 Shimizu, I., 278, 343 Shinohara, K,, 229, 231, 343 Shore, M. G. T., 271, 343 Shore, V. C., 293, 338 Shorter, S. A., 303, 328, 343 Stipikitcr, V. O., 118, 188 Shunsuke, M., 96, 105 Siepmann, E., 242, 346 Sikorski, J., 288, 298, 299, 308, 309, 316, 319, 324, 334, 338, 343 Silber, R., 2, 35 Silva, E., 257, 334 Silverberg, A,, 259, 340 Simmonds, D. H., 216, 227, 228, 229, 230, 231, 232, 233, 234, 235, 238, 339, 335, 336, 342, 343 Simon, G., 6 , 32 Simpson, W. S., 299, 343 Sinesi, S. J., 240, 340 Singer, B., 60, 97, 101 Singer, S. J., 160, 188 Sinn, L. G., 65, 81, 100 Skertchly, A., 295, 297, 299, 305, 321, 343 Skertchly, A. R. B., 328, 343 Skinner, B. B., 206, 215, 216, 259, 343 Slabin, L. T., 97, 106 Slobodian, E., 189 Smillie, L. B., 51, 106 Smith, A. L., 259, 283, 337, 343 Smith, E. L., 38, 39, 55, 57, 59, 61, 63, 65, 66, 69, 72, 83, 85, 88, 89, 90, 92, 93, 95, 97, 99, 100, 101, 106, 103, 104, 106,

107, 144, 177, 178, 187, 188, 189, 220, 337

Smith, L. F., 55, 105, 206, 214, 216, 242, 244, 330, 342 Smith, J. C., 324, 342 Smithers, M. J., 64, 102 Smyth, D. G., 41, 57, 61, 62, 67, 69, 89, 106

Snaith, J. W., 282, 306, 309, 327, 335, 337 Snell, E. E., 59, 103 Snell, N. S., 228, 237, 345 Snoke, J. E., 65, 106 Snyder, E. R., 93, 101 Sobue, H., 322, 343 Sokal, G., 6, 8, 10, 21, 22, 35 Sokolovsky, M., 318, 341 Solomons, C. C., 138, 189 Sookne, A. M., 257, 259, 311, 343 Sorm, F., 66, 107 Sotiriou-Provata, M., 280, 282, 343 Spackman, D. H., 44, 55, 91, 102, f06, 227, 237, 343 Speakman, J. B., 211, 226, 235, 236, 254, 256, 257, 258, 259, 265, 266, 269, 271, 276, 277, 279, 284, 303, 304, 306, 307, 308, 310, 311, 317, 318, 320, 323, 326, 327, 328, 331, 332, 333, 334, 337, 339, $41,342, 343, 344, 346 Speakman, P. T., 115, 118, 119, 175, 188, 189, 318, 320, 321, 324, 327, 331, 344 Spies, J. R., 39, 58, 106 Spiro, R. G., 93, 106, 177, 178, 189 Springell, P. H., 194, 196, 216, 224, 225, 248, 256, 336, 338, 339, 344 Spurr, 0. K., 184 Srinivassan, V. R., 280, 309, 346 Stacey, M., 181 Stainsby, G., 115, 116, 117, 183, 188 Stam, P. B., 279, 280, 281, 331 Stanislawski, F., 3, 34 Stanley, W. M., 81, 107 Stark, G. R., 61, 62, 67, 106 Statham, K. W., 226, 283, 286, 336 Stnub, A,, 65, 81, 100 Steber, A,, 132, 148, 162, 170, 184 Steele, R., 282, 337 Steihm, E. R., 97, 100 Stein, W. H., 39, 40, 41, 44, 55, 57, 59, 61, 64, 69, 72, 75, 88, 89, 92, 100, 101,

AUTHOR INDEX

361

Tan, W., 59, 60, 107 Tanaka, N., 223, 344 Steinhardt, J., 226, 256, 259, 260, 261, 262, Tang, J., 78, 106 263, 264, 265, 266, 269, 273, 275, 276, Tanner, C. E., 94, 96, 103 Tarbell, D. S., 254, 346 281, 344 Stephen, J. M. L., 96, 100 Thomas, J., 110, 176, 188, 189 Steuerle, H., 214, 226, 242, 344 Thompson, A. R., 53, 60, 107 Strven, F. S., 60,106 Thompson, E. 0. P., 38, 52, 55, 57, 59, Stillhardt, H., 6, 33 60, 63, 66, 69, 72, 75, 105, 106, 107, Stockell, A,, 59, 106 143, 180, 188, 196, 197, 198, 201, 203, Stokes, R. H., 278, 342 204, 205, 206, 207, 214, 215, 216, 217, Stone, B. D., 327, 344 220, 227, 228, 242, 243, 248, 336, 341, Stone, D., 144, lS3 345 Stone, W. K., 324, 342 Thompson, J. F., 122, lS7 Stoner, R. E., 179, 183 Thorne, C. B., 124, 149, 156, 189 Stott, E., 256, 257, 258, 265, 344 Tietze, F., 68,107 Stoves, J. L., 309, 344 Tiffany, M. L., 222, 301, 345 Strandbcrg, B. E., 293, 338 Tiler, E. M., 223, 230, 339 Strassner, E., 26, 36 Tiselius, A., 94, 107 Street, A., 300, 304, 331 Toda, H., 97, 107 Stretton, A. 0. W., 53, 103 Todd, J., 224, 231, 253, 335 Tokuyasu, K., 96, 107 Stricks, W., 223, 344 Tolgyesi, E., 318, 320, 331 Stringer, H. C. W., 112, 189 TomLZek, V., 66, 107 Strinivassan, V. R., 346 Tomimatsu, Y., 203, 331, 345 Sturtevant, J. M., 65, 68, 103 Sugae, K., 132, 160, 181 Tomlin, S. G., 189 Sugawara, T., 282, 337 Tong, W., 90, 107 Sullivan, M. X., 225, 344 Toperd, G., 230, 337 Tower, D. B., 58, 90, 91, 92, 107 Sutherland, G. B. B. M., 292, 334 Sutton, D. A,, 140, 187 Towne, K., 324, 342 Swallow, D. L., 56, 106, 189 Traumann, K., 224, 346 Swan, J. M., 208, 223, 249, 250, 251, 254, Tristram, G. R., 60, 106 285, 339, 340, 345 Tritch, H., 67, 99 Swanbeck, G., 295, 299, 346 Troshko, E. V., 182 Trotter, I. F., 292, 331, 332 Swart, L. S.,207, 211, 337 Sweetman, B. J., 203, 210, 334 Truscott, T. G., 41, 43, 44, 48, 104 Swenson, R. T., 87, 89, 102 Tsai, L., 8, 33 Sykes, R. L., 137, 138, 140, 141, 189 Tsiganos, C., 252, 334 Synge, R. L. M., 37, 40, 45, 47, 49, 50, 53, Tsugita, A., 81, 107, 132, 160, 177, 178, 102, 104, 106, 223, 227, 230, 237, 333, 341, 343, 344

101, 104, 106

181, 189

Synman, J. G., 231, 345 Szent-Gyorgyi, A. G., 13, 18, 23, 35, 248, 331

Szorenyi, B., 67, 101 Szucht, E., 323, 324, 846

T Takahashi, K., 96, 106 Talka, M., 67, 107 Tan, M., 96, 106

Tucker, D. J., 283, 287, 340 Tuppy, H., 51,52, 66, 69, 81, 93, 106, 107 Turner, K. J., 189 Tuttle, L. C., 155, 187 Tyler, F. H., 91, 105

U Uchio, H., 65, 103 IJhlig, H., 69, 75, 99, Underwood, D. L., 279, 280, 281, 331, 346

362

AUTHOR INDEX

IJntlerwootl, G. E., 107 IJrnes, P., 293, 345

V Vand, V., 292, 338 van der Wyk, A. J. A., 310, 340 Van Ordcn, H . O., 65, 107 V>in Overbckc, M., 229, 230, 253, 3?1, 334, 34G

Van Scott, E. J., 240, 345 Van Slyke, D. D., 39, 40, 107 Van Vunakis, H., 107 Vasquez, J. J., 2, 35 Vassalli, P., 6, 32 Vassiliadis, A. G . P., 280, 282, 309, 320, 343, 345, 346

Veis, A., 114, 116, 118, 119, 160, 161, 180, 183, 189

Veldsman, D. P., 226, 3.45 Verztir, F., 112, 18.9 Vickrrst:tff, T., 257, 259, 275, 939, 343, 546 Vickery, H. B., 37, 38, 41, 107 Vinograd, J. B., IS1 Virtanen, A. I., 122, 187 Virtanen, IJ. K., 187 Vithayatbil, 1'. J., 97, 98, 101, 105 von Bergen, W., 283, 345 von Hcyl, C;. S., 140, 190 Von Hippel, P. H., 95, 97, 102, 107, 113, 115, 116, 117, 186, 189 von Muralt, A. I,., 20, 35

W Waaler, B. A,, 8, 32 Wacker, O., 150, 153, 156, 157, 187 Waelsch, H., 57, 103 Wagman, J., 96, 107 Wagner, A., 253, 342 Wainfon, E., 96, 102 Walden, M. K., 287, 342 Waldron, R. D., 282, 345 Waldschmidt-Leitz, E., 121, 127, 189 Waley, S. G., 63, 99, 107, 122, 124, 182, 189

Walford, R. I,., 110, 145, 189 Walsh, K. A., 66, 107 Walter, W., 49, 102 Warhurton, F. I,,, 310, 328, 345 Ward, A. G., 115, 116, 141, 143, 180, 18G, 188, 189

Ward, W. H., 191, 192, 203, 209, 221, 228, 237, 288, 296, 238, 299, 331, 339, 346 Wartkoff, H. K., 228, 238, 239,336 Watson, J., 63, 107 Watt, I. C., 326, 327, 335, 345 Waykole, P., 115, 119, 18G Weaver, E. S., 116, 184 Wcbh, E, C., 124, 183 Weber, E., 69, 75, U9 Weber, H. IT., 18, 23, 35 Wridinger, A., 280, S38 Weigmann, H. D., 225, 346 Weil, L., 67, 107 Weinmann, F., 51, 99 Weisiger, J. R., 123, 137, 186 Weiss, E., 188 West, G. W., 326, 337 West, P. W., 189 Westheinier, F. H., 62, 10.4 Weston, G. C., 316, 336 Restover, C. J., 222, 301, 346 Whcrrett, J. R., 58, 90, 91, 92, 107 Whewell, C. S., 279, 280, 282, 283, 308, 309, 319, 320, 331, 339, 344, 345, 346 Wliitaker, J. R., 47, 107 ' White, F. H., 248, 346 White, H. J., 271, 279, 280, 331, $45, 346 White, W. F., 97, 107 Whiteley, K. J., 311, 320, 346 Whitfield, R. E., 48, 107 Whitwell, J. C., 233, 234, 336 Wibaux, G., 229, 230, 346 Wiederhorn, N. M., 112, 113, 144, 173, 189 Wieland, T., 189 Wilcox, P. E., 59, 61, 100, 107 Williams, V. A., 273, 346 Williams, J. W., 97, 98, 105 Williams, W. J., 124, 149, 156, 189 Wilson, H . R., 299, 346 Wilson, I,., 51, 107 Winitz, M., 37, 38, 39, 55, 101 Wirtschaftcr, Z. T., 119, 190 Wiseblatt, I,., 51, 107 Witkop, B., 38, 107, 225, 336 Witte, S., 3, 8, 35 Wolf, D. E., 136, 188 Wolf, K., 163, 164, 166, 167, 184 Wolff, G., 138, 139, 185 Wolfram, L. J., 319, 320, 321, 398, 34G Wolfrom, M. L., 190

363

AUTHOR INDEX

Wolman, V., 64, 108 Wood, G. C., 117, 118, 190 Wood, H. N., 156, 181 Woodhouse, J. M., 308, 320, 331 Woodin, A. M., 214, 221, 243, 303, 346 Woods, E. F., 197, 204, 208, 211, 216, 217, 218, 221, 222, 223, 237, 238, 244, 302, 303, 335, 336, 337, 346 Woods, H. J., 280, 282, 288, 291, 295, 297, 299, 300, 305, 306, 307, 308, 309, 310, 311, 316, 318, 319, 321, 324, 328, $31, 338, 341, 343, 345, 346

Woods, K. R., 227, 346 Woods, P. B., 291, 299,341 Wortmann, V., 240, 241, 341 Wright, J. H., 2, 35 Wright, M. I,., 260, 274, 346 Wronski, M., 224, 346 WU, Y.-C., 177, 178, 187 Wyckoff, R. W. G., 291,333 Wyman, J., 97, 99

Y Yajima, H., 64, 89, 92, f02 Yakel, H., 292, 346 Yale, H. L., 128, 149, 154, 190 Yamanaka, T., 97, 105

Yamashina, I., 177, 178, 186, 190 Yamashita, T., 65, 103 Yanaihara, C., 64, 89, 92, 108 Yanari, S. S., 74, 100, 127, 190 Yang, J. T., 213, 341 Yanihara, N., 64, 106 Yashpe, J., 155, 156, 190 Yates, J. R., 256, 316, 329, 335 Yonetani, T., 80, 108 Young, E. G., 118, 181, 190 Young, J., 81, 107

Z Zacharias, J., 163, 164, 170, 172, 188 Zahn, H., 113, 140, 190, 194, 224, 225, 230, 242, 253, 254, 256, 280, 308, 310, 311, 320, 323, 325, 334, 337, 346 Zaiser, E. M., 260, 261, 262, 275, 344 Zarcharius, R. M., 122, 187 Zelmenis, G., 169, 183 Ziegler, K., 230, 245, 318, 334, 338, 346 Zimmer, E., 115, 119, 186 Zimmerman, M., 126, 134, 135, 186 Zingsheim, M., 240, 346 Zito, R., 97, 99 Zuber, H., 224, 346 Zucker, M. B., 3, 5, 8, 9, 35

SUBJECT INDEX enzymatic hydrolysis of, 93 glycopeptide study of, 177 Chromatium heme protein, enzymalic: hydrolysis of, 93 Chymotrypsin, enzymatic hydrolysis of, 96 protein hydrolysis by, 68-74, 96-97 Clot retraction, blood platelets and, 9-10 Collagen, acid-soluble, denatured, 117-119 aldehyde links of, 179 e-amino group availability in, 138, 140 arginine cross-links, 179-180 carbohydrate linkages in, 162-169 B periodic acid study of, 165-169 Bacillus anthrczcis, poly-b-ylutamic acid carbohydrate removal from, 162-164 from, 123-124, 126 collagenase study of cross-links in, 173 Bacillus subtilii;, a-component of, 173 “Nagarsc” from, 80 cross-links, 10 poly-y-glutamic acid in, 123, 126 carbohydrak and ester interrelationsubtilisin from, 80 ships in, 169-178 Bacitracin A, P-aspartyl linkage in, 123 physical evidence for, 111-114 Bacterial proteinases, prot,ein hydrolysis enzymatic hydrolysis of, 97 by, 80-83 ester-like linkages in, 144-162 Blood platelets, hytirazine use on, 158-161, 173 clot retraction and, 9-10 hydroxyluinine use in, 149-158, 170 essential factors for, 7 lithium borohydridc use for, 146-149 in hemostasis, 7-9 -gelatin transformation, 114115 metabolism of, 6-7 y-glutamyl peptidc linkages in, 131-136 origin and morphology of, 2-6 hexoses in, at various ages, 174 “rrlaxing factor” of, 20 insoluble, ester linkages of, 175 rctractozyme from, 9-10 lathyritic, 119-120 role in blood coagulation, 7 linkages (unusual) of, 109-190 thrombin effects on, 3, 5-6 peroxide cross-links of, 180 viscous metamorphosis of, 3, 5-6 phosphate-mediated cross-links of, l3otulinuni toxin, enzymatic hydrolysis 180-181 of, 96 procollagen compared to, 117 protein-carbohydrate link of, 178 C relationship to gelatin, 114-116 Callus, N-terminal amino acids in, 243 soluble, 116-119 Carboxypeptidasrs, protein hydrolysis trypsin attack on, 165 by, 87-88, 97 Collagenase, collagen hydrolysis by, 97 Casein, enzymatic hydrolysis of, 82 Contractile proteins, Catitlase, enzyrriiitic hydrolysis of, 96 actomyosin type of, 22-27 Chondroitin sulfate complex, enzymatic activities of, 30-31 364

A

Adrenocorticotropic hormone, enzymatic hydrolysis of, 96, 97 Aldolase, enzymatic hydrolysis by, 96, 97 Amidrs, hydrolysis of, 41 Amino acids, photochrmical changes in, 285-287 e-Aniinopeptide linkages, incorporation of, 142 in proteins, 136-144 Aspartic acid peptides, 120-136 hydrolysis of, 51-52 naturally occurring, 122-123

365

SUBJECT INDEX

from smooth muscle, 24-26 from striated muscle, 23-24 blood platelet, see Thrombosthenin muscle structure and, 28-30 quantitative distribution of, 25, 28 rat sarcoma cell, 2627, 31 Corticotropin, chymotryptic hydrolysis of, 72 Crotoxin, enzymatic hydrolysis of, 97 Cystine, reactivity of in keratin, 247-256 Cytochrome c, enzymatic hydrolysis of, 66, 72, 93, 97

D Dentine collagen, phosphate cross-links of, 180-181 Dinitrophenyl proteins, hydrolysis of, 60 Diphtheria antitoxin, enzymatic hydrolysis of, 96 Disulfide bonds, in proteins, hydrolysis of, 54-55

E Edestin, y-glutamyl links in, 124-125 hydrolysis studies on, 51 Egg albumin, aldehyde links in, 179 peptide-carbohydrate links of, 176 Egg shell keratin, amino acid composition of, 238-240 Elastin, cross-links of, 110, 176 Enolase, enzymatic hydrolysis of, 96, 97 Ester linkages, in proteins, 145

F Feather(s), keratin, amino acid composition of, 236235 N-terminal amino acids in, 244 structure of, 301-303 protein, amino acid composition of, 222-223 end groups of, 214-215 extraction of, 220-223 sequence studies on, 245-247 Fetuin, enzymatic hydrolysis of, 93 glycoprotein study of, 177, 178

Fibers (animal), disulfide bond rupture in, 322-323 extension in nonaqueous solutions, 323324 plasticity, creep, and stress relaxation of, 307-310 set of, 317-322 stress-strain relationships of, 303-305, 322-326 stretched, retractive forces of, 310-311 supercontraction of, 312-317 and set of, 305-307 thiol group block of, 324 torsional properties of, 310 Fibrinogen, enzymatic hydrolysis of, 96

G Gelatin, aldehyde links in, 179 €-amino group availability in, 138 -collagen transformation, 115-116 ester linkage determination in, 149-158 y-glutamyl links in, 131-136 relationship to collagen, 114-116 Gliadin, isoglutaminyl residues in, 126 Globin, dinitrophenyl, hydrolysis of, 60 y-Globulin(s), enzymatic hydrolysis of, 93, 95 glycopeptide study of, 177, 178 peptide-carbohydrate links of, 176 Glucagon, trypsin hydrolysis of, 65-66 y-Glutamyl peptide linkages, 120-136 identification of, 125, 126 naturally occurring, 121-122 Glutathione, y-glutamyl peptides in, 121 Glycoprotein, al-,glycoprotein study of, 177, 178 protein-carbohydrate links of, 176 structures of various, 178 Growth hormone, enzymatic hydrolysis of, 96

H Hair, human, amino acid composition of, 232 N-terminal amino groups in, 243 Hemoglobin, A*, structural analysis of, 53-54 chymotrypsin hydrolysis of, 71-73 enzymatic hydrolysis of, 97

366

SUBJECT INDEX

y-glutamyl links in, 124-125 pcpsin hydrolysis of, 74, 76-77, 7'3 tryl)tic hydrolysis of, 66-67 Hemostasis, platclet role in, 7 Heparin sulfate complcx, glycopeptide sludy of, 177 Hofmann rearrangement, y-glutamyl peptide identification using, 125, 126 Horn, acid-binding capacity of, 260 amino acid composition of, 238, 239 Horsehair, N-terminal amino groups in, 243 Hydrazine, use t o determine ester linkages, 158-161 Hydrolysis of proteins, see Protein hydrolysis Hydroxylamine, use to determine ester linkages, 149-158

I Ich thyocol, aldehyde links of, 179 carbohydrate of, 168-169 y-glutamyl peptides in, 131 hexose linkage in, 173-174 Insulin, chymotryptic hydrolysis of, 72 enzymatic hydrolysis of, 96 partial acid hydrolysis of, 52-53 side reactions in, 55-57

K Kcratins, 191-346 a-7

electron microscopy of, 288 structural models of, 292-300 structure of, 287-292 X-ray diffraction of, 291 amino acid composition of, 227-242 amino end group determination in, 226 analytical methods for, 223-227 ,&, structure of, 300-301 characterization of, 193-223 by oxidation, 206-207 I)y reduction, 193-206 chemical modification of, 311-330 chcmical reactivity of, 247-287 composition of, 223-247 rystcine determination in, 224-225

cystine determination in, 223-224 cystine reactivity in, 247-256 end groups of, 242-244 fiber histology of, 1%-193 cortex, 193 cuticle, 192-193 medulla, 193 infrared spectra of, 291-292 ionized basic groups in, 226-227 isolation of, 19%223 using bisulfite, 207-208 by oxidation, 206-207 by reduction, 193-206 lanthionine determination in, 225-226 lanthionine-forming reactions of, 252255 molecular structure of, 287-303 oxidation of, 251-252 photochemical reactivity of, 283-287 physical properties compared to chemical structure, 303-330 reaction with ions, 256-283 reduction of, 247-248 setting, mechanism of, 320 stress-strain relationship of, 304-305, 330 sulfitolysis of, 248-251 supercontraction of, reagents producing, 308-309 set of, 305-307 swelling and chemical reactivity of, 277-280 torsional properties of, 310 tryptophan determination in, 226 (See also Feather, Fibers, Hair, Horn, Quill, Wool) Keratoses, amino acid composition of, 215-216 from wool, 2-207 sequence studies on, 246-247

L Lanthionine-forming reactions in keratins, 252 Lathyrism, of collagen, 119-120 Leucine aminopeptidase, protein hydrolysis by, 88-89, 97 Iipovitellin, enzymatic hydrolysis of, 97 Lithium borohydride, as reagent for esters, 146-149

SUBJECT INDEX

Lossen rearrangement, y-glutamamyl and P-aspartyl residue identification by, 128-129,154 Lysozyme, chymotryptic hydrolysis of, 72 hydrolysis studies on, 51

367

Phosphothreonine ester bonds, hydrolysis of, 60-61 Plasma albumin, enzymatic hydrolysis of, 96 Procollagen, compared t o collagen, 117 cross-links of, 171 M Pronase, protein hydrolysis by, 80 Protein(s), Micrococcus lysodeikticus mucopeptide, carboxymethyl derivatives of, hydrolcross-links of, 127 ysis of, 61 Metals, peptide bond hydrolysis by, 62 conjugated, enzymatic hydrolysis of, 93 Mohair, amino acid composition of, 232 contractile, see Contractile protein Myosin, enzymatic hydrolysis of, 96 cyanate derivatives of, hydrolysis of, 61 N derivatives, hydrolysis of, 60-61 Nagarse, protein hydrolysis by, 80 ester links in, 145 hydrolysis of, see Protein hydrolysis 0 Protein hydrolysis, 37-107 Ovalbumin, acid, 39-61 enzymatic hydrolysis of, 93, 97 amide hydrolysis in, 41 glycopeptide study of, 177, 178 complete, 57-60 Ovine submaxillary gland mucoprotein, electrostatic effects in, 41-45 peptide carbohydrate links of, 176 partial, 40, 52-57 Ovomucoid, glycopept.ide study of, 177, side reactions in, 55-57 178 specificity of, 40 Oxytocin, enzymatic hydrolysis of, 96, 97 steric effects in, 4S50 of sulfide bonds, 54-55 P alkaline, 60-61 Pancreatin, protein hydrolysis by, 91 by bacterial proteinases, 80-83 Papain, by carboxypeptidases A and B, 87-88, chymotryptic hydrolysis of, 72 97 enzymatic hydrolysis of, 97 by chymotrypsin, 68-74, 96-97 protein hydrolysis by, 83-87, 96 of derivatives, 60-61 trypsin hydrolysis of, 66 by leucine aminopeptidnse, 88-89, 97 Pepsin, enzymatic, 63-99 enzymatic hydrolysis of, 96 advantages of, 63-64 protein hydrolysis by, 74-83, 96 “all or none,” type, 94 Peptide bond, total, 89-94 hydrolysis of, 38-39 “zipper” type, 94 acid, 46-47 by papain, 83-87, 96 Peptides, photochrmical changes in, by pepsin, 74-83, 96 285-287 peptide bond hydrolysis in, 38-39 Periodic acid, collagen carbohydrate by metals and metal chelates, 62 study using, 165-169 hy S. gnkeus protease, 97 Phosphoiylase, enzymatic hydrolysis of, by subtilisin, 97 93 by trypsin, 64-68, 96 Phosphoserine ester bonds, hydrolysis of, Psoriasis scales, N-terminal amino 6081 acids in, 243

368

SUBJECT INDEX

Q Quill keratin, amino acid composition of, 238, 239

R Resilin, crow-links of (nondisulfide), 110 Ribonuclease, chymotrypsin hydrolysis of, 72 enzymatic hydrolysis of, 9 6 9 9 residue release from, 52

5 Serine peptides, hydrolysis of, 50-51 Serum albumin, enzymatic hydrolysis of, 96 Serum globulins, enzymatic hydrolysis of, 97 Skin keratin, amino acid composition of, 238-242 Soybean trypsin inhibitor, enzymatic hydrolysis of, 97 Streptomyces griseus, “Pronasc” from, 80 protein hydrolysis by, 92 Subtilisin, protein hydrolysis by, 80,97 specificity of, 81

T Taka-amylase, enzymatic hydrolysis of, 97 Taka-amylase A, glycopeptide study of, 177, 178 Threonine peptides, hydrolysis of, 50-51 Thrombosthenin, 1-35 A, 18-20 ATPase activity of, 14-18, 22 isolation of, 10-12 M, 18-20 other contractile proteins and, 28 physiological significance of, 21-22 “relaxing factor” and, 20 solubility of, 11-12 superprecipitation of, 12-14 Thyroglobulin, enzymatic hydrolysis of, 97 Tobacco mosaic virus, enzymatic hydrolysis of, 97

Tropomyosin, enzymatic hydrolysis of, 96 Tiypsin, enzymatic hydrolysis of, 96 hydrolysis studies on, 51 protein hydrolysis by, 64-68 in synthetic substrates, 65 protein-derivatives hydrolysis by, 6768, 91

W Wheat protein, y-glutamyl link in, 127 Wool, acid-alkali capacity for, 259-261 amide content of, 229 amino acid composition of, 227-236 nutrition and weathering effects on, 234 blocking of thiol groups of, 324 breaking disulfide bonds of, 195-208 charged groups in, 258-259 copper-deficient, amino acid composition of, 218 covalent linkage formation of, 317-319 creep and stress relaxation of, 311-312 cystine reactivity of, 255-256 extracted proteins of, origin, 209-210 fiber, models of, 328-330 fibers of different strains, etc., composition of, 231-233 high-sulfur protein derivatives SCMKB, 198-200 hydrogen bond formation in, 319-320 internal solution of, 27&277 isoelectric point of, 257-258 keratoses from, 2W207 loading rate and “transition temperature,” 324 low-sulfur protein derivatives SCMKA, 195, 198, 202 morphological components of, amino acid composition of, 233-234 N-terminal amino acids of, 242 oxidation of, 251 peptide bond hydrolysis of, 208-209, 325-326 photochemical reactivity of, 283-285 prevention of photochemical degradation of, 287 protein extraction from, 194-223

SUBJECT INDEX

protein interaction during, 220 by reduction, 195-206 proteins, see Wool proteins reaction with ions, 256-283 Peters-Speakman theory of, 271-275 Steinhardt-Harris theory of, 265-276 Vickerstaff-Delmenico-Peters theory of, 275-276 reduction of, 24EL249 roots, protein extraction from, 204 sequence studies on, 244 set of, 317-322 mechanism of, 320 sorption and swelling of, 326-328 adsorbates effect on regain, 327 pore size of keratin, 327-328 of water, 326

369

sulfitolysis of, 248-251 sulfur balance for, 229 supercontraction of, 312-317 in salt solutions, 280-283 set of and, 305-307 titration data for, 256, 261-265 torsional properties of, 310 (See also Fibers) Wool protein, amino acid composition of, 215-219, 227-236 conformation of, 213 end groups of, 213-215 molecular wcight of, 210 propertlies of, 210-220 root, amino acid composition of, 219 sequence studies on, 244-247

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